CA2191603A1

CA2191603A1 - Antifungal-wound healing compositions and methods for preparing and using same

Info

Publication number: CA2191603A1
Application number: CA002191603A
Authority: CA
Inventors: Alain Martin
Original assignee: Individual
Current assignee: Warner Lambert Co LLC
Priority date: 1994-07-22
Filing date: 1995-07-07
Publication date: 1996-02-08
Also published as: US5663208A; MX9606501A; WO1996003149A1; EP0773795A1; PH31465A; JPH10503200A; NZ290029A

Abstract

This invention pertains to therapeutic antifungal-wound healing compositions. The compositions comprise a therapeutically effective amount of an antifungal agent and a wound healing composition. In one embodiment the wound healing composition comprises (a) pyruvate; (b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids. The therapeutic antifungal-wound healing 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 antifungal-wound healing compositions and the pharmaceutical products in which the compositions may be used.

Description

Wo96/03l49 r~l/o. s ANTIFUNGAL-WOUND HEALING COMPOSITIONS
AND METHODS FOR PREPARING AND USING SAME

BACKGROUND OF THE INVENTION

1. Field of the Invention This invention pertains to therapeutic antifungal-wound healing ~ q~ More Li~uldlly, the antifungal-wound healing ~ q ,S ~: ."c comprise an antifungal S agent and a therapeutic wound healing c~ andlor its n7,.t9l~nljt~C Thisinvention also pertains to methods for preparing and using the antifungal-wound healing ~ and the 1~ I products in which the therapeutic ~.. q.n ~~;...,s may be used.

A preferred ~ .o~ 1 of the therapeutic wound healing ~ q~ of this invention comprises (a) pyruvate selected from the group consisting of pyruvic acid, lly acceptable salts of pyruvic acid, and mixtures thereof, (b) an qr~tinYi/~ t and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular ' and lS ~ 1;.. of " ",, l;-- cells.

wo 96103149 2 1 9 1 6 0 3 2. Descripffon of the l~a(.
Wound Healing Wounds are intemal or extema] bodily injuries or lesions caused by physical 5means, such as ' I chemical viral, bacterial, or thermal means, which disrupt the normal continuity of stluctures. Such bodily injuries include contusions, wounds in which the skin is un~roken, incisions, wounds in which the skin is broken by â
cutting instrument, and lacerations, wounds in which the skin is broken by a dull or blunt instrument. Wounds may be caused by accidents or by surgical procedurcs.
Patients who suffer major wounds could benefit from an rll} ~ l.. s in the wound healing process.

Wound healing consists of a series of processes whereby injured tissue is repaired, specialized tissue is ~ J, and new tissue is ~cul~~ ,d. Wound 15healing consists of three major phases: a) an n phase (0-3 days), b) a cellular proliferation phase (3~12 days), and (c) a remodeling phase (3 days-6 months).

During the ~ phase, platelet a23~ aliu~ and clotting form a 20matrix which traps plasma proteins and blood cells to induce the influx of various types of cells. During the cellular ~ a;~ 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 fornnation of scar tissue.
When cells are injured or killed as a result of a wound, a wound healing step is desirable to resuscitate the injured cells and produce new cells to replace the dead cells. The healing process requires the reversal of ~iylulw~ the DUE~JICDD;UII of i,,n-,,,, -~;ull, and the stimulation of cellular viability and proliferation. Wounds 30require 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 fommation by fbroblasts.

RECrIFIED SHEET (RULE 91 ISA/EP

wo96/03149 21 q1 603 .

~ ' ' cells are . 'y e~cposed to activated oxygen species such as superoxide (~2-)' hydrogen peroxide (H2O2), hydroxyl radical (OH ), and singlet oxygen (102). In vrvo, these reactive o~rygen ~ ' are generated by cells in response to aerobic . " ~ " catabolisrn of drugs and other ~ hi~ltir s~ ultraviolet and x-ray radiation, and the respiratory burst of phagocytic cells (such as white blood cells) to kill invading bacteria such as those introduced through wounds. Hydrogen perc-xide, for example, is produced during respiration of most living organisrnsespecially by stressed and injured cells.

These active oxygen species can injure cells. An irnportant exarnple of such damage is lipid p~ ;,... which involves the oxidative ArgrqA~n of l ~
lipids. Lipid p~ ", ;A - ;. ., is highly detrimental to mernbrane structure and function and can cause numerous cy~ effects. Cells defend against lipid p ..~ l- ;.. by producing radical scavengers such as superoxide dismutase, catalase, and peroxidase. Injured cells have a decrersed ability to produce radical scavengers.
Exccss hydrogen peroxide can react vith DNA to cause backbune breakage, produce mutations, and alter and hberate bases. Hydrogen peroxide can also react with ~,~.IIIIIUhl~D to open the 5, 6-double bond, which reaction inhrbits the ability of ~ ' to hydrogen bond to ~ - - ~ bases, Hrllaender et al. (1971). Such oxidative l.:.. ~ .,.. ~l injury can result in the loss of cellular membrane integrity, reduced enzyme activity, changes in transport kinetics, changes in membrane lipid content, and leakage of potassium ions, amino acids, and other cellular material.

Anti~iAqntc 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 ~ .,Al,y with hydrogen peroxide to protect cells from cytolytic effects, O'Donnell-Tormey et aL, J. Exp. Med., 165, pp. 500-514 (1987).

United States Patents 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, 1~;D~ L~ which consist of applying to the affected area a topical ~
comprising from about 1% to about 20% of a lower aliphatic compound containing wo 96/03149 2 T 9 1 6 0 3 P~

from two to six carbon atoms selected from the group consisting of ~Ipha-Ly Lu~ "iL, Alpha-ketoacids and esters thereof, and 3-Ly~Lu~y~ , acid in a .. _ll.y acceptable carrier. The aliphatic compounds include pyTuvic 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 ~ ~c~-,.,Liv.,l~, for treating dry skin which consists of applying to the affected area a topical r~ comprising from about 1% toabout 20% of a compound selected from the group consisting of amides and =onium salts of .41pha-Ly~Lu~ ,;L, ~B-L~u7.~;L~ and ~Ipha-ketoacids in a IJ .,..-.. .. lly acceptable carrier. The compounds include the amides and =onium salts of pyruvic acid and lactic acid.

United States Patent No. 4,234,599, issued to Van Scott ei al., discloses a method for treating actinic and nonactinic skin keratoses which consists of applying to the affected area a topical ~ comprising an effective amount of a compound selected from the gr~up consisting of ~1~7ha-L~..AyG4;L, ~ Ly~Lu~Ly~.;L, and Alpha-ketoacids in a lJ " ~ ly acceptable calrier. The aciLc .
include pyruvic acid and lactic aciL
United States Patent No. 4,294,852, issued to Wildnauer et al., discloses a for treating skin which comprises the Alpha-hy Lu,.y~";L"B-hydroxyacids, and ~Ipha-ketoacids disclosed above by Van 5cott et aL in ~ ;-... with C3-Cg aliphatic alcohols.
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, .".,~.,.,Li ~ , or a mixture of D-,B-Ly~Lu~ and ~r~tr ~ ' ' , in a ratio from 6:1 to 0.5:1, .~ ,L~
Sodium pyruvate has been reported to reduce the number of erosions, ulcers, and h.. ,.1. ~ on the gastric mucosa in ~unea pigs and rats caused by WO96/03149 2 1 9 1 6~3 P_IIIJ..,~.,. I

a~.,L,~IDal;~ , acid. The analgesic and antipyretic properties of ac~,Ly' ' ~, acid were not unpaired by sodium pyruvate, Pi ' ,A,, ~ . lr..,~.' -- ~ 33, pp. 410-415 and 415-416 (1983).

Pyruvate has been reported to exert a positive inotropic effect in stunned uu~. ' which is a prolonged ventricular d~Drull~,Luu following brief periods of coronary artery occlusions which does not produce irreversible damage, Mentzer et al., Ann. Surg., 209, pp. 629-633 (1989).

Pyruvate has been reported to produce a relative ~ .. , of left ventricular pressure and work pararneter and to reduce the size of infarctions. Pyruvate irnproves resurnption of s, beating of the heart and restoration of normal rates and pressure d~ ,L~u~~ Bunger et aL, J. Mol. Cell. Cardiol., 18, pp. 423-438 (1986), Mochizuki ef al., J. Physiol. (Paris), 76, pp. 805-812 (1980), Regitz et al., Cardiovasc. Res., 15, pp. 652-658 (1981), Giarmelli et aL, Ann. Thorac. Surg., 21, pp. 386-396 (1976).

Sodium pyruvate has been reporkd to act as an antagonist to cyanide ' ' ' ' ' (~I~DIIIIW~ tbrough the formation of a ~ L-.I) and to protect against the lethal effects of sodium sulfide and to retard the onset and d.,.,' . of functional".,..,l.lmln~; -1, and hirrh--mir~l measures of acrylamide ne:lropathy of axons,Schwartz etal.,Toxicol. Appl. Pharrnacol.,50,pp. 437-442(1979),Sabri etal., Brain Res., 483, pp. 1-11 (1989).

A 1 ~ h .~ cure of advanced L1210 leul~nia has been reported usmg sodium pyruvate to restore ~ , deformed red blood cells to normal. The deformed red blood cells prevented adequate drug delivery to tumor cells, Cohen,Cancer Chcmother. Pharmacol., 5, pp. 175-179 (1981).

Primary cultures of heterotopic tracheal t~ansplant exposed in uvo to 7, 12-dimethyl-b~7(~ ' were reported to be ! " ~l~y maintained in enrichment medium ~ , .,..e..l with sodiurn pyruvate along with cultures of ~ ~ 2 WO96/03149 21 91 603 r.~

stimulated peripheral blood ly~L.J~"yLD, and PI~-YV.~D and L~L~;du~ pig embryos, and hurnan blastocysts, Shacter, J. Immunol. Methods, 99, pp. 259-270 (1987), Marchok etal., Cancer Res., 37, pp. 1811-1821 (1977), Davis, J. Reprod. Fer~l. Suppl., 33, pp. 115-124 (1985), Okamoto et aL, No To Shinlcei, 38, pp. 593-598 (1986), Cohen et al., J. In Vitro Fert. Embryo Transfer, 2, pp. 59-64 (1985).

United States Patents Nos. 4,158,057, 4,351,835, 4,415,576, and 4,645,764, aU issued to Stanko, disclûse methods for preventing 6e 2 ' of fat in the liver of a mammal due to the ingestion of alcohol, for controlling weight in a mammAl, for inhlbiting body fat while increasing protein ~ ~ .S.AI;.". in a mammal, and for controlling the deposition of body fat in a living being, I~D~ The methods comprise A l g to the mammal a therapeutic mixture of pyruvak and d;Lylllu/~y~ and optionally nboflavin. Unikd States Paknt No. 4,548,937, issued to Stanlco, discloses a method for controlling the weight gain of a mammal which comprises ' _ to the mammal a ' "~I effective amount of pyruvate, and optionaUy nboflavin. United States Patent No. 4,812,479, issued toStanko, discloses a method for controlling the weight gain of a mammal which comprises ' ~ to the mammal a ' , 'b, effective arnount of '~ u....... , and optionally n~oflavin and pynlvate.

Rats fed a calcium-oxalate lithogenic diet including sodium pyruvate were reported to develop fewer minaly calculi (stones) than control rats not given sodiurn pyluvate, Ogawa et aL, Hinyok;ka Kiyo, 32, pp. 1341-1347 (1986).
United States Patent No. 4,521,375, issued to Houlsby, discloses a metbod for sterilizing surfaces which come into contact with living tissue. The method comprises steriLzing the surface with aqueous hydrogen peroxide and then, s...l,,..,p the surface with pyruvic acid.

WO96/03149 21 91 603 r~

United States Patent No. 4,416,982, issued to Tauda et al., discloses a method for ~ ".g hydrogen peroxide by reacting the hydrogen perwcide with a phenol or amline derivative in the presence of peroxidase.

5United States Patent No. 4,696,917, issued to Lindstrom et al., discloses an eye irrigation solution which cornprises Eagle's Minimum Essential Medium with Earle's salts, choDdroitin sulfak~ a buffer NlutiOn, 2~ and a pyruvate.
The irrigation solution may optionally contain ascorbic acid and ~Ipha-tocopherol.
United States Patent No. 4,725,586, issued to Lindshom et aL, discloses an irrigation 10Nlution which comprises a balanced salt solution"~ hn suhàk~ a buffer Nlution,2~ sodium ~' ' or dexhrose, a pyruvate, a sodium phosphate buffer system, and cystine. The irrigation solution may optionally contain ascorbic acid and gamma-tocopherol.

15United States Paknt No. 3,887,702 issued to Baldwin, discloses a for hreating fingemails and toeDails which consists essentially of Nybean oil or sunflower oil in ' ' ' with Vitamin E.

United States Patent No. 4,847,069, issued to Bissen et aL, discloses a20~Lulu~lu;~,~,L;ve ~ comprising (a) a ~u.l,oh~Lu~,, acid, (b) an anti-A y agent selected from steroidal - n ' y agents and a natmal anti-- n ' y agent, and (c) a topica] carrier. Fahy acids may be present as an omoilient. United States Paknt No. 4,847,071, issued to Bissett et aL, discloses a u~ uv~ comprising (a) a tocopherol or tocopherol oster radical scavenger, (b) an anti n- .. ~ .y agent seleckd from steroidal anti-;~.ll- . . -.. y agents and a natm.~l anti n ' y agent, and (c) a topical caiTier. United States Patent No. 4,847,072, issued to Bissett et aL, discloses a topical ~ ~- y...- '~;....
comprising not more than 25~/o tocopherol sorbate in a topical cairier.

30United 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 hpophilic nutrient, and SelUm albumin, and wo96/03149 r~ s,, I I

c.y.,lOP The lipophilic substances include, ' fatty acids and lipophilic vitamins such as Vitamin A, D, aod E. Ascorbic acid may also be present United Kingdompatentapplication no. 2,196,348A, to Kovar et ai., discloses a synthetic culture medmm which comprises inorgaoic salts, ",~ amino acids, vitamins, buffering agents, and optionally sodium pyruvate adding .., _ - "
hydroxide or _ oxide to the emulsion The oil phase may include chicken fat.

United States Patent No. 4,284,630, issued to Yu e~ aL, discloses a method for stabilizing a water-in-oil emulsion which comprises adding rn~E~r~~Tm hydro~ide or g oxide to the emulsion The oil phase may include chicken fat Preparation HTM has been reported to increase the rate of wound healing in artifcially created rectal ulcers The active ingredients in Preparation HTM are skin respiratory factor and shark liver oil, S ' ~ et al., Digestive Diseases and Sciences, 29, pp 829-832 (1984) The addition of sodium pyruvate to bacterial and yeast systems has been reported to inhibit hydrogen per~cide production, enhance growth, and protect the systems against the toxicity of reactive oxygen ' The, I fatty acids and saturated fatty acids contained within chicken fat enhanced membrane repair and reduced ~,y~uLu~ y. The ' glutathione and IlLu~lyl l- reduced the injury induced by oxygen radical species, Mar~n, Ph.D. thesis, (1987-89).
United States Patent No. 4,615,697, issued to Robinson, discloses a controlled release treatment ~ complising a treating agent and a l,io~;h_Div~
agent comprising a \ s~ l,k but water-insoluble, fibrous çross l _b~-~ carboxy-functional polyrner European patent apphcation no 0410696Al, to Kellaway et al, discloses a " ~ , delivery system comprising a treating agent and a polyacrylic acid cross-WO96/03149 2i 91603 r~l,.Jes.~ I

Lnked with from about 1% to about 20a/o by weight of a pGlhLyJ~u~.y compound such as a sugar, cyclitol, or lower polyhydric alcohol.

al ~fections s Ol.l...u ~ fimgal infections are genera~lly caused by nolmally n( . ' ,, orgsnisms in patients whose host defense ~ have been -- d Hostdefense.. ~ L~. ' g anatomic,or '), may be altered or breached by disease or trauma, or by procedures or agents used fordiagnosisortherapy. Thus,v~.lJ.. ~ infectionrnayoccurif A~ U~ therapy alters the normal ,~ . between host and microbe, or if the host defen~e . . --.~ have been altered by bums, anemia, neoplasms, metaboLc disordcrs, irradiation, foreign bodies, ... ..-~ ~l.l..cu~.;v.. or cytotoxic drugs, c ~ - .";.l~, or diagnostic or therapeutic ~ The underlying alteration ~I~La~u.._a tbe patient to infections from ... r;~ microflora or from organisms acquired by contact with other L.d;v-' r, : r I women often suffer from atfophy in the vaginal area. This atrophy generally leads to cellular thinnmg of tissue which causes the tissue to break easily, to form scabs, and to lose elasticity. ~, , ' women also suffer with a diminished rate and amount of vaginal lubrication and decreased acidity of thevaginal cllvilulr~.ll~ which pemmits ~lulcDu~blc bacteria and pathogens to flourish.
r~rh~lululc~ CUII~r~,llLiUu~l douches contain strong ~ , .ul lc that kill normal flora allowing pathogens to grow and invade the genib~l area. As a f---, , many women suffer with Candida infections, caused primarily by the yeast Candida albi,,ans.
Candida infections are genelally treated with vaginal inserts or s ~ containing an antifungal agent such as .. :... .. 1. or ~

SUMMARY OF Tl~ INVEN~ON
This invention pertains to therapeutic antifungal woundhealing f~ " "1" '- 'I ~ ''' -The f~ ;.. "c of this invention comprise a ' 'lSr effective amount of an WO96/03149 2191603 r~l~u l I

antifungal agent and a wound healing ~ A preferred r J.~p1; .. of the wound healing ~ 6~ of this invention comprises (a) pyruvate solected from the group consisting of pyruvic acid, lJ~ ; lly acceptable salts of pymvic acid, andmixtures thereof; (b) an A.,~ and (c) a mixture of saturated and ~ ' fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular ~ ibl~ ., and.~ . of ' cells. The therapeutic antifungal-wound healing c~ of this invention may be utilized in a wide variety of ~' - ' products. This invention also relates to methods for preparing and using the therapeutic ' ~ ' ..JUIId healing rA~Y~ and the 1.1,- ...- .~ ~1 products in which the therapeutic rA~ ~-U~ - may be used.

This invention further comprises augmented therapeutic antifungal-wound healing ''~ comprising antifungal agents and a therapeutic wound healing Y~ in ~ J~ I with one or more additional ,., ~1 r - Il- .'~ This invention also relates to methods for preparing and using the augmented therapeutic antifungal-wound healing ~ and the rL 1 products in which the augmented CA~YI~ may be used BRlEF DESCRIPTION OF TUE DRAWINGS

Figure I depicts in bar graph forrnat the viability of U937 monocytic cells following exposure of the cells to various A~ Examples 1-5).
Figure 2 depicts in bar graph format the viabllity of U937 monoc.~tic cells following exposure of the cells to various ~.. ' ---l;.. ~ of ' (Examples 6-13).

Figure 3 depicts in bar graph format the levels of hydrogen peroxide produced by U937 monocytic cells following exposure of the cells to various Anhr~ l (Examples 14-18).

21 9~ 603 wo 96/03149 Figure 4 depicts in bar graph format the levels of hydrogen peroxide produced by U937 monocytic cells following exposure of the cells to various ~ of ' (Examples 19-26).

5Figure 5 depicts in bar graph format the levels of hydrogen peroxide produced by U937 monocytic cells following exposure of the cells to various ~ c of _ ~ l ,t~ with and without a mixture of saturated and, fatty acids (Examples 27-32).

10Figure 6 depicts in bar graph format the levels of hydrogen peroxide produced by epidermal keratinocytes following exposure of the cells to various ' with and without a mixture of saturated and, ' fatty acids (Exarnples 3342).

15Figure 7 depicts in bar graph forrnat the levels of hydrogen peroxide produced by epidermal I ~D following exposure of the cells to various - of ~ with and without a mixture of saturated and fatty acids (Examples 43-52).

20Figure 8 depicts in bar graph format a summary analysis of the levels of hydrogen peroxide produced by epidermal kclalhlu~,J ~D following exposure of the cells to the individual c.. ~l.u~ of the wound healing ~ ~J~ .. to various ".. 1. -.- ;.,.. ~ of the wound healing ~ . and to the wound healing f u~
Figures 9A-9D are ~ .a~hs of wounded mice after 4 days of treatmcnt with:
no ~ c~ n (Figure 9A, control); a petrolatum base r..,,,...l- ;..- containing live yeast cell derivative, shark oil, and a mixture of sodium pyruvate, vitamin E, and chicken fat (Figure 9B); a petrolatum base r.."., ~ containing live yeast cell ,30 derivative and shark oil (Figure 9C); and Preparation HlM (Figure 9D).

RECTIFIED SHEET (RULE 91) ISAIEP

wo 96/03149 Figure 10 is a photograph of a wounded mouse after 4 days of treatment with a petrolatum base r...,., ~ ", only.

DETAILED DESCRn'TlON OF TIE INVENTION

Applicant has discovered therapeutic wound healing ~ ." q - ,- ~ for preventing and reducing injury to ' cells and increasing the 1. rate of injured ~"~,"" l cells. Cells treated with bhe tberapeutic wound healing ~u~ v~ c of this invention and/or their metabolites show decreased levels of hydrogen peroxide production, increased resistance to cytotoxic agents, increased rates of l..ul;~,.uliu.., and increased viability. Cellular cultures containing the therapeutic wound healing c..,..~ ;....c showed enhanced dir~ id~iull and p.~.l;r~,,,.~;.~.. over control cultures and rapidly formed An -- l...,..,:- or tight junctions between the cells to form an epidermal sheet. Wounded mammals treated with the therapeutic wound healing .. q.. - ~;.. --~ show S~ l;ri~,,.. ~y improved wound closing and healing over untreated mammals and mammals breated with .,u,. ~ . -' healing .. , .1 .v~3 ;.. ~ The wound healing '~""'1'~~ may be used alone or in ~ulllb;lluliull with other ~" l: - ..- ,1- More specifically, the therapeutic wound healing ~ .q"r ~ of this invention are used in ...... 1. - -~;.. l with therapeutic amounts of antifungal agents. The antifungal-wound healing ~ - of this invention may be further combined with addibonal ",..l;..-..,.., ~ for enhanced bherapeutic efficacy.

The therapeutic wound healing c..".l..~ of this invention are disclosed as F.~hv~ One (1). There are several aspects of Fr-'-- ' One. In a first aspect (I.A), the therapeutic wound healing l,(.(.q,.,~;l;.." comprises (a) pyruvate selected from the group consisting of pyruvic acid, pl "~, , ; ,~lly acceptable salts of pyruvic acid, and mixtures thereof, (b) an ~nti~ nt, and (c) a mixture of saturated and ~ ,d fatly acids wherein the fatLy acids are those fatty acids required for the repair of cellular .~ .,l,.. ~ and ~ ;u~ of ' cells. In a second aspect of F.",l"~.l;.. ~l One (I.B~, the therapeutic wound healing ~u~q~O-~~;u~ comprises (a) pyruvate selected from the group consisting of pyruvic acid, pl ".~ lly RECI IFIED SHEET (~ULE ~1) ISAIEP

WO 96/03149 2 l q 1 6 0 3 r~.,o~ ~
.

acceptable salts of pyruvic acid, and mixtures thereof, (b) lactate selected from tbe group consisting of lactic acid, I,L_ . ; "v acceptable salts of lactic acid, and rnixtures 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 .... -31-A -' ' and ~- ~ of ' cells. ~n a third aspect of T~ v-l ~. '' One (I.C), the therapeutic wound healing r, ''l" '~ ... cornprises (a) an antioxidant and (b) a mixture of saturated and ' fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular ' and of ' cells.
~n a fourth aspect of F~ One (lD), the therapeutic wound healing r,.. ~ .... comprises (a) lactate selected from tbe group consisting of lactic acid, .Ally acceptable salts of lactic acid, and mixtures thereof, (b) an Y~ t and (c) a mixture of s,aturated and I ' fatty acids wherein the fattyacids are those fatty acids required for the repair of cellular ~" ,3, ~ and of Amm l cell s The therapeutic wound healing c~ of this invention are further combined with a i' r " lly effective arnount of an antifimgal agent ~) to foIm therapeutic ,...iîu~ .. ' healing ~A )-- Il" ~"1 - ~-- - (TA D + X) The antiiung~
healing ~.. l.v~ may be used alone or in ,v - ~ ;.... with other This invention also pertzins to methods for preparing and using the ~u.. ~,.d healing ~ C and the PI--...-~ I.A1 products in which the therapeutic r~ may be used.

The therapeutic Antifi~n.g7l .. ' healing ~ of this invention are further combined with additional ". 1: -- - ~ for treating wounds to folm augmented ~ntiifi-ngA~ wound healing ~ This invention also pertains to methods for preparing and using the augmented therapeutic antifungal-wound healing ~ " "l" ~ ' ;- ~- - -and the ~ products in which the therzpeutic ~ may be used.

The t~rm "injured cell" as used herein means a cell that has any activity disrupted for any reason. For example, arl rnjured cell may be a cell that hrs injured =branes or damaged DNA, RNA, and ribosomes, for example, a cell which has (a) WOg6/03149 2 19 1603 r~.~u~ I

injured membranes so that transport through the mernbranes is dimirlished resulting in an increase in toxins and normal cellular wastes inside the cell and a decrease in nutrients and other rA.~ necessary- for cellular repair inside the cell, (b) an increase in of oxygen radicals inside the cell because of the decreased abllity of the cell to produce ~ and enzymes, or (c) damaged DNA, RNA, and ribosomes which must be repaired or replaced before norrual cellular functions can be resumed. The term "~ G~ Ir of injured ' cells as used herein means tbe reversal of l..yLULUAU,;Ly~ the ~ U.~ of the cellular =brane, an increase inthe l""1:S u,.. . rate of the cell, and/or the ~ of cellular functions such as the secretion of growth factors, ho~nes, and the hke. The term "~;yluLJai~,;Ly" 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 ~ -.1;~ ~Ai;

The herm "prodrug", as used herein, refers to compounds which undergo l,;-h, ~ ;..., prior to exhlbiting their ~ 1 effects. The chemical "..~ of drugs to ovemome 1 ' problems has also been h~rmed "drug I Drug latentiation is the chemical ,...~ ., of a ILulu~
active compound to form a new compound which upon h vlvo enzymalic attack will hberate the parent compound. The chemical alterations of the parent cornpound are such that the change in ~h~ ' ' properties will affect the absorption, dish ibution and enzyrnatic . 1 ~ .. The definition of drug latentiation has also been oxtended to include ~ of the parent cornpound :~ g n~i~m takes place as a ~ of hydrolytic, dissociative, and other reactions not necessanly enzyme mediated. The terms prodmgs, latentiated drugs, and I1JIe derivatives are used , ' "~. By inference, latentiation implies a time lag element or time component involved in _ _ the bioactive parent molecule in vivo. The term prodrug is gene}al in that it includes latentiated drug derivatives as well as those substances which are converted after r ' ' to the acnual substance which cornbines with receptors. The term prodrug is a generic term for agents which undergo l.;..~ r.,. ~;.... prior to exhibiting their pl - " .~i.,~;. 1 actions. In the case where the ' ' drug is not the active agent, but rather is WO 96/03149 2 1 ~t l 6 0 3 r~ m s~

r~ to the active agent, the term "prodrug" also includes compounds which may not necesr,anly undergo l....l,- r"", :;"" to the ~ 1 drug but may undergo 1~ . r " to the active agent which ex~ubits the desired ~,u " .~ gj~l effect.
s The term "~;d ol;L,", as used herein, refers to any substance produced by or by a metabolic process. r~ as used herein, refers to the various chemical reactions involved in the ~,, r~ ", of molecules or chemical c~rlmflc occurring in tisrue and the cells therein.

L Wound Healin~ C . ''' A. r One ~ D) The cells which may be treated with the therapeutic wound healing in the present invention are m~mTn~ n cells. Although applicant will describe the present therapeutic wound healing cl., .l --'l --- as useful for treating ...-- ....~1:- epidermalkud~illu~ LD and. - ,., l monocytes,applicant, - ~' that the therapeutic wound heahng ., ... ~ - - - . c may be used to protect or resuscitatc all " "" ll-, cells. KC~ILi11U~D are '~ :vc of nom~al ". ".1.-1- cells and are the fastest l""l r . .I,..g cells in the body. The correlation between the reaction of kc.~l;..u.,~ ~.D to ingury and therapy and that of . ~ - cells in general is very high.
Monocytes are Ic~lcDc..~ of specialized -- ~l: --- cells r,uch as the white blood cells in the immune rystem and the organ cells in liver, kidney, heart, and brain. The ' cells may be treated i~z vivo and Ln vib o.

Epidem~al kcla~illu~D are the r,pecialized epitbelial cells of the epidemmis which synthesize keratin, a ~ which is the principal constituent of epidermis, hair, nails, homy tisrue, and the organic matri~c of the enamel of teeth.
r~ ~ epidermal kcl.lLil-o~"~D conrtitute about 95% of the epidemlal cells and together with 11I~I~UIO~ D fomm the binary syr,tcm of the epideImis. In its various w0 96l03149 2 1 9 1 6 ~ 3 .

successive stages, epidermal hc~dLillu~D are also known as basal cells, prickle cells, and granular cells.

Monocytes are ' ~ u~,~ . ., leukocytes which undergo respiratory bursting and are involved in reactive oxygen mediated damage within the epidermis.
T J ~ ,D are white blood cells or corpuscles which may be classified into two main groups: granular leukocytes (~, ' ~D) which are leulrocy tes with abundant granules in the cytoplasm and ~ Ieukocytes (. .,.~ ~D) which are leukocytes without specific granules in the cytoplasrn and which include the l~ U~J~D and monocytes. Phagocyte cells are cells which ingest ., ,. ,. y,r~ or other cells and foreign particles. Monocytes are also known as large ' leukocytes, and hyaline or transitional leukocytes.

Epidermal kcldLillu~,y~ic cells and monocytic cells have multiple oxygen generating .". 1, " ,.~ and the degree to which each type of mechanism functionsdiffers in each type of ccll. In ~nocytes, for example, the respiratory burstingprocess is more ~ f 1 than in epidc~mal LclaLillu~,~D. Hence, the ~ a in the therapeutic wound healing ~ of the present invention may vary depending upon the types of cells involved in the condition being treated.
~0 As set out above, in a first aspect of F.."l,o.l..~ One (I.A), the therapeutic wound healing ~ for treating ", "". l cells, preferably epidermal LCI~LIIIU~ comprises (a) pyruvate selected from the group consisting of pyruvic acid, 1ll -- .. - . ..;;, Ally acceptable salts of pyruvic acid, and mixtures thereof, (b) an _~ltirly~ and (c) a mixture of saturated and ' fatly acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and Ir~ of ", "" . 1 , ceDs. In a second aspect of r., . ll u l . ..1 One (I.B~, the therapeutic wound healing ...,..l..,- ;.... for treating ' ceDs, preferablyepidermal l~cldLulu~y~, comprises (a) pyruvate selected fr~m the group consisting of pyruvic acid, pl - ", ~ .. 1. lly acceptable salts of pyruvic acid, and mixturcs thereof, (b) lactate selected from the group consisting of lactic acid, ~' 'Iy acceptable salts of lactic acid, and mixtures thereof, and (c) a mixture of saturated and WO 96/03149 2 19 16 0 3 r~lm~

~1 ~ 5.1 fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular . .l .~ and Ir 3~1;..., of " ~ cells. In a third aspect of r...,l.UJ .. l One (I.C), the therapeutic wouTTd healing c-- q-~- l;- . for treating ". ."...~T ~ cells, preferably epidermal ~ " comprises (a) an antioxidant and (b) a mixture of saturated and 1 fatty acids wherein the fatty acids are those fatty acids required for the repair of celh~lar mernbranes and of . . - - -, -~: cells. In a fourth aspect of F~ J ~ One ~.D), the therapeutic woTmd healing r~ ~, for Ireating ' cells, preferably monocytes, compIises (a) lactate selected from the group consisting of lactic acid, ~ acceptable salts of lactic acid, and mixtures thereof, (b) an ~ntinYi~l -t, and (c) a mi,xture of saturated and, ' fatty acids wherein the fatty acids are those fatly acids required for the repair of cellular ,., ~l ~ and .~ ;-- of ' cells.

Pyruvicacid(2~ r~ k~ acid,CH3COCOOH) or pyruvate is a r ' ' ' in protein and c~ul,uhJ.' ~ l-J 1:- ., amd in the citric acid cycle. The citric acid cycle (l~ lJvA~ acid cycle, Kreb's cycle) is the major reaction sequence which executes the reduction of oxygen to generate adenosine , ' , ' (ATP) by oxidizing organic compounds in respiring tissues to provide electrons to the transport system. Acetyl coenzyme A ("active acetyl") is oxidized in this process and is lilereafter utilized in a variety of biological processes and is a precursor in the b;ua~l~u~ of many fatty acids and sterols. The two major sources of acetyl coenzyme A are derived from tbe m~t~Jlnlicrn of glucose and fatty acids. Glycolysis consists of a series of ~ S"": wherein each glucose molecule is r ~ in the cellular cytoplasm into two ~lecules of pyruvic acid.
Pyruvic acid may then enter the ' ' where it is oxidized by coenzyme A in the presence of enzymes arld cofactors to acetyl coenzyme A. Acetyl coenyme A can then enter the citric acid cycle.

In muscle, pyruvic acid (derived fmvm glycogen) can be reduccd to lactic acid during anervbic m-t~n1icrn which cau occur during exercise. Lactic acid is reoxidized and partially ~c~ 1 to glycogen durmg rest. Pymvate can also act as an 2 1' 9 1 603 WO 96/03149 P~,ll LJ~. ~/l .

antioxidant to neutralize oxygen radicals in the cell and can be used in the r ~ oxidase sysfem to reverse ~;ylu~u~u~,;ly.

The pyruvate in the present invention rnay be selected from the group consisting of pyruvic acid, ~1 " ~ .. 1:. .11,~ acceptable salts of pyruvic acid, prodrugs of pyruvic aciL and rnixtures thereof. ~ general, the Pl . _ 1~ /f 1~ A11Y 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, lithi~-fm pyruvate, sodium pyruvate, potassium pyruvate, ~ pyruvate, calcir-fm pyruvate, zinc pyruvate,manganese pyruvate, methyl pyruvate, Alpha-l -t,~"' - acid, and mixtures thereo~More preferably, the pyruvate is selected from the group of salts consisting of sodir-fm pyruvate, potassium pyruvate, ~ ~ pyruvate, calcmm pyruvate, zinc pyruvate, manganese pyluvate, and the like, and mixtures thereof. Most preferably, the pyruvate is sodium pyruvate.

The amount of pyruvate present in the therapeutic w~-md healing '~""~1"~--~;""~ of the present invention is a ;'.~ AP~ effective amount. A
lly effective arnount of pyruvate is that amount of pyruvate necessary for the inventive ~ 1 -; to prevent and reduce injury to mammalian cells or increasethe ~ ;"" rate of injured ' cells. The exact amount of pyruvate is a matter of preference subject to such factors as the type of condition being treated as well as the other ingredients in the ~ " In a preferred r ~ 5, pyruvate is present in the therapeutic wound healing If ....1 ~ ~ ;.... in an amount from about 10%
to about 500/D, preferably from about 20% to about 45%, and ~re preferably from about 25% to about 40C/o, by weight of the therapeutic wound heahng ~ -- llnl- ~

~Lactic acid ((S}2-L,~dlu~,~r r ' acid, (+),41pha-l,yfLJ,.~.u~;u~., acid, CH3CHOHCOOH) or lactate occurs in small quantities in the blood and musclefiuid of marnmals. Lactic acid ~ increases in muscle ard blood afrer vigorous activity. Lactate is a component in the cellular feedback mechanism andinhibits the natural respiratory bursting process of cells thereby s ~ g the production of oxygen radicals.

2 1 9 ~ 603 wo 96/03149 The lactate in the present invention _ay be selected from the group consisting of lactic acid, pl,- ., - . ~,. il~ acceptable salts of lactic acid, prodrugs of lactic acid, and mixtures thereo~ In general, the ~ X ~lly acceptable salts of lactic acid may be aDcaii salts and allcaiine earth salts. Preferably, the lactate is selected from the group consisting of lactic acid, lithium lactate, sodium lactate, potassium lactate, m~ci ~ lactate, calcium lactate, zinc lactate, manganese lactate, and the l~ce, and mixtures thereo~ More preferably, the lactate is selected from the gloup consisting of lactic acid, sodium lactate, potassium lactate, , lactate, calcium lactate, zinc lactate, manganese lactate, and mixlures thereof. Most preferably, the lactate is lactic acid.

The amount of lactate present in the therapeutic wound heaiing ~, ... q... 1.. ,..-of the present invention is a ~ t, lly effective amount. A ~
effective amount of lactate is that amount of lactate necessary for the inventive .,- q.. - ~-.. to prevent and reduce injury to ' cells or increase the rate of injured ' ceDs. For an ingeshble q~ a y effective amount of lactate is that amount necessary to suppress the respiratory bursting process of wbite blood ceDs to protect and resuscitate the r~-~nrl;~- cells. In general, a ~ ly effective amount of lactate in an ingesùble .. ~ ;,.. is from about 5 to about 10 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 ~ In a proferred ~1 o l; ~ ~1 lactate is present in the therapeutic wound healing ~ in an amount ~m about 10% to about 50~/c, preferably from about 20% to about 45%, and more preferably ~rom about 25% to about 40~/O, by weight of the therapeutic wound healing ,~.,,,1...- ~; .

~ntimri/i ntc are substances which inlubit oxidation or suppress reactions promoted by o:sygen or peroxides. .A ' especiaDy hpid-soluble r ~- ~
can be absorbed into the ceDular membrane to neutralize o~ygen radicals and thereby protect the membrane The ' useful in the present invention may be selected from the group consisting of aD forms of Vitamin A (retinol), ail forms of w0 96/03149 2 19 16 0 3 r~l~o~ l .

Vitamin2 ~3, 4~ hL~dlule~ all forrns of carotene such as A/ '-~~trn~
carotene (~eta, ,B-carotene), gamma-carotene, delta-carotene, all forms of Vitamin C
(D-ascorbic acid, L-ascorbic acid), all forms of tocopherol such as Vitamin E (.4Ipha-tocopherol, 3,4-dihydro-2,5,7,8-LeL-~.LL~1-2-(4,8,l2-LIilll~. hylLIi-decyl)-2H-l-benzopyran-6-ol), ,tl-tocopherol, gamma-tocopherol, delta-tocopherol, I , -, tocotrienol, and Vitamin E esters which readily undergo hydrolysis to Vitamin E such as Vitamin E acetate and Vitamin E succinate, and Lh ~ 1'~, acceptable Vitamin E salts such as Vitamin E phosphate, prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E, l'~ ,.. .. ,I l. _lly acceptable salts of Vitamin A, carotene, Vitamin C, and Vitamin E, and the hlce, and mistures thereo~ Preferably, the antioxidant is selected fiom the group of lipid-soluble ' consisting of Vitamin A, ,B-carotene, Vitamin E, Vitamin E acetate, and mixtures thereof. Morepreferably, the anioxidant is Vitamin E or Vitamin E acetate. Most preferably, the anioxidant is Vitamin E acetate.

The amount of antioxidant present in the therapeuic wound healing of the present invenion is a ~'..._I....:,._lly effecnve amounL A
i' . 'l~, effecive amount of anioxidant is that amount of antioxidant necessary for the inventive ~ to prevent and reduce inju~y to " ceDs or increase the rate of injured m~ ceDs. The exact amount of antioxidant is a matter of preference subject to such factors as the tgpe of condition being treated as well as the other ingredients in the c.~.,q.~ In a preferred 1 the antioxidant is present in the Lllerapeutic wound healing 4..1.1...- l;....in an amount from about 0.1% to about 40%, preferably from about 0.2~/c to about30%, and more preferably from about 0.5% to about 20%, by weight of the therapeutic wound healing ~ ""l"~~ ' ' The mixture of saturated and unsaturated fatty acids in the present invention are those fatLy acids required for the repair of ~ - . cellular membranes and the production of new cells. Fatty acids are carboxylic acid compounds found in animal and vegetable fat and oil. Fatty acids are classified as lipids and are composed of chains of allyl groups containing from 4 to 22 carbon atoms and 0-3 double bonds and ~ 1 9 1 603 W096103149 I.~
.

i by a terminal carboxyl group, -COOH. Fatty acids may be saturated or ' 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). TTnc~h~t, ~l fatty acids are usuaDy derived from vegetables and consist of alkyl ch~ains containing from 16 to 22 carbon atoms and 0-3 double bonds with the ~ h- ~ tenninal carboxyl group. The most common ' fatty acids are oleic acid, linoleic acid, and linolenic acid (all C18 acids).

In general, the mixture of saturated and unsaturatod fatty acids required for the repair of m~ T; l~ cellular, .. ,l.. ~, .4 in the present invention may be derived from animal and vegetable fats and waxes, prodrugs of saturated and I ' fatty acids useful in the present invention, and mrb turos thereof. For example, th~e fatty acids in the therapeutic wound healing ~ may be in the foml of mono-, di-, or tli~yh ~lidcs, or free fatty acids, or mixtures theroof, which are readlly available for the repair of injured ceDs. Cells produce tho chemical ~ and the energy required 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 ~ sirnilar to that of human fat and the fat contained in human breast milk. The preferred animal fats and wa~ces may be selected from the group consisting of human fat, chiclcen fat, cow fat (defined herein as a bovine domestic animal regardless of sex or age), sheep fat, horse fat, pig fat, and whale fat. The ~re preferred animal fats and waxes may be selected fromthe group consisting of human fat and chicken fat. The ~st preferred animal fat is hurnan fat. Mixtures of other fats and waxes, such as vogetable wa~es (especiaDysunflower oil), marine oils (especially sharlc liver oil), and synthetic waxes and oils, which have a fatty acid c ~ .,. similar to that of animal fats and waxes, and preferably to that of human fats and waxes, rnay also be employed.

~ In a preferred ~,-"l,o~ , tho mixture of saturated and I ' fatty acids has a ~ . similar to that of human fat and comprises thc following fatty acids: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, rnyristoleic acid, palmitic acid, pahmitoleic acid, stearic, oleic acid, linoleic acid, WO96/03149 2 1 9 1 6 03 lr~ J.. ,SI. I
.

Iinolenic acid, arachidic acid, and gadoleic acid. Preferaibly, butyric acid, caproic acid, caprylic acid, capric acid, lamic acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic, oleic acid, I;noleic acid, linolenic acid, arachidic acid, and gadoleic acid are present in the mi~ure in about the foDowing p~ ~c ~ by weight,S ~CD~ Y (carbon chain number and nurnber of I - are sbown D~ Y) 0.2/c--0.4/o (C4), 0.1 /o (C6), 0.3/o--0.8/o (C8), 2.2/o--3.5/o (C10), 0.9%-55% (C12), 2.8~/c-85% (C14), 0.1~/c-0.6~/c (C14 1), 23.2%-24.6% (C16), 1-8~/c-3 0% (C16 1), 6.9%-9.9% (Clg), 36.0%-36.5% (Clg 1), 20%-20.6% (Clg 2), 7.5-7.8% (Clg 3), 1.1%-4.9% (C20), and 3.3~/c-6.4% (C20 1).
In anotber preferred ~ ,o~ , the mixture of saturated and I
fatty acids is typically chicken fat comprising tbe following fatty acids: lauric acid, mylistic acid, myristoleic acid, ~ acid, palmitic acid, palmitoleic acid, margaric acid, ",~ .1 :. acid, stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, and gadoleic acid. Preferably, lauric acid, myristic acid, myristoleic aciL ~ ~ - aciL palmitic aciL palmitoleic aciL margaric aciL ." .~,,....7~il acid, stearic, oleic aciL linoleic aciL linolenic aciL arachidic acid, and gadolac acid are present in the mixture in about the following .IJ~ D by weight, ICD~
0.1 /o ~C12), 0-8 /o (C14), 0 2/o (C14 1), 0.1 /o (C15), 253 /D (C16)~ 7 '~ /o (C¦6 1), 0.1%
(C17), 0 1% (C17 1), 6 5% (C18), 37.7% (C18 1), 20 6% (C18 2), 0 8% (cl8 3)~ 0 2%
(C20), and 0.3% (C20 1)~ all p~ +/-10%.

In another preferred ~",1..~.1:".. . :, the mixture of saturated and, fat~y acids comprises lecithin. Lecithin (~ u~ ) is a pl ~ found in all living organisms (plants and anirnals) and is a significant constituent of nervous tissue and brain substance. Lecithin is a mixture of the d;gl~.~i.le6 of stealic, palmitic, and oleic acids, linked to the choline ester of phosphoric acid. The product of commerce is ~ ~ hJ soybean lecithin obtained as a by-product in the ' ,, of soybean oil. Soybean lecithin contains palmitic acid 11.7%, stearic 4.0%, palmitoleic 8.6%, oleic 9.8%, linoleic 55.0%, linolenic 4.0%, C20 to C22 acids (includes arachidonic) 5.5%. Lecithin may be represented by the formnla:

WO96/03149 2~1 ql 603 p~"~
.

I

CHOCOR

CH20-P(0)2-OCH2CH2N~(CH3)3 wherein R is selected from the group consisting of stearic, palmitic, and oleic acid.
The above fatLy acids and p ~ ~L ' thereof present in the fatty acid raixlure are given as an example. The exact type of fatty acid pre~ent in the fat~y acid mixture and the exact amount of fatty acid employed in the fatly 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 ~ r The amount of fatty acids present in the therapeutic wound heahng c~ of the present invention is a i' , "y effective amount. A
Il . 'ly effective amount of fatly acids is that amount of fatty acids necessaryfor tbe inventive ~ to prevent and reduce injury to ~ :, cells or increase the I G~ . rate of injured ~ cells. The exact amount of fatty acids employed is subject to such factors as the type and distnbution of fatty acids employed in the mixture, the type of condition being treated, and the other ingredients in the ~ In a preferred ...,~ the fatty acids are present in the therapeutic wound healing c~ 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 wound healing ~,-~ 'l ~ ';

In accord with the present invention, the therapeutic wound heahng c~ of F~ l--r1; : One ([A-D) for treating 1 cells maY be selected from the group consisting of WO g6/03149 2 1 9 1 6 0 3 r~

~LA)(a) pyruvate selected from tbe group consisting of pyruvic acid, lly acceptable salts of pyruvic aciL and mixtures thereof;
(b) an ' ~ and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatly acids required for the repair of cellular =branes and ~ II of m~nmql cells;

~B)(a) pyruvate selected from the group consisting of pyruvic acid, pl; " -- . Ily acceptable salts of pyruvic acid, and mixtures thereof;
(b) lactate selected from the group consisting of lactic acid, I,1. ", . .. :,. AnY
acceptable salts of lactic acid, and rnixtures thereof; and (c) a m~ture of saturated and, ' fatty acids wherein the fatty acids are those fatty acids requircd for the repair of celhllar membranes and of mq~Tr q~ cells;
(LC) (a) an -- ~ and (b) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular ~ and of mq~mql~ cells;
(LD) (a) lactate selected from the group consisting of lactic acid, p~ ;, Ally acceptable salts of lactic acid, and mixtures thereof;
(b) an ' '., and (c) a mixture of saturated and I ' fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular .. ~.. ,1.,. ~ and of mf mmalian cells.

Preferably, the woundhealing ~ of r ~ ~ One(I) for treabng, .. - .. ~ - cells, preferably epidermal LC1~L;.. ,~ ~, rnay be selected from the group consisting of.

WO96/03149 21 91 603 P~

~A) (a) pyruvate selected from the group consisting of pyruvic acid, ,1. ", . ~..I;. Aily acceptable salts of pyruvic acid, and rnDctures thereof;
(b) an ~ and (c) a mixture of saturated and i ' fatty acids wherein 5the &ny acids are those fatty acids required for the repair of cellular ' and , of",--,....~l:-, cells;

a.B) (a) pyruvate selected from the group consisting of pyruvic acid, y acceptable salts of pyruvic acid, and mixtures thereof;
10(b) lactate selected from the group consisting of lactic acid, acceptable salts of lactic acid, and mixtures thereof; and (c) a mixture of saturated and ~ 7 fany acids wherein the fatty.acids are those fatty acids required for the repair of cellular ...- ..,1...- ~ and Of m~mm~ n cells; and~5 ~C) (a) an "1;~ and (b) a rnixture of saturated and unsaturated fatty acids wherein the fatty acids are those fany acids required for the repair of cellular 1.. ~ C and ,_ .. . ::.i~... Of m mm~ n cells.
More preferably, the wound healing c ~ of r l u-l .1 One (1) for treating m~mm~ cells, preferably epidermal l ~, m y be selected from the group consisting of:

25(LA) (a) pyruvate selected frorn the group consisting of pyruvic acid, y acceptable salts of pyruvic acid, and m~tures thereof;
(b) an ' ~, and (c) a mixture of saturated and ' fatty acids wherein the fatty acids are those fatty acids required for the repair of cellu1ar ' and of m ~-nm~ l cells; and WO96/03149 2 19 16 03 r~l,u~ l ~

(LC) (a) an A..~ , and (b) a i~cture of saturated and unsaturated fatty acids wherein the fatly acids are those fatty acids required for the repair of cellular ,.- 6 arld of m --n~ lj ..~ cells.

More preferably, the wound healing ~ of r...~ ., ... . ; One O for treating mAmm-A~ cells, preferably epidemmal }~ila61llu~ ,D, may be selected fmm the group consisting of:

(LA) (a) pyruvate selected from the group consisting of pyruvic acid, acceptable saltc of pyruvic acid, and m~ures thereof;
(b) an ~ l and (c) a i~ture of saturated and unsaturated fatty acids vherein the fatly acids are those fatty acids required for the repair of cellular m~m~r~n~C and of mammalian cells; and (LB) (a) pyruvate selected from the group consisting of pyruvic acid, acceptable salts of pyruvic acid, and mr1ctures thereof;
(b) lactate selected from the group consisting of lactic acid, l~ y acceptable salts of lactic acid, arrd mixtures thereof; aud (c) a mixhire of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of ceDular .. ~ ..II.,A.,~ . and r~ ;.... of 1 cells.

Most preferably, the wound healing ~ u- ;- ~ of F.. ~l,u 1,~.. 5 One (I) for treating ' ceDs, preferably epidermal ~clalil~u~"~D, cornprise:

(LA) (a) pyruvate selected from the group consisting of pyruvic acid, pt~ y acceptable salts of pyruvic acid, and mi7~tures thereof;
30 (b) an ~ - and WO 96103149 2 1 9 1 6 0 3 PCT/U'igSI08551 (c) a mixture of saturated and I ' fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and of ~ - l cells.

Most preferably, the wound healing c~ of r~ One O for treating ' cells, preferably monocytes, comprise:

(LD) (a) lactate selected from the group consisting of lactic acid, ~h 11~ acceptable salts of lactic acid, and mixtures thereof, (b) an ' ', and (c) a mixture of saturated and I ' fatty acicls wherein the fatty acids are those fatty acids required for the repair of cellular membranes and 1~ of ~ cells.

Throughout this disclosure, applicant will suggest various theories or by which applicant believes the ~ in the therapeutic wound healing c~ - and the antiviral agent function together in an unexpected synergistic marner to prevent and reduce injury to " " 1 ~ cells, increase the .. - -- :'-: ~~.. rate of injured m~mm~ cells, and reduce viIal titcrs. While applicant may offer various ,.. ~ to explain the present invention, applicant does not wish to be bound by theory. These theories are suggested to better understand the present invention but are not intended to lirnit the effective scope of the claims.

In the first aspect of r.".~ One (IA), applicant believes that r,5 pyruvate can be transported inside a cell where it can act as an antioxidant to neutralize oisygen radicals in the cell. ~yruvate can also be used inside the cell in the citric acid cycle to provide energy to increase cellular viability, and as a precursor in the synthesis of important 1~ L . ~8~ ~ to promote cellular 1 T r In addition, pyruvate can be used in the ~..r oxidase systern to reverse iy~ ,;iy. ~nfi~Yi~~~fc especially lipid-soluble ---:;,,~,1- a can be absorbed into the cell mernbrane to neutralize oxygen radicals and thereby protect the membrane. The saturated and - - ' fatty acids in the present invention are those fatty acids required for the WO96/03149 2 1 9 1 6 0 3 F~IIIJ~,. _.'l I

; " ~ of ", "", ~l . cells and are readily ava~able for the repair of injured cells and the ~ ;r,,,.l;.." of new cells. Ce11s injured by oxygen radicals need to produce ' fatty acids to repair celluLr m~h~n~-c However, tbe production of ' fatty acids by cells requires oxygen. Thus, the injured cell needs _igb levels of oxygen to produce unsaturated fatty aciLc 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, ' fatty acids needed for repair, the need of the cell for ' fatty acids is reduced and the need for higb oxygen levels is also reduced.

The ~.. ".l. -~;.". of pyruvate inside the cell and an antiw~idant in the cellular membrane functions in an unexpected synergistic marmer to reduce hydrogen peroxide production in the cell to levels lower tban can be achieved by use of either type of component alone. The presence of mixtures of saturated and unsaturated fatty acids in the therapeutic wound healing .A~ si~lirl~.~ily enhances the ability of pyruvate and the antioxidant to in~ubit reactive oxygen production. By stabilizing the cellular mernbrane, ' fatty acids also improve membrane function and enhance pyruvate tr..nsport into the cell. Hence, the three ~ - in the therapeutic wound healing ç~ - c~ of the first aspect of ~.I.l,.rl...,...l One (IA) function together in an, , ' synergistic =er to prevent and reduce injury to " ~ ; - cells and increase the rate of injured ' cells.

In the second aspect of r ~- One (LB), lactak is employed inskad of an ' .An~ Si~qrtc react with~ and neutralize, oSygen radicals after the radicals are already formed. Lacrate, on the other hand, is a component in the cellular feedback mechanism and in~ubits the respiratory bursting process to suppress the production of active oxygen species. The .,.,.~l - -:;..,. 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 componer~t alone. The presence of mixtures of saturated and unsaturated fatty acids in the therapeutic wound healing 4 ~ q~ " S;~ llly enhances the ability of pyruvate and lactak to inhrbit reactive oxygen production. Hence, the three . I in the therapeutic wound healing - =
WO96103149 21 91 603 r~

4""1"'-'' "' in the second aspect of F~ One a.B) function together in a synergistic manner to protect and resuscitate I~ - cells.

In the third aspect of F~One (I.C), the presence of mixtures of saturated and unsaturated fatty acids in the therapeutic wound healing ~
in this r 1 U~ s;~firl~,a.lily enhances the ability of the Antinsi~l-...t to inlubit reactive oxygen production. The .,..,.l. - ;.... of an antioxidant to neutlalize active oxygen species and fatly acids to rebuild cellular m~3nbranes and reduce the need of the cell for oxygen functions in a synergistic rnanner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved by eitber lype of co_ponent alone. Hence, the ~ in the therapNtic wound healing c. ", ~L r.- ~ - ", in the third aspect Of r ' '' ' One (I.C) function together in a synergistic m~nner to protect and resuscitate m---nmAli~n cells.

In the fourth aspect of F.. ,l.~l;.. l One (I.D), lactate is employed because the respiratory bursting process is ~re ~ ' in monocytes than in epidemmal kc~d~ u~"~.. The c.. l. -:.. of lactate to suppress the respiratoly bursting process and an antioxidant to neut~alize active oxygen species functions in a synergistic manner to reduce hydrogen peroxide prcduction in the cell to levels lower than can be achieved by either component alone. The presence of mixtures of saturated and, ' fatLy acids in the therapeutic wound healing ~ in this ~ ";.l.,~.-l~ enhances the ability of lactate and the antioxidant to ir~ubit reactive oxygen production. Hence, the three I , in the therapeutic wound healing ~u,. l ~ ~;-. - in the fourth aspect of F~ -l--~-l; - -' One (LD) function together in an unexpected synergisic manner to protect and resuscitate l, "", cells.

Accordingly, the ~, .l -- ~:- of ingredients set out in the above ~l ~l; l~ funcions together in an enhanced manner to prevent and reduce injury to ... ~l:- cells and increase the .~ ;--" rate of injured " --, l; cells.
The therapeuic effect of the . ' of the c -" l. ~-- t~ in each of the abovee ~ ' is markedly greater than that expected by the mere addition of the wo 96/03149 2 1 9 1 6 0 3 F~

individual therapeutic ~ Ience, applicant's therapeutic wound healing 'f ""m'~'~ for treating ' cells have the ability to decrease ~1 ~
levels of hydrogen peroxide production, in= e cellular resistance to cytotonic agents, increase rates of cellular ~ F,~ " and increase cellular viability.

B. Methods For Mal~g The Therapeutic Wonnd ~Iealing ~' . ' ' Of r ~ ~ One (LA-D) The present invention extends to rnethods for rna1~ing the therapeutic wound healing ~ -- of F~hv~ . .1 One (IA-D). In general, a therapeutic wound healing c. ~ " is rnade by forrning an adrnixture of the ~ of the ... In a ~rst aspect of r.~hv~ .1 One (IA), a therapeutic wound healing ~ is rnade by forming an adrnixture of (a) pyruvate selected f~n the group consisting of pyruvic acid, ~ , acceptable salts of pyruvic acid, and rnixtures thereof, ~b) an qn~i~nirTq..t, and (c) a mixture of saturated and, fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular ' and 1c of ' cells. In a second aspect of r.. ,l,O l; .. 1l One a.B), a therapeutic wound healing ~ is made by forming an adrnixture of (a) pyruvate selected from tbe group consisting of pyruvic acid, "~ acceptable salts of pyruvic acid, and mixtures thereof, (b) lactate selected from the group consisting of lactic acid, IP ~ ; lly acceptable salts of lactic acid, and rni~tures thereof, and (c) a rni~ture of saturated and ' fatty acids wherern the fat~ acids are thosc fatLy acids requu~d for the repair of cellular membranes and ~ Of ' cells. In a third aspect of F...~l..~l:...~..,l One (I.C), a therapeutic wound healing ~f J"'l"'-' ;' "' is rnade by forn~ng an adrnixture of (a) an antioxidant and (b) a i~cture of saturated and unsaturated fiatLy acids wherein the fatty acids are those fatty acids required for the repair of cellular m~mhrqn~ c and ~r--.-- .1_1;~ll Of m~ rmo~ cells. In a fourth aspect of r~.~h~T;" -: One (I.D), a therapeutic wound healing c~ is m ade by fomming an adm~xture of (a) lactate selected from the group consisting of lactic acid, 1'7 ~" 1; tl,r acceptable salts of WO96/03149 21 91 603 r~ x~

lactic acid, and mixhlres thereof, (b) an ~nh~ nt and (c) a mi chme of sahlrated and ' fahy acids wherein the fahy acids are those fahy acids required for the repair of cellular .,...,1. . ~ and .~ ;.. of ' cells.

For some l,~Ti - ;.. ., the admistore may be formed in a solvent such as water, and a surfactant may be added if required. If necessary, the pH of thesolvent is adjusted to a range f~m about 3.5 to about 8.0, and preferably from about 4.5 to about 7.5, and ~re preferably about 6.0 to about 7.4. The admixh~re is then stenle filtered. Other ingredients may also be ~ ~ into the therapeutic wound healing ~ as dictated by the nahare of the desired c ~ as well known by those having ordinary slcill in the art. The ultimate therapeutic wound healing are readily prepared using methods generally known in the pn, - ~ artS.

In a prefelred e~ the invention is directed to a method for preparing a therapeutic wound healing ~.,. l...- ~ . ~A) for preventing and reducmg injury to ,., --.,,,._l:-" cells, and increasmg the rate of injured m~mm~l;~
cells, which comprises the steps of admixing the following ,, (a) pymvate selected from the group consisting of py~uvic acid, y1 -- ~ .s acceptable salts of pyruvic acid, and mixhures thereof;
(b) an ,,..1 ;. .~ .1, and (c) a mixture of saturated and ~' fahy acids wherein the fahy acids are those fany acids required for theof injured m~mm~ n cells.

C. Methods ~or r The Tl , Wo~d E~ealing ~' Of F ~ One (LA-D) The present invention extends to methods for employing the therapeutic wound healing ~ of F."~l.~l;."~ One (I) ih vrvo and ~n vilro. In general, a therapeutic wound healing c~ :c~ is employed by contacting the therapeutic with ' cells.

In a first aspect of F...d.. 1 ~- -: One (IA), the invention is directed to a method for preventing and reducing injury to ' cells, and increasing the - rate of injured l cells, which comprises the steps of (A) providing a therapeutic wound healing c. ~ .. which comprises (a) pyruvate selected from the group consisting of pyruvic acid, l ' ~ lly acceptable salts of pyruvic acid, and rruxtures thereof, (b) an -~ ioYi~ , and (c) a mixture of saturated and, 1 fatty acids wherein the fatty acids are those fatty acids required for the of injured 1 cells, and (B) contacting the therapwtic wound healing c. .l...- ~.-- with the ' cells.

In a second aspect of r~ One (LB), the invention is directed to a method for preventing and reducing injuly to n~ cells, and increasing tbe rate of injured ~ cells, which comprises the steps of (A) providing a therapeutic wound healing ~ which comprises (a) pyruvate selected from the group consisting of pyruvic acid, pl - ~ acceptable salts of pyruvic acid, and rnixtures thereof, (b) lactate selected from the group consisting of lactic acid, l,l -. " l ;. _lly 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 .r~ ;. . . of injured ' cells, and (B) contacting the therapeutic wound healing c~ . with the m ~ nm~ r cells.

In a third aspect Of r ' " One (I.C), the invention is directed to a method for preventing and reducing injury to m~~n~ l cells, and increasing the~c- - :l-l;... rate of injured ' cells, which comprises the steps of (A) providing a therapeutic wound healing ~ . q.. .- ~ which comprises (a) an ~nti~Yi~ t and (b) a mixture of saturated and, ' fatty acids wherein the fatty acids are those fatty acids required for the .~ - of injured n~ n cells, and (B) contacting the therapeutic wound healing ~ with the ' cells.

W096103149 21 91 603 r ~

In a fourth aspect of r - One (I.D), the invention is directed to a method for preventing and reducing injury to ~ cclls, and increasing the rate of injured ." "" 1;, ceLs, which comprises the steps of (A) providing a therapeutic wound healing c. ,. . ~ which cornprises (a) lactate selected from the group consisting of lactic acid, L~ ,y acceptable salts of lactic acid, and mi~tures thereof, (b) an ~lltir~Y~ t and (c) a mixture of saturated and ' fatty acids wherein the fattv acids are those fatty acids required for the of injured ." "", 1 , cells, and (B) contacting the therapeutic wound healing c.~ ;.... with the, "" ~1:, cells.
In a preferred nl.,l.~l;.... the invention is directed to a method for healing a wound in a mammal which coraprises the steps of:
(A) providing a therapeutic wound healing c~ (I A) which comprises:
(a) pyluvate selected from the group consisting of pyruvic acid, pl --, .~.,,1l;, lly acceptable salts of pyruvic acid, and mixtures thereof;
(b) an - -';. .~ and (c) a mixture of saturated and ~' fatty acids wherein the fatty acids are those fatly acids required for theof injured ~- cells;
and (B) contacting the therapeutic wound healing ~; - with the wound.

The types of wounds which may be healed using the wound heaLng ,,_ of r ..1.~.. ;.... One (IA-D) of the present invention are taose wbich result from an injury which causes epidermal darnage such as incisions, wounds in which the skin is broken by a cuthng instrument, and lacerations, wounds in which the skin is broken by a dull or blunt instrnment. The therapeutic r ''' rnay also be used to treat various r1. .",~l ~lr~ disorders such as L.~L ' l' aL;~g, burns, donor site wounds from skin transplants, ulcers (cutaneous, decubitus, venous stasis, and diabetic), psoriasis, skin rashes, and sunburn l' ~ , processes. Thetopical therapeutic c~ - may also be used orally in the form of a ~uth wash or spray to p}otect and accelerate the healing of injured oral tissue such as mouth sores and bums. The topical therapeutic ~A~.'q"~- 1''"'' may futther be used in WOg6/03149 21 91 603 r~"J~ ~

, ' '' ' ~ o ~ iUlls to treat wounds such as hhose which result from corneal ulcers, r~ y~ comeal hansplants,,, I ~ and ohher surPOically induced woimds in hhe eye. The topical therapeuic 4'~ may in addihon be used in anorectal creams and ~ t~ to treat such condiions as prurituS ani, procitis, anal fissiires, and l .. ,l.l .;.k In a preferred rl - u~; l, the iherapeutic ~A ,l - - ~ ;- -- - are used io treat wounds such as incisions and lacerations.

The wound hoaiing - ,~ , of r ~ ' One (I.A-D) of the present invenion may be uhlized in topical pi~ducts, ingeshble products, and issue culhire medium to protect ' cells and increase hhe ~r~ ate of inJured m~mm~ cells. For example, the therapeuic wound healing c ..l.-- :~;...,~may be used in topical skin care products to protect and increase the rate of skin issile such as in the heahment of various d. .~ I disorders such as L~ , g and sunbmn ~ . pwcesses. Injmy to skin can occiir for a variety of reasons. Injury often occurs to individuals who wash their hands often, to individuals who are exposed io stressful e,.v ' conditions ; to sun or chemicals), or to the elderly or individuals with an ull~ll;.. g disease. The addiion of the wound healing ~f~ of the present invention to a loion pwvides a soDe of ~ ~' to the skin which would protect uhe skin from the ha~ l effects of W light, chemicals, and severe d~ying. The wound healing ',~ can be used for hhe following indicahons: a) 1~f ~ ~ ~ and pwtecing; b) HeaLng diy cracked skin; c) Treahng irritated skin such as diaper rash;
d) Healing severe dry skin due to other diseases (venous demnaiis); e) Treahng psoriasis and other L~ ;rrl l;v~ diseases; fJ Pwtecing skin from W Lght damage (~nfinlriri~nf skin Ir~ ), g) Treating seborrheic conditions; and h) Treaing shav;ng wounds in an after shave lotion.

The topical therapeuic wound healing ~A- Il- - l ~ may also be used orally in the form of a rnollth wash or spray to pwtect and accelerate the healing of injured oral fissue such as ~uth sores and bums. The topical therapeutic wound healing c~ ;- -- - may further be used in ~ - r~ C such as eye care products to neuhalize hydrogen pewxide used in the cleaning of contact lenses.

WO g6~03149 2 1 9 1 6 0 3 r~ c l The topical therapeutic wound healing ~ may in addition be used in anorectal creams and ~ to Treat such conditions as plUTitUs am, proctitis, anal fissures, and k.. l,l.. ;.k Initially as white blood cells enter a wound site, the cells release oxygen radicals, depleting the ....:;-.,.;~-.:- at the wound site, thus impauing the healing process. I , _ the wound heaiing c ~ of the present invention into a wound healing ll..,.,./l-:;.". would facllitate healing by providing the site with usable - :~ ., and a source of fatty acids needed for mernbrane repair. The wound healing .,~ - -; - - can be used for the following indications: a) Healing of cuts and scrapes; b) Bums (heals bums with less scaTing and scabbing); c) Decubitus ulcm; d) Bed sores, pressure ulcers; e) Fissures, TT .. 1,.. ;.1~
f) Use in ~.. ." .' . - I: ", with ~ ~ (simulated healing in healing def cient people); g) Post surgical wounds; h) Bandages; i) Diabetic ulcers; j) Venous ulceration;
and k) Use in ,~....3. - ;.. with wound cleansing agents.

The therapeutic wound healing ~ may also be used in ingeshble products to protect and increase the Tate of erosions, stomach ulcers, and k.-...,.,.l...~,. ' in the gastric mucosa. Other ingestible therapeutic products include: stroke ",.1:. ~;.,..~ .s..;", ..~ disease,, ~ , arthritis ,.,..li...,,...~
ulcer ,,,..1;. -~: ...~, cancer "...1;. -l;.... (cytotoxic agents); heaTt medication to improve regional venTricular function and restore no~mal heart rate and pressure functions; lung medication to repair in3ured tissue; liver medication to suppress lipogenesis of alcoholic origin and prevent hepatic steatosis; kidney medication to suppress urinaly calculi (kidney stones); ~1....;1~. l;..,. medication to antagonize heavy mePI poisoning, cyanide poisoning, sodium sulfide poisoning, other types of poisoning. ; and reduce and neuhalize the produchion of oxygen Tadicals which produces injury to hissue, to protect and fwther enhance the . c : 1;- - rate of the mjured ~ "--.. l: -, cells. The therapeuhc wound healing ~ --- may be used in ingeshble products to heat ~ n ' ,~ diseases such as hepahihs, gast~ihis, colihs, e~phq.~ric~ arthTihis, and ~ p.. ~q~

The therapeuhc wound healing u-, ,~ of the present invention may also be used in hissue culture media and organ h,qnsplant media to prevent and WO96/03149 21 91 603 P~I/U~ J1 reduce injuly to m-mm~ l cells and increase the ,~ I l; rate of injured 7;-- cells. Tissue cultures and transplant organs encounter reachve o~ygen species generated in the cuiture media by the injured cells. Organs paTticularlysuscephble to oxidahve damage during transport and i , ' due to reperfusion injury follov,/ing ischemia are corneas, livers, hearts, and kidneys. The theTapeuhic wound hcaling ~ --- rnay be usefill to abwgate reperfusion injuly to such transplant organs.

In a specific l".l..vl:.... ~, the invenhon is directed to a method for preserving ", "," 1; cells in a culture medium which comprises the steps of:
(A) providing a therapeuhc wound healing t, ~ - :'; - selected fiom the gwup of consisting of:

~A) (a) pyruvate selected f~m the gwup consishng of pyruvic acid, pt~ hl~ y acceptable salts of pyruvic acid, and mixtures thereof, (b) an ., ~ t, and (c) a mrZcture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids req~ired for the repair of cellular membranes and of",--, ,~l: cells;
(LB) (a) pyruvate selected from the g~up consisring of pyruvic acid, i~-- 111-- .~6~11y acceptable salts of pyruvic acid, andm~tures thereof;
(b) lactate selected from the graup consishng of lachc acid, "v acceptable salts of ]achc acid, and mixtures thereof; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatl~ acids are those fatty acids required for the repair of cellular membranes and of '~ cells, ~C) (a) an ' ~ and (b) a mixture of saturated and unsaturated fatty acids wherein the fat~y acids are those fatty acids required far the repair of cellular membranes and of ", "" ,~l: -- - cells;

WO 96/03149 2 1 9 1 6 ~ 3 (LD) (a) lactate selected from the group consisDDg of lactic acid, "~ .T~y acceptable salts of lactic acid, aDd mixtures thereof;
(b) aD: , aDd (c) a mixture of saturated and ' fatty acids wherein the fatty acids ar~ those fatly acids required for the repair of cellular .. , . ~ and ;.", of r~ cells; and (b) aD ' , and (c) a mi~ure of satmated and ' fatty acids wherein the fatty acids are those fatty acids required for the ~ of iDjured cells;
(B) p}oviding 1, "", l , cells in a culture medium; and (C) corltacting the therapeutic wound healiDg ~ q.---~~; from step (A) with the ,., .,.... 1 , cells in the culture medium from step (B).

D. r; Of l he n , WouDd ~ealiDg ~ .
Of r One (L~-D) ODce prepared, the iDventiVe therapeuic wouDd healing, of r ~ ~ One (IA-D) may be stored for future use or may be formulated iD
effective arnounts with ~,: -- ", ~ acceptable carriers to prepare a wide vaTiety of ~ q~ Exarnples of 1.1, -- ., -- ~ acceptable carrierri are ~1 -,.,- :; 1 appliances, topical vehicles (non-oral and oral), and ingestible vehicles.

Examples of pl.-.",- ~ l appliances are sutures, staples, gauze, bandages, burn dressings, artificial skins, liposome or micell r.. l-,;
aqueous vehicles for soaking gallze dressings, and the like, and mixtures thereo~ Non-oral topical ., ~.-- :--- - employ non-oral topical vehicles, such as creams, gels r.,.".. l.l,.. ,,~, foarns, ointments and 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 ~ u~ ~ employ oral vehicles, such as ~uLL~L~, rinses, WO96/03149 2 1 9 1 6 03 P~
.

oral sprays, ~ and dental gels, which aro intended to be talcen by mouth but are not intended to be ingested. Ingestlble ~ ernploy ingestlble or partly ingestible vehicles such as ~ . y bulking agents which include hard and soft '' y such as lozenges, tablets, toffees, nougats, c- .y .~ , chewy candies, and chewing gums.

In one form of the invention, the therapeutic wound healing ~
is ~ I ' into a ~ - t ~1 appliance which rnay be in the form of sutures, staples, gauze, bandages, burn dressings, artificial slcins, liposorne or rnicell r.. ".,l ;.. ~ ".;.. Jr .~ . aqueous vehicles for soaking gauze dressings, and the like, and mi~res tbereof. A variety of traditional ingredients may optionally beincluded in the ~.h-.,,.~ ;1... in effective amounts such as buffers,Ci?Cl~' LUV~,~, tonicity adjusting agents, '' "~ t~, polymers for adjusting viscosity or for use as e~tenders, and excipients, and the l~ce. Specific illustrative e~amples of such traditional ingredients include acetate and borate buffers; thimerosol, sorbic acid, methyl and propyl paraben and rhlt rob ~ ?1~ L~ LL~~ , socmm chloride and sugarsto adjust the tonicity; and excipients such as mannitol, lactose and sucrose. Other ~ .IILWLI~II pl. .~, ' -;;' ~1 additives known to those having ordinary skill in the yl ~ i arts may also be used in the 1 In accordance with this invention, ~ lly effective amounts of the therapeutic wound healing c~ - 6 - - of the present invention may be employed in the 1,l ", . . x. ~1 appliance. These amounts are readily determined by thosesl~illed in the art without the need for undue ~ . The exact arnount of the therapeutic woumd healing r~ n~ employed is subject to ruch factors as the type and .~ .,. of the therapeutic wound healing ~ and the type of yl ,l, ~ -; ~1 appliance employed. Thus, the amount of therapeutic wound healingC~ "''1- - ~- ... may be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those slcilled in the art without the need forundue~ y ;~ . Jnapreferred t-~ , the~1 "
will comprise the therapeutic wound healing ~ y~ in an amount from about 0.1% to about 5%, by weight of the yl~ x ~1 "" y - ,~:1 ;. In a ~re preferred ~ 21 ql 603 . ' ~ ' ~ the ~will cornprise the therapeutic wound healrng r~ in an arncmnt from about 0.1% to about 3%, by weight of the ~ In a most prcferred ~ ' ~ the ~ r.. ~
wll comprise the therapeutic wound hcaling ~ ;..., in an amount from about 0.1% to about 1%, by weight of the Thcprescntinventioncxtcndstomcthodsforrnakingthepl;-...-~,..:;. l c~ In gencral, a ~ l l CA~ is rnade by contacting a i- . '1~/ effective amount of a therapeutic wound healing ~ . with a ~ appliance and the other ingrcdients of the final desired pl .. -- ~.. l;. l Y~ The therapeutic wound healing ~ ~- ;- ~-~ rnay be in a solvent and rnay be absorbed onto a l.l.-.... .,..l;. -l appliance.
.

Other ingredients will usually be ~ r 1 into the c~ as dictated by the nature of the dcsired ~ as well known by those having ordinary skill in the art. The ultirnate ~l "., ,~ .n- l;....c are readily prcpared using methods generally known in the ~ arrs.

In another form of the invention, the therapeutic wound healing ~ - c~ is h.~.l ' into a non-oral topical vehiclc which may be in thc form of a crcarn, gel, foarn, ointment, spray, and the l~e. Typical non-toxic non-oral topical vchicles known in the l.h- ... ,l;, ~l arts may be used in the present invention. The prefcrrcd non-oral topical vehicles arc water and ~l..". ...,;, ~11y acceptable water-misclble organic solvents such as ethyl alcohol, isopropyl alcohol, propylene glycol, glyccrin, and thc lilce, and mixturcs of these solvcnts. W , 1 1 ~ mixlures arc p~uLil,ul~uly preferred and are generally employcd in a weight ratio from 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~ y.

The non-oral topical therapeutic wound healing ~ may also contain ~ additivcs employed in those products. Conventional additives include hllrn~rt~rltc emollients, lubricants, stabilizers, dyes, and perfumes, providing WO 96/03149 2 1 9 1 6 0 3 PCTIUS951085!i1 the additives do not interfere with the therapeutic properties of the hherapeutic wound healing ~

Suitable humect2nts useful in the non-oral topical therapeutic wound healing c~ ....... c include glycerin, propylene glycol, pfJ~ k~ glycol, sorbitan, fructose, and the l~e, and rnixtures thereof. TT when employed, rnay be present in amounts from about 10% to about 20%, by weight of the topical therapeutic wound healing ~

The coloring agents (colors, colorants) usefi~l in the non-oral topical therapeutic wound healing f..- ~ ;- are used in arnounts effective to produce the desired color. These coloring agents include pigments which may be ~ r ' ~ in =ts up to about 6% by weight of the non-oral topical therapeutic wound healing .., A preferred pigment, titzniurn dioxide, rnay be I ' in arnountc up to about 2%, and preferably less than about 1%, by weight of the non-oral topical therapeutic wound healing c~ The coloring agents may also include natural food colors and dyes suisable for food, drug and cosrnetic ~ These coloring agents are known as F.D.& C. dyes and lakes. Thc rnaterials acceptable for the foregoing uses are preferably water-soluble. Illustrative ~ uhug examples include she indigoid dye known as FD & C. Blue No.2, which is sbe disodium salt of 5,5 .~, ~ ~r ~ acid. Sirnilarly, she dye known as FD.& C. Green No.1 comprises a hi~h~ .f~ue dye and is the . ---~ -I ,. salt of 4-[4-(N-ethyl-p-! ~r ' ~ y~ lu-o) ~ ,uy~ C]-[I-~N-et~lyl~N-~-~"Ir~ yl)-delta~
2,5~iy. 1..1.. .~ ;.,r] A full recitation of all F.D.& C. coloring agents and their ~ " '~'l" ' I ,, chemical structures rnay be found in the K~ic-Oshmer l~u~ of Chernical Technology, 3rd Edition, in volurne 5 at pages 857-884, which te~s is ~ . ' herein by reference.

In accordance with this invention, ' r 'ly effective amounts of the therapeutic wound healing f.. ~ of the present invenion may be admiced with a non-oral topical vehicle to form a topical therapeutic wound healing -~- ... These amounts are readily deterrnined by those skilled in the art without WO96/03149 21 91 603 r~.,u~

the need for undue ~ -,- In a preferrcd ~ ...u, the non-oral topical therapeutic wound hea]ing r.. ~ will comprise the therapeuic wound healing ~A q...-;~ ... in an amount from about O 1O/D to about 10% and a non-oral topical vehicle in a quantity sufficient to bring the total amount of ~ q~ to 100%, by S weight of the non oral topical therapeutic wound healing ~ In a more preferred ~ 3.u.1~ , the non-oral topical therapeuic wound healing ~
wiD comprise the therapeuic wound healing .1- .l...- ,..., in an amount from about 0.1% to about 5%, and in a most preferred e ' ' ~ the non oral topical therapeutic wound healing . , will comprise the therapeutic wound healing c ,1. ~- u . .. , in an amount from about O.1% to about 2%, and a non-oral topical vehicle in a quantity sufficient to bring the total amount of ~ q-.-S;.... to 100%, by weight of the non-oral topical therapeutic wound healing ~ q -~

The present invention extends to methods for preparing the non-oral topical therapeutic wound hea]ing ~ - '; - - - In such a method, the non-oral topical therapeutic wound healing (.. q.. - u.. l is prepared by admixing a i' L '' ~
effective amount of the therapeutic wound healing ~ q ~ - ~ of the present invention and a non-oral topical vehicle. The final ~-- .q ~- ~; --- are readily prepared using standard methods and apparatus generaDy known by those sl~lled in the I ' arts. The apparatus useful in accordance with the present invention comprises mixing apparatus well known in the ~ l arts, and therefore the selection of the specific apparatus will be apparent to the artisan.

In another form of the invention, the therapeutic wound heahng ~ q~ is ~ into an oral topical vehicle which may be in the forrn of a mouthwash, rinse, oral spray, , dent~l gel, and the like. Typical non-tmcic oral vehicles known in the 1' ~ ' arts may be used in the present invention.
The preferred oral vehicles are water, etbanol, and water-ethanol mixtures. The water-ethanol mixtures are generally employed in a weight ralio f~m about 1:1 to about20:1, preferably from about 3:1 to about 20:1, and ~st preferably f~m about 3:1 to about 10:1, ~ ly. 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 wo96/03149 21 91 603 r~

a pH value below about 4 is generally irritating to dhe orAi cavity and an oral vehicle having a plI value greater alan about 7 genera~ly results in an unpleasant mouth feel.

The oral topical aherapeutic wound healing ~ - may also contain w.l~Lu~l~l additives norrnally ernployed in dhose products. Conventionaladditives include a fluorine providing compound, a sweetening agent, a flavoring agent, a coloring agent, a humectant, a buffer, and an emulsifier, pronding dhe additives do not interfere wia, ahe aherapeutic properties of tbe dherapeutic woond healing ~'""1"'-'';""

The coloring agents and l and ahe amounts of these additives to be employed, set out above as useful in the non-oral topical therapeutic wound healing ~ ...., may be used in ahe oral topical aherapeutic wound healing Fluorine providing cornpounds may be fully or slighay water soluble and are .1..,.. ~ ..I by their abilily to release fluoride ions or fluoride containing ions in water alld by their lack of reaction with oaler c- .. ~.. - .- - ~ in the .. l.. - ~ .. Typical fluorine providing compounds are ino~ganic fluoride salts such as water-soluble alkali metal, aLcaline ear~ metal, and heavy metal salts, for example, sodium fluoride,potassium fluoride, arnrnonium fluoride, cup~ous fluoride, zmc fluoride, stannicfluoride, stannous fluoride, barium fluoride, sodium .~ ~ =onium ~~- - sodium rll sodium n ~ ~ ', aluminummono-anddi-n ,' ~' and fluorinated sodium calciurn ~ r 1 ' Alkali metal fluorides, tin fluoride and n 1~ ~ ~ ' , such as sodium and stannous fluoride, sodium n ~ ' and mLlctures thereof, are preferred.

The amount of fluorine providing cornpound present in the present oral topical aherapeutic wound healing ., ~ l u-~~ is dependent upon the type of fluorine providing compound employed, ahe solubility of ahe fluorine compound, and the nature of the final oral therapeutic wound healing ~...,.~-.-:~-." The amount of fluorine WO96/03149 2 19 1603 r~lm~ ~1 providing compound used must be a nontoxic amount. In general, the fluonne providing compound when used will be present in an arnount 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 wound healing When sweetening agents (sweeteners) are used, those sw=ers well known in the art, including both natuIal and artificial sweeteners, may be employed.
The sweetening agent used may be selected from a wide range of materials including water-soluble sweetening agents, water-soluble ar~ficial sweetening agents, water-soluble sweetening agents derived from naturally occurring water-soluble sweetening agents, dipeptide based sweetening agents, and protein based sweetening agents, including miY~tures thereof. Wthout being limited to particular sweetening agents, ;v~ categories and examples include:
(a) water-soluble sweetening agents such as .,... ~
~1;-- i ;r1~ and pol.~ ' ' such as ~ylose, 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 solids, r'~ yd ' ' - monellin, steviosides, and ~ ,y~lL;~, and mixtures thereof;(b) water-soluble artificial sweeteners such as soluble saccharin salts, i.e., sodium or calcium saccharin salts, cyclamate salts, the sodium, ~mm~ m or r~~ .f3,4-dihydro-6-methyl-1,2,3~dlL;~Ic 1 one-2,2-dioxide, IL~I
salt of 3,4-dihydro-6-methyl-1,2,3~Y~~ ~~ 1 one-2,2-dioxide (Acesulfame-K), the free acid form of saccharin, and the like;
(c) dipeptide based sweeteners, such as ~aspartic acid derived sweeteners, such as ~aspartyl-L-. ' ,' ' ~thyl ester (Aspartame) and materials described n~ United States Patent No. 3,492,131, L-~lp~ rl-N-(2,2,4,4-- yl-3-thietanyl)-D ~~ lr hydrate (Alitame), methyl esters of L-aspartyl-L-~L~... r l~"ly~,~,l ule andL-aspartyl-L-2,5~1~1y~vlJII~l..~ . glycine, L-aspartyl-2,5-dihydro-L-aspartyl-L~l~;y, ' ' )-alanine, and the l~ce;
(d) water-soluble sweeteners derived from naturally occurring water-soluble ~... such as chlorinated derivatives of ordinary sugar (sucrose), e.g., ~,LI~ ~v~y _ derivaives such as derivatives of cLluludcv~.y~u~,lv~i or chlu~uu_~.y2;~1- r.~...,J~ known, for escample, under the product designation ofSucralose; es~unples of ~1IIUIUdW~Y~U~U:~G and uhlul~d~y~ sucros2 derivatives include but are not limited to: l-chloro-l'-duu~-y~u~u~e, 4-chloq~4-deoxy-Alpha-D-galacto-pyranosyl-Alpha-D-r.... ~..r. ,...~ ~k or 4-chloro-4~,.y~ . 4-S chloro 4~eo~ry-Alpha-D-galacto-pylanosyl-l-chloro-l-deoxy-B-D-fructo-faIanoside~ or 4,1'-dichloro-4,1'~i;dw..y~,e1- 1..- ....~ 1',6'-dichlor~1',6'-diuwA.yau~u~., 4-chloro-4-deoxy-Alpha-D-galacto-py~anosy~ 6-dichloro-l~6-dideo~y-~3-D-fructo-furanoside~ or 4~1 ',6'-trichloro-4, 1 ~6~-4;d~uAny~cLLL~ sucrose; 4,6-dichloro-4,6-dideoxy-Alpha-D-galact~pyranosyl-6-chloro~ deoxy-B-D-r~. ~ ~r. ~ . or 4,6,6'-trichloro-4,6,6'-Il;dw~yE~ 6,1',6'-trichloro-6,1',6'-~1;dw~.~ , 4,6-dichloro-4,6-dideoxy-Alpha-D-galacto-pyrauosyl-1,6-dichloro-1,6-di~ y-B-D-r~ r,A",.~ . or 4,6,1',6'-te~rachloro-4,6,1',6'-~ Adw~y~ .,lu-sucrose; and 4,6,1',6'-tetrachloro-4,6,1',6'-tehadeoxy-sucrose; and (e) protein based sweeteners such as i' danielli Crhaumahn I and 11) ~n general, an effechve amount of sweetening agent is uhlized to provide the level of sweetness desired in the pa~ticular oral topical therapeuhc wound healing y ~ and hhis amount will vary wihh the sweetener selected and hhe fmal oral therapeuhc product desired The amount of sweetener nom~ally present is in the range from about 0.0025% to about 90~/0, by weight of hhe oral topical therapeuhc wound healing ~ 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.
The flavoring agents (flavors, flavorants) which rnay be used include those fiavors known to the sl~lled alhsan, such as nahual and artificial flavors.
Suitable flavoring agents mclude mints, such as p~rp~:-t, cihus flavors such as orange and lernon, ar~ficial vamlla, cinnamon, various fruit flavors, both individual and mixed, and the like.

w096/03149 2 1 ~ 1 6~.3 P~ ~-The arnount of flavoring agent employed in the oral topical therapeutic wound healing ~,. " . y.. l- 6 . .., is normally a matter of prcference subject to such factors as the type of final oral therapeutic wound healing L ~" , the individual flavorernployed, and the strength of flavor desired. Thus, the 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 for undue ;.." 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 wound healing .,.- q -~
Suitable buffer solutions useful in the non-oral topical therapeutic wound healing ~.",l,.. :~;....~ include citric acid-sodium citrate solution, phosphoric acid-sodium phosphate solution, and acetic acid-sodium acetate solution in amounts up to about 1%, and preferably frorn about 0.05~/0 to about 0.5% by weight of the oraltopical therapeutic wound healing 4~

ln accordance with this invention, e -r ' 1~/ effective amounts of the therapeutic wound healing ~ of the present invention rnay be admixed with an oral topical vehicle to forrn a topical therapeutic wound healing ~
These arnounts are readily determined by those sl~led in the art without the need for undue ~ 1. ;",. - ;. .,. In a preferred ~ l o~ , the oral topical therapeutic wound healing ~ will comprise the therapeutic wound healing ~ 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 ~....~...-:~1- to 100%, by weight of the oral topical therapeutic wound healing 4""'l"' - In a more preferred, ~ ' ~ the oral topical therapeutic wound healing c~ : -- will comprise the therapeutic wound healing ~A...Il.. ~ ;. .., in an amount from about 0.1% to about 5%, and in a rnost preferred r~ the oral topical therapeutic wound healing ~ will cornprise the therapeutic wound healing ~ in an arnount from about 0.1%
to about 2%, and a oral topical vehicle in a quantity sufficient to bring tbe total amount of ~ to 100%, by weight of the oral topical therapeutic wound heali-ng . .

W096/03149 2 l 9 1 6 03 P~

The present invention extends to methods for preparing the oral topical therapeutic wound healing .,.... q...~ In such a rnethod, the oral topical therapeutic wound healing ~ f-- s.... is prepared by adrr~ix ng a ' r - "y effective amount of &e therapeutic wound healing ~ of the present invention and an oral topical vehicle. The final ~ are readily prepared using standard methods and apparatus generally known by tbose skilled in the ~ A1 arts. The apparatus useful in accordance with the present invention comprises rmxing apparatus well known in the 1 1 arts, and therefore the selection of the specific apparatus wlll be apparent to the artisan.
In a preferred ~,.,1.~1;. ~:, an oral topical therapeutic wormd healing f~ q~u~ ~ l is rnade by first dissolving coloring agents, sweetening agentS, and sirnilar additives in water. The therapeutic wound healing ~ 1 l q~ ~- ~: ., . is then adrnixed with the aqueous solution. Then sufficient water or ethanol, or rnixtures of water and ethanol, are added to the solution with mixing until the final solution volume is reached In a more preferred ~., ,l "~,li . .. the therapeutic wound healing ~ ~ '1" ~~:1 ;. .1.
is added to the solution as the final ingredient. The final oral topical therapeutic wound healing ~""1'''- ;" - are readily prepared using methods generally known in the 1.l.- . --.~..l;. ~1 arts.
The oral therapeutic wormd healing ~ ;- ." rnay also be in the form of dental gel. As used herein, the term "gel" means a solid or semisolid colloid which contains ~1 :1 Al,k quantities of water. The colloid particles in a gel are linlced together in a coherent meshwork which ;..,.,..-~ the water contained inside the 2~ rneshwork.

The dental gel ~ of the present invention may contain the ou..~ u.,~.l additives set out above for oral topical therapeutic wound healing f~q~ such as '~ ' rinses, oral sprays, and ~ and, in addition, may contain additional additives such as a polishing agent, a d .. ~ ;,I.. g agent, and the l~ce, providing the additional additives do not interfere with the therapeutic proper~ies of the therapeutic wound healing ~ f-- ~ -...

WO 96/03149 r~
~ 21 91 6~J3 In a dental gel c. .,. q..~- ~ ;. - the o;al vehicle generally comprises water, typically in an amount from about 10% to about 90%, by weight of the dental gel ';" PulJ~ hyL~l~, glycol, propylene glycol, glycerin, and mixtures thereof rnay also be present in the vehicle as humectants or binders in amounts from about 18% to about 30%, by weight of the dental gel ~ . q.- -~ Particularly preferred oral vehicles comprise mixtures of water with pol~. ~ylLI.~, glycol or water with glycerin and pol.~ u~yk,~c glycol.

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 hyL~ 1 celhllose, methyl ceLulose, glycerin, ~Uh~ ulr~L~ " and gelatin and the like, and mixtmes thereof rn~y be used. The preferred gelling agent is L~ Lyl cellulose. Gelling agents may be used in arnounts from about 0.5%
to about 5%, and preferably from about 0.5% to about 2%, by weight of the dental gel ,"",l", """

The dental gel ~ q~ - - of the present invention may also include a polishing agent. In clear gels, a polishing agent of colloidal silica and/or alkali metal complexes is preferred since these materials have refractive indices close to the refractive indices of thc gelling systerns cormmonly used in dental gels.
In non-clear gels, a polishing agent of calcium carbonate or calciurn dihydrak may be used. These polishing agents rnay be used in amounts up to about 75~/O, and preferably in amounts up to about 50%, by weight of the dental gel ~ n~

The dental gel may also contain a ~ - -'; .g agent such as a r ' of citric acid and sodium citrate. Citric acid may be used in an =t from about 0.1% to about 3%, and preferably from about 0.2% to about 1%, by weight, and sodium citrate rnay be used in an amount from about 03~/O to about 9%, and preferably from about 0.6% to about 3%, by weight of the dental gel r~ q In accordance with this invention, - r ' ~l!/ effective amounts of the therapeutic wound healing ~,.... q.. ~- ~ of the present invention may be admixed into the dental gel ~ These amounts are readily determined by those slcilled in the art without the need for undue ~ In a preferred . .,1 the dental gel ~ will comprise the therapeutic wound healing ~~- q -- ~: ... 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 ~ to 100%, by wcight of the dental gel ~ In a more preferred ~ the dental gel c ...q...- l;.l..~ wlll comprise the therapeutic wound healing ~ in an amount from about 0.1% to about 5%, and in a most preferred _ ~l o l' - ~-1, the dent~l gel q~-; ...c will comprise the therapeutic wound healing ~ ; ~ 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 r..---q--.- --- to 100%, by weight of the dental gel C- " "l "~- ' ;~ .. .

The present invention ~tends to methods for preparing the therapeutic dental gel c~. .l.. ~-~~;~.. ,c In such a method, the dental gel Iv~ q l-~l;l is prepared by admixing a ~ effective amount of the therapeutic wound healing ~- q~- ~;... of the present invention and an oral topical vehicle. The final c,".~ are readlly prepared using methods generally known by those skilled in the dental and i' ' arts. The apparalus useful in accordance with the present invention comprises rrixing apparatus well known in the ~ 1 arts, and therefore the selection of the specific apparatus will be apparent to the artiran.

In a preferred rl I ~ -, a therapeutic dental gel ~ ~ ~ q-- ~- ~;- ~- is made by first dispersing a gelling agent in a humectant or water, or a mi~ture of both, then admixing to the dispersion an aqueous solution of the water-soluble additives such as the fluorine providing compound, sweeteners and the l~ce, then adding the polishing agent, and lastly admL~ing the flavoring agent and the therapeutic wound healingThe final gel mi~ture is then tubed or otherwise paclsaged. The liquids and solids jn a gel product are I ~ ' to form a creamy or gelled mass which is extludable from a pressurized container or from a collapslble tùbe. The finaltherapeutic wound healing c ~ q ~ - are readily prepared USing methods generallyknown in the ~,1 ",~ 1 arts.

Wo 96/03149 2 1 9 1 6 Q 3 E~

~ yet another foml of the invention, the therapeutic wound healing r-Ty is . '~ l into an ingestble vehicle. The ingestible vehicle may be a tA~.Irr. I~ y bulking agent in the folm of lozenges, tablets, toffees, nougats, chewy candies, chewing gums, and the like. The pl~ lly acceptable carriers may be prepared from a wide range of matelials including, but not limited to, diluents, binders and adhesives, lubricants, ~ coloring agents, bulking agents, flavoring agents, sweetening agents and " rnaterials such as buffers and adsorbents that may be needed in order to prepare a particular therapeutic confection.
Thepreparation of ~ ,.,r, ~; -- Y fr~~ innc ishistorically wellknown and has changed little through the years. (~ r y items have been classified as either "hard" C....r, ~ Y or "soft" r----F ~,1;..-- y. The therapeutic wound healing ~" q~ of the present invention can be ;..--.,~ .l into ~....F . ~;..,,..y ~, y.. ~~ -. - by admixing tbe inventive ~ into ~Il~. ~ ' hard and soft rAnf~chr~nc As used herein, the term ~ ~ y material means a product containing a bulking agent seleckd from a wide variety of makrials such as sugar, com syrup, and in the case of sugarless bulking agents, sugar alcohols such as sorbitol and mannitol and mixtures thereof C~-~r~ ~;- Y material may include such exemplary substances as lozenges, tablets, toffec, nougat, ~ chewy candy, chewing gum and the l~ce. The bulking agent is present in a quantity sufficient to bring tho total amount of r~ y~ to 100%. In general, the bulking agent will be present in amounts up to about 99.98%, preferably in amounts up to about 99.9%, and more preferably in amounts up to about 99%, by weight of the ingestble therapeutic wound healing ~ y~

Lozenges are flavored medicated dosage fomss inknded to be sucked and held in the mouth. Lozenges may be in the fomm of various shapes such as flat, circular, octagonal and biconvex fomms. The lozenge bases are generaUy in two forms:
hard boiled candy lozenges and ~,. .., q.. . -~ .1 tablet lozenges.

Wo96/03149 21 q1603 r~,o... ~ ~

Hard boiled candy lozenges may bo processed and forrrrulated by cu..v~.iull~ll means. In general, a hard boiled candy lozenge has a base cormpûsed of a mixture of sugar and other ~,~bullyl' bulking agents kept in an amorphous or glassy condition. This amorphous or glassy forrn is considered a solid syrup of sugars generally having from about 0.5% to about 15% moismre. Such materials Do~ ly contain up to about 92% corn syrup, up to about 55% sugar and from about O.lC/o to about 5% water, by weight of the final r, ., ..~ ., The syrup component is generally prepared from com syrups high in fructose, but may include other materials. Further ingredients such as flavoring agents, sweetening agents, acidulants, coloring agents and the like may also be added.

Boiled candy lozenges may also be prepared from non-r ~ "
sugars such as sorbitol, mannitol, and LylL ,, ' com syr~p. Typical L~
com syrups are Lycasin, a ,,.. ,~, -117 available product ., r . ~ by Roquette ('nrp~tinn, and Hystar, a . . . 'ly available product ~ ' by Lonza, Inc.
The candy lozenges may contain up to about 95% sorbitol, a mixture of sorbitol and rnannitol in a ratio from about 95:0.5 up to about 7.5:25, and ;.~ " ' corn syrup up to about 55%, by weight of the solid syrup component.

80iled candy lozenges may be routinely prepared by w~liullal methods such as those involving fire cookers, vacuum cookers, and scraped-surface cookers also referred to as high speed: . ' cookers.

Fire cookers involve the traditional method of making a boiled candy lozenge base. In tbis method, the desired quantity of ~bull~l' bulking agent is dissolved in water by heating the agent in a keffle until the bulking agent dissolves.
Additional bulking agent may tben be added and cooking continued until a final t 1~l~. 0. ~ of 145~C. to 156~C. is achieved. The batch is then cooled and worked as a plastic-like mass to incorporate additives such as flavors, colorants and the like.
A high-speed ~ I,h ;~ cooker uses a h~ surface which involves spreading a film of candy on a heat exchange surface, the candy is heated to WO 96/03149 2 1 9 1 6 0 3 r~m ~ .

165~C. to 170~C. in a few minutes. The candy is then rapidly cooled to 100~C. to120~C. and worked as a plastic-like mass enabling ;~ of the additivos, such as flavors, colorants and the like.

In vacuum cookers, the c~l,~,h~,' bulking agent is boiled to 125~C.
to 132~C., vacuum is applied and additional water is boiled off without extra heating.
When cooking is complete, the mass is a semi-solid and has a plastic-like y.
At this point, flavors, colorants, and other additives are admi~ed in the mass by routine mechanical rnixing operations.
The opti= mixing reqmred to uniforrnly mi~c the flavoring agents, coloring agents and other additives during ~UI~.iUlldl m~mlf~rhlring of boiled candy lozenges is detennined by the time needed to obtain a uniforrn distribution of the materials. Normally, mixing times of from 4 to 10 minutes have been found to be acceptable.

Once the boiled candy lozenge has been properly ternpered, it may be cut into workable por~ions or formed into desired shapes. A variety of fonning techniques may be utilized depending upon the shape and size of the final product desired. A general discussion of the ~ ; - and preparation of hard cnnf~ctinnc may be found in ~A. Lieb=, ~S l Dosa~e Forms: Tablets, Volume I
(1980), Marcel Dekker, Inc., New York, N.Y. at pages 339 to 469, which disclosure is i-- ' herein by reference.

The apparatus useful in accordance witb the present invention comprises cooking and mixing apparatus well lrnown in the ~ ~ . y ~ g arts, and therefore the selection of the specific apparalus will be apparent to the artisan.

In contrast, compressed tablet confections contain particulate materials and are formed into structures under pressure. These confections generally contain sugars in amounts up to about 95%, by weight of the c~ and typical tablet wo 96/03149 2 1 9 1 6 0 3 F~,l/v... ~

excipients such as binders and lubricants as well as fhvoring agents, coloring agents and the lilce.

In addition to hard ~-,.rr.,~ y rnah rials, the lozenges of the present inverltion rnay be rnade of soft r y mah~rials such as those contairled m nougat. The preparation of soft c.-~f~rh~ns, such as nougat, involves w~lv~dvllal rnethods, such as the . ' of hwo primary ~ , namely (1) a high boiling syrup such as a com syrup, Ly~' , ' starch hydrolysate or the hke, and (2) a relatively light h~stirred frappe, generaUy prepared from egg albumin, gehtul,vegetable proteins, such as soy derived r ~5~ sugarless millc derived cv~pourds such as rmDc proteins, and rnrshures thereof. The frappe is generaLy relatively hght, and may, for example, range rn density from about 0.5 to about 0.7 grams/cc.

The high boiling syrup, or 'rbob Sylup" of the soft ~ is IS rehtively viscous and has a higher density tb~n the frappe component, and frequently contains a substantial amount of ~ buhy.- bullcing agent such as a Lr' O
starch Ly~llul~. C~,1.~, 'l~/, the final nougat ~ is prepared by the addition of the "bob symp" to the frappe under agitation, to forrn the basic nougat mrsture. Fur~her rngredients such as flavoring agents, additional ~4l1u~LyL4ic bulking agent, coloring agents, ~1~ia~ 4~ , mishmes thereof and the lilce may be added thereafter also under agitation. A general discussion of the ~ and preparation of nougat confections may be found in B.W. Minifie, Chocolate. Cocoa and C.. f. 1;.. ~C Science and TechnoloFY. 2nd edition, AVI Publishing Co., Inc., Weshpor~ Conn. (1980), at pages 424-425, which disclosure is r 1 herein by refaence.

The procedure for preparing the soft c f ~ y involves ~nown In gend, the frappe component is prepared first and thereafter the syrup component is slowly added under agitation at a ii.~llt)~4LlU~ of at least about 65~C., and pxferably at least about 100~C. The mi~cture of u "''1" - : - is wntinued to be mi~ed to form a unifortn rnrshlre, after which the mixtux is cooled to a t ~1 ~.t~ ~: below 80~C., at which point, the flavoring agent rnay be added. The mistux is further mr~ed WO 96/03149 2 1 ~ 1 6 0 3 P~ c ~

for an addihonal period unhl it is ready to be re~ved and formed into suitable ,...~ . ;;. ,....y shapes.

The ingeshble therapeuhc wound healing ~-I q ~ c may also be in the folm of a p~ . a ,. 1 Cllcr~ci,.n ~ ."C of this invenhon may be prepared by cul.~, ' ' methods long estabLshed in the art of 13 - - . ., - -: . -l ~ / ' g SllCr~;~C may contain adjunct materials employed in ~ ' ~ the of the art. The , of the present invention can comprise:
(a) ~ D~IYau~D such as butylated Ly~hu~. '~ (13HA), butylated LY~ Y~L~ ; (BHT), benzoic acid, ascorbic acid, methyl paraben, propyl paraben, rl ~ and the hke, and mixtures tbereo~ I'~ D~,lvali~ are generally present in amounts up to about 1%, and preferably from about 0.05% to about 0.5%, by weightof the suspension;
(b) buffers such as cihic acid-sodium citrate, phosphoric acid-sodium phosphate, and acehc acid-sodium acetate in amounts up to about 1%, and preferably from about 0.05% to about 0.5%, by weight of the ---r (c) suspending agents or thickeners such as cellulosics Lke ~...jl~,..lL.lose, ~ - . hke alginic acid and its derivahves, xanthan gums, gelahn, acacis, and llfi.,lu~ " cellulose in amounts up to about 20%, and preferably from about 1% to about 15%, by weight of the . ~
(d) ~ . a l r. . - . ", .g agents such as dimethyl p~ ' in amounts up to about 02%, and preferably from about 0.01% to about 0.1%, by weight of the (e) sweetening agents such as those sweeteners well known in the art, including both nah~ and ar~ficial sweeteners. S~.~. ~g agents such as ~ 1 .1 - - . 1, ;.1. ~ and pOIJ~ -- .1.-.,.1. - such as ~cylose, nbose, glucose (dextrose), =ose, galactose, fmctose (levulose), sucrose (sugar), maltose, invert sugar (a mixture of fructose and glucose derived from sucrose), parhaLy hydrolyzed starch, corn syrup solids, '~ J-ll~ , monellin, steviosides, ~ ,y ' ~ and sugar alcohols such as sorbitol, mamritol, malhtol, Lyl' ~ ' starch Ly~
and mixtures thereof rnay be uhLzed in amoumts up to about 60%, and preferably from about 20% to about 50%, by weight of the suspension. Water-soluble arhficial WO 96/03149 2 1 9 1 6 0 3 r~

sweeteners such as soluble saccharin salts, i.e., sodium or calcium saccharin salts, cyclamate salts, the sodium, ammonium or calcium salt of 3,4~ihydro-6-methyl-1,2,3-' ~ one-2 7-dioxide, the potassium salt of 3,4-dihydro-6-methyl-1,2,3-- ' ' A one-2, ~-dioxide (Acesulfame-K), the free acid form of saccharin, and the like may be utilized in amounts from about 0.001% to about 5%, by weight of the CIlCrr~nCi~
(f) flavoring agents such as those flavors weD known to the skiDed artisan, such as natmal and ar~ficial flavors and mints, such as ~ ' menthol, citrus flavors such as orange and lemon, ar~ficial vaçilla. cinnamon, various fruit flavors, both individual and mixed and the like may be utilized in amounts from about 05% to about 5%, by weight of the suspension;
(g) coloring agents such as pigments which may be l ' in amounts up to about 6%, by weight of the suspension. A preferred pigrnent, titanium dioxide, may be r ' ~ in amounts up to about 2%, and preferably less than about 1%, by weight of the s~er~neir~n The coloring agents may also include natural food colors and dyes suitable fo} food, dmg and cosrnetic ..~ These colorants are known as FD.& C. dyes and lakes. The materials acceptable for the foregoing uses are preferably water-soluble. Such dyes are generally present in amounts up to about 0.25%, and preferably from about 0.05% to about 0.2%, by weight of the 1, (h) ~lrr~l...;; Iy agents such as sodiurn , I-l i ~t, ascorbic acid and the like may be l ~ into ~e suspension to prevent color changes due to aging.
In general, ~i.,,.,l",;,;..g agents may be used in amounts up to about 0.25%, and preferably fçom about 0.05% to about 0 2%, by weight of the , and (i) solubihzers such as alcohol, propylene glycol, pol ~ glycol, and the like rnay be used to solubilize the flavonng agents. In general, ~"l..l,;l;,:.,g agents may be used in amounts up to about 10%, and preferably from about 2% to about 5%, by weight of the suspension.

The ~ of the present invention may be prepared as follows:

W096/03149 2 1 9 1 6 03 P~,ll~J,,,_. I

(A) admix the thickener with water heated from about 40~C. to about 95~C., preferably from about 40~C. to about 70~C., to form a dispersion if the thickener is not water soluble or a solution if the thickener is water soluble;
~3) adrnix the sweetening agent with water to forrn a solution;
(C) adrnix the therapeutic wound healing ~ y~ with the L~L~.~,. .. admixlure to form a unifolm thickener ;h.Aa~/.,.lliG wound healing ~)) cornbine the sweetener solution with the thickener t~p~ ir wound healing ~ and mix until unifom~; and O admix the optional adjunct rnaterials such as coloring agents, fiavoring agents, Ar~rl~lr~r~nts, ~ ;;f~ agents, buffers and additional water with the mixture of step (D) to forrn the - ~

The ingesùble therapeutic wound healing ~ of this invention rG/~y also be in chewable form. To achieve acceptable stability and quality as well as good taste and rnouth feel in a chewable r ~ several ~ are important. These ' include the amount of active substance per tablet, the flavoring agent employed, the degree of ' r "'-'~/ of the tablet and the properties of the ~"~I"~- ';""
Chewable therapeuic candy is prepared by procedures similar to those used to rnake soft ,-.. r,.. ~;.. ~. ~ a typical procedure, a boiled sugar-com syrup blend is fom~ed to which is added a frappe mixture. The boiled sugar-com syrup blend may be prepared from sugar and com symp blended in parts by weight ratio of about 90:10 to about 10:90. The sugar-com syrup blend is heated to t~y, above about 120~C. to remove water and to form a molten rnass. The frappe is generallyprepared from gelatin, egg albumin, milk proteins such as casein, and vegetable proteins such as soy protein, and the like, which is added to a gelatin solution and rapidly mixed at ambient ~ .AIIIIG to fomm an aerated sponge like rnass. The frappe is then added to the ~Iten candy mass and mixed until 1.. ,.. c. - ~ - at h--r between about 65~C. and about 120~C.

WO 96/03149 2 1 9 1 6 ~ 3 l ~ 5/~ ~

The ingesùble therapeutic wound heaEng ~ of tbe instant invention can then be added to the 1.. ",.n~ -- v - mi~ture as the ~ = is lowered to about 65~ C.-95~ C. whereupon additional ingredients can then be added such as davoring agents and coloring agents. The fn~-lqfi~n is further cooled and formedinto pieces of desired ~liml ncirmc A general discussion of the lozenge and chewable t_blet forrns of ___r_~ y rnay be found in E~A Lieberman and L Lachman, pl -". ..~.. I
Dosa~e Forms: Tablets Volume I, Marcel Del~cer, Inc., New Yo}~, N.Y. at pages 289 to 466, which disclosure is il1w1r ' herein by referencc.

In accordance with this invention, i' "~ effective arnounts of the therapeutic wound healing ~ of the present invention rnay be adrnL~ ed into the hard and soft A ~ y products. These amounts are readily determined by those skilled in the art without the need for undue r ~ a preferred .I o~ the ingesùble therapeutic wound healing ~ y..~ wll comprise the therapeutic ~round healing ~ in an amom~At from about 0.1% to about 10%
and an ingestlble vehicle, that is a 1' "y acceptable calrier, in a quantity sufficient to bring the total am~nt of c.. .1 ~- 1: .., to 100%, by weight the ingestlble therapeutic wc~und healing ~ In a rnore preferred l-rl -- the ingestlble 11. .'1" .- :~i .. will comprise the therapeutic wound healing c~ - o ~ in an amount from about 0.1% to about 5%, and in a most preferred L '.~l:., . . l the ingestible r,~ . will comprise the therapeutic wound healing ~ in an amount from about 0.1% to about 2~/n, and an ingestlble vehicle in a quanity sufficient to bring the total amount of ~ - I ." to 100%, by weight the ingestlble therapeutic wound healing ~

The present invention e~tends to methods of m. king the ingeshble therapeutic wound healing ~ In such rnethods, an ingestlble therapeutic wound healing ~ / ""1 - ~: ~ is prepared by adm~ing a 1~ Ally effectivo amount of the therapeutic wound healing ~ with a ~ ly-acceptabie carrier. The apparatus useful in accord nce with tbe present invention cornprises mixing and heating apparatus well known in the .,~ y arts, and therefore the selection of the specific apparatus will be apparent to the altisan. The final ingesLble therapeutic wound heahng ~, .. . q.. .~ are readily prepared using meLhods generally known in the ~- f.. l; ... y arts.

The therapeutic wound healing c~ q ~- ';-- may also be ~ r ' into chewing gums. In this forrn of the invention, the chewing gum ~
contains a gum base, a bulking agenL the inventive therapeutic wound healing ;.... and various additives.
The gum base employed will vary greatly depending upon vanous factors such as the type of base desired, Lhe r ~ y of gurn desired and the other rA ~ used in the rA ~ to make the final chewing gum producL The gum base may be any water-insoluble gum base known in the arL and includes Lhose gumbases utilized for chewing gums and bubble gurns. ~lustrative e~camples of suitable polymers in gum bases include both natmal and synthetic elastomers and mbbers. For example, those polymers which are suitable as gum bases include, without limitation, substances of vegetable origin such as chicle, crown gum, nispero, rvsadinha, jelutong, perillo, niger gutta, tunu, balata, gutta-percha, lechi-capsi, sorva, gutta lcay, mixtures thereof and the like Synthetic elastomers such as b ' styrene ~;ul~vly~
POI~ JUIY~ C~ iDUhU~ylUllC isoprene w~uly~u~l~, pol~ lu..c, mixtures thereof and the like are particularly useful.

The gum base may include a non-toxic vinyl polyrner, such as polyvinyl acetak and its partial LyLul~. polyvinyl alcohol, and mixtures thereof. When utilized, the molecular weight of Lhe vinyl polymer may range from about 2,000 up to and including about 94,000.

The amount of gum base employed will vary greaUy depending upon various factors such as the type of base used, Lhe w .,;~ .,y of the gum desired and the other r..., q. ~ used in the c-- q - ~ to make the final chewing gum product.
In general, the gum base will be present in amounts from about 5% to about 94~/O, by w096/03149 2 1 9 1 6 ~3 weight of the final chewing gum c~ 3;~ , ard preferably in amounts from about 15% to about 45%, and more preferably in amounts from about 15% to about 35%, and most preferably in amounts from about 20% to about 30~/c, by weight of the final chewing gl~m c.~

The gum base ~ may cont~un Cuu~,uL;uu~l elastomer solvents to aid in softening the elastomer base componerlt. Such elastomer solvents may comprise te~pinerle resins such as polymers of Alpha-pirlene or 13-praene, methyl, glycerol or ~ La~,~yau;Lul esters of rosins or modified rosms and gums, such as LrLI " 1, dimerized or pulru~.~ rosins or mi~res thereof. Examples of elastomer solvents suitable for use herein include the p~,uk~ rauiLul ester of partially LrLu~, ' wood or gum rosin, the L~L~.~ ~iiul ester of wood or gum rosir., the glycerol ester of wood rosin, tbe glycerol ester of parlially dirnerized wood or gum rosin, the glycerol ester of pU.J.ll~ wood or gum rosin, the glycerol ester of tall oil rosin, the glycerol ester of wood or gum rosin and the partiaay 1.~.' " ' wood or gurn rosin and the partially l.rl' ~ ' methyl ester of wood or rosin, mixtures thereof, and the like. The elastoraer solvent may be employed in amounts from about 5% to about 75%, by weight of the gum base, and preferably from about 45% to about 70%, by weight of the gum base.

A variety of traditional ingredients may be included in the gum base in effective amounts such as plasticizers or softeners such as lanolin, palmitic acid, oleic acid, stearic acid, sodium stearate, potassium stearate, glycelyl triacetate, glyceryl lecithia, glyceryl propylene glycol acetylated ~Oly~,.,.;de, glycerine, mibctures taereof1 and the like raay also be hl~UII ' ' iato the gum base to obtaia a variety of desirable textures and consistency properties.
Waxes, for example, natural and syathetic waxes, Lyd c. ' vegetable oils, petroleum waxes such as pul~.,.c' waces, pol~.,ihylh..~, waxes, paraf~ia waxes, . u~,.u.,.~,,;Gllhle waxes, fat~y waxes, sorbitan , tallow, propyleae glycol, mixtures thereof, and the like may also be r ' ~ iato the gum base to obtaia a vatiety of desirable textures and ~,uu~;~L--~,y properties. These traditional additional materials are generally employed in amounts up to about 30%, by weight of the gura ~ WO 96/03149 2 1 9 1 6 0 3 r~

base, and preferably in amaunts from about 3C/o to about 20%, by weight of the gum base.

The gum base may include effective amounts of mineral adjuvants such S as calcium carbonate, magn~ n carbonate, alumina, aluminum hydroxide, aluminum silicate, talc, tricalcium phosphate, dicalcium phosphate and the like as weD asrnixtures thereo~ These mineral adjuvants may serve as fiDers ard textural agents.
These fiDers or adjuvants may be used in the gum base in various amounts. Preferably the amount of filler when used wiD be present in an amount up to about 60%, by weight of the chewing gum base.

The chewmg gum base rnay ~LI;JULL~1IY include the GUII~,ILLLU ~LI
additives of coloring agents, Al.~ LLL~.. and the IDce. For example, titanium dioxide and other dyes suitable for fooL drug and cosmetic ~
known as F.D. & C. dyes, may be utihzed. An amtioxidant such as butylated hyd~u~.yi ' ~ (BE~I'), butylated L~Lu.~y~l;svl~ (BHA), propyl gallate, and mi~tures thereof, may also be included. Other GU1L~LLiIULL~I chewmg gum additives known to one having ordinary shll in the chewing gum art may also be used in the chewing gum base.

The gum c--"q- - ~;-- may include effective amounts of L~ULL~ILLLLUILa1 additives selected from the group consisting of sweetening agents (sweeteners), pl~-~iri7~s~ softeners, emulsifiers, waxes, fiDers, bulking agents, rnineral adjuvants, flavoring agents (flavors, flavorings), coloring agents (colorants, colorings), : ' ' , acidulants, thickeners, mixtures thereof and the like. Some of these additives may serve more than one purpose. For example, in sugarless gum ~-- q ~ , the sweetcner, e.g., sorbitol or otber sugar alcohol or mixtures thereof, may also function as a bullcing agent. Similarly, in sugar contair~ing gum c-- q.. ~ the sugar sweetener can also function as a bulking agenL
The p~ i7~c softeners, mineral adjuvants, coloranLs, waxes and discussed above as being suitable for use in uhe gum base may also be WO96/03149 21 91 603 r~

used in the gum ~ 6. ~ amples of other ~,uuv~..rhullrJl additives which may be used include emulsifiers, such as lecithin and glyceTyl ~ thickeners, used alone or in .. 1.. - ;.. with other softeners, such as methyl cellulose, alginates, xanthan gum, gelatin, carob, tragacanth, locust bean, and carbo~y methyl cellulose, acidulants such as malic acid, adipic acid, citric acid, tartaric acio, fumaric acid, and mixtures thereof, and fillers, such as those discussed above under thecategory of mineral adjuvants. The f~lers when used may be utilized in an amount up to about 60%, by weight of the gum ~

Bulking agents (carriers, extenders) suitable for use in chewing gums include sweetening agents selected from the group consisting of ~ r . .i.l., poly ' ' sugar alcohols. and mixtures thereof; polyl~hu~
.. '-.~. .I,;.,c minerals, such as calcium carbonate, talc, titanium dio~ide, dicalcium phosphate, and the l~ce. Bulking agents may be used in amounts up to about 90%, by weight of the final gum v- .~ ; ~ with amounts from about 40% to about 70%, by weight of the gum ~ " being preferred, with from about 50% IO about 65%, by weight, being more preferred and from about 55% to about 60%, by weight of the chewing gum ~ being most preferred.

The sweetening agent used may be selected from a wide range of materials including water-soluble sweeteners, water-soluble artificial sweeteners, water-soluble sweeteners derived from naturally occurring water-soluble sweeteners, dipeptide based sweeteners, and protein based sweeteners, including mixtures thereof. Without being limited to particular sweeteners, ~ categories and examples include:
(a) water-soluble sweetening agents such as .. - - . l._. ;.1. ~, .1-_ .1._.;.1. ~ and ~ul~ ' ' such as ~ylose, nbulose, glucose (de~trose), mannose, galactose, f~uctose (levulose), sucrose (sugar), maltose, invert sugar (a mixture of fiuctose and glucose derived from sucrose), panially hydrolyzed d, com sympsolids,.'~ u' I monellin,steviosides,oly~yllL~u,andsugaralcohols such as sorbitol, mannitol, maltitol, L~uh~o ' starch L.~LulJ~ and mixtures thereof;

WO 96/03l49 P~

(b) water-soluble artificial sweeteners such as soluble saccharin salts, i.e., sodium or calcium saccharin salts, cyclamate salts, the sodium, s~njll~ orc~lrillmcslfnf~4-dihydro-6-methyl-l~2~3~ ~2,2-dioxide, il~ ut~;u~
salt of 3,4-dihydro-6-methyl-1,2,3- ' 1 one-2,2-dioxide (~ ''' K), the free acid form of saccharhn, and the like;
(c) dipeptide based sweeteners, such as L-aspartic acid denved sweeteners, such as L-aspartyl-L-~ ' ~ methyl ester (Aspartame) and materials dcscnbed in United States Patent No. 3,492,131, L-Alpha-aspartyl-N-(2,2,4,4-t~ha~ ,lh,~l-3-thiefa}lyl)-D el~ ~e~iA~ hydrate (Alitame), methyl esters of L-aspartyl-L-l ' yl~;l.y~ .,IL.. andL-aspar~yl-L-2,5~.~Lu!,L.,.. J' glycine, L-aspartyl-2,5-dihydro-L-~JL~ 4. e; L,aspartyl-L-(1-cyclohexen)-a]anine, and the like;
(d) water-soluble sweeteners derived from naturaLy occurring water-soluble sweeteners, such as chlorinated derivatives of ordinary sugar (sucrose), known, for example, under the product designation of Sucra4ose; and (e) protein based sweeteners such as i' _ daniehi (Tha-umatin I and I[).

In general, an effective arnount of sweetener is utilized to provide the level of bulk and/or sweetnoss desired, and this amount wll vary with the swee4-ner selected. This arnount of sweetener will normally be present in amounts from about 0.0025% to about 90%, by weight of the gum ~ depending upon the sweetener used. The exact range of arnounts for each type of sweetener is weL known in the art and is not the subject of the present invention. The amount of sweetener ordinarily necessary to achieve the desired level of sweetness is ', ' from the flavor level achieved from flavor oi4s.

Preferred sugar based .. _re sugar ~sucrose), corn syrup and mixtures thereo~ Preferred sugarless sweeteners are the sugar alcohols, arti~icial sweeteners, dipeptide based sweeteners and mixtures thereof. Preferably, sugar alcohols are used in the sugarless ~ because these sweeteners can be used in amounts which are sufficient to provide bulk as weh as the desired level of sweetness. Preferred sugar alcohols are selected from the group consisting of sorbitol, WO96/03149 2 1 9 1 6 Q3 i~ J~l ~

ylitol, rnaltitol, mannitol, and rni~tures thereof. More preferably, sorbitol or a rni~cture of sorbitol and rna~mitol is utilized. The gamrna form of sorbitol is preferred. An artificial sweetener or dipeptide based sweetener is preferably added to the gum ~ q~ which contain sugar alcohols.
s The coloring agents useful in the gum , are used in amounts effective to produce the desired color. These coloring agents include pigrnents which may be ~ ~ I in arnounts up to about 6% by weight of the gum c. ~I~h~
A preferred pigrnent, titanium dio~ide, may be ~ ~ ' in arnounts up to about 2Q/o~ and preferably less than about 1% by weight of the ~- . q -- ~;-- The colorants rnay also include natural food colors and dyes suitable for food, drug and cosrnetic Al.~.l;. ;--llc These colorants are known as F.D.& C. dyes and lakes. The rnaterials acceptable for the foregoing uses are preferably water-soluble. Illustraùve nonlirniting examples include the indigoid dye known as FD.&: C. Blue No.2, which is the disodiurn salt of 5,5 ~ lig ~ '~ ~' ~ acid. Similarly, the dye known as F.D.& C.Green No.l comprises a lfil.h~ ., dye and is the " ,. . .~ i,, . salt of 4-[4-(N-ethyl-p ~' ~ ~ld. f,.,o~h.,.ly~.;L;l~e] [I~N-ethyl-N-p ~
delta-2,5~y.1..1.. ~ ]. A full recitation of all FD.& C. colorants and their w~ )u~ o cherr!ical structures may be found ir. the Kirl~-Othmer E~l..~.,l",,_Ld of Cherrfical Technolo~. 3rd Edition, in volume 5 at pages 857-884, which text is r ' herein by reference.

Suitable oils and fats usable in gum r.- ~ include partially L~,' o ' vegetable or ani;Dal fats, such as coconut oil, palm kemel oil, beef tallow, lard, and the l~ce. These ingredients when used are generally present inarnounts up to about 7%, by weight, and preferably up to about 3.5%, by weight of the gmn f..".l.u~ u....

In accordance with this invention, ~ ly effective amounts of the therapeutic wound healing ~ of the present invention may be admixed into a chewing gmn These amonnts are readily detem~ined by those skilled in the alt without the need for undue l. ".,., -:;- ~ In a preferred ~ the final W096,03,4g 2 1 q 1 6 0 3 F~,l/u ~S'l chewing gum ~ u~ will comprise the therapeutic wound healing ~
in an amount from about 0.1% to about 10% and a chewing gum ~ y ~ m a quanity sufficient to bring the total amount of f .... q...- :~; to 100%, by weight of the chewing gum, - y...- l;.... In a more preferred c ' ~ t, the final chewing gum ~.. ~1.. - ;.. will comprise the therapeutic wound healing ~.. y.. -:l .. in an amount from about 0.1% to about 5%, and in a most preferred ~ ' ~ tbe final chewing gum c.- .~ ;.... Wlll comprise the therapeutic wound healing c~ y~ i;n an amount from about 0.1% to about 2%, and a chewing gum ~ y~ in a quantity sufficient to bring the total amount of ~ .... y. ~- ~ to 100%, by weight of the chewing gum ~

The present invention extends to methods of making the therapeutic chewing gum ~ The therapeutic wound healing ~ y.. -~ may be ' into an otherwise .,u...~,..Lu~l chewing gum ~ y~ using stmdard techmiques amd equipment known to those skilled in the art. The apparatus useful m accordance with the present invention comprises mixing amd heatmg apparatus wellknown in the chewing gum ..,--.--r~..,...;..g arts, and therefore the selection of the specific apparatus will be apparent to the artisam.

For example, a gum base is heated to a t.,lll~.,l,l,~C sufficiently high enough to soften the base without adversely effectmg the physical amd chemical make up of the base. The optimum i r ' utihzed may vary depending upon the y~ - of the gum base used, but such ' ~y ~ are readily detem~med by those skilled in the art without undue ~ r ~ ' '-The gum base is cu.~ / melted at i r ' that r;mge from about 60~ C. to about 120~ C. for a period of time sufficient to render the base ~Iten.
For example, the gum base may be heated umder these conditions for a period of about thirty minutes just prior to being admi~ed; . ~ with the remaiumg mgredients of the base such as the plasticizer, fillers, the bul~ing agent amd/or sweeteners, the softener and coloring agents to plasticize the blend as well as to modulate the hardness, v;, ~,. ~ and formability of the base. The chewing gum base is then blended wo s6l03l49 2 1 9 1 6 0 3 P~

with the therapeutic wound healing ~ of the present invention which may have been previously blended with other traditional in~i.~ntc ~ing is Nntinued until a uniform mixture of gum ~ 1 is obta~ed Thereafter the gum i o- ~ ~ mixture may be formed into desirable chewing gum shapes En a specific ~ tbe invention is directed to a therapeutic for preventing and reducing injury to " cells, aDd increasing the ~ l rate of injured o, 1" l; --- cells, which comprises(A) a ~ I, t; 'l~ effective amount of a therapeutic wound healing c""l"~ of r~ One (I) selected from the group consisting of (LA) (a) pyruvate selected from the group consisting of pyruvic acid, yl --, - ; lly acceptable salts of pyruvic acid, and mixtures thereof;
(b) an F ~' '~ and (c) a mi~rture of saturated and, 1 fatty acids wherein the fatly acids are those fatty acids required for the repair of cellular membranes and - of ' cells;

~B) (a) pyruvate selected from the group consisting of pyruvic acid, 20pl --~-- ; lly acceptable salts of pyruvic acid, and mixtures thereof;
(b) lactate selected from the group consisting of lactic acid, ~"" - ,, ; ily acceptable salts of lactic acid, and mixtures tbereof; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids ale those fiatLy acids required for the repair of cellular membranes and 25~ ~ of ' cells;

~LC) (a) an ' ~ and (b) a rnixture of saturated and ~ ' fatty acids wherein tbe fatty acids are those fatty acids required for the repair of cellular In~l~n' c and 301 of m ~ cells;

WO96/03149 2 1 9 1 603 P~

(LD) (a) lactate selected from the group coosisting of lactic acid, pl ~ Y acceptable salts of lactic acid, and mixtures thereof;
(b) an ' ~., aod (c) a mi~ture of saturated and I ' fatty acids wherein the fatly acids are those fatty acids required fo} the repair of cellular ' and t ~' - of " "", 1 , cells; and (B) a rl -. ~ acceptable carrier.

The ~ ; Al~y acceptable carrier roay be selected from the group consisting of ~' - ,.. - ~ .,1,.. ~l appliances, topical vehicles, and ingestlble vehicle In anotber specific ~",l..,A I the invention is directed to a method for preparing a therapeutic 1,l "" ~ l;-- for preventing and reducing injury to mq~m~liqn cells, and increasing the ~ ~ rate of injured ...~....,..1- -cells, which comprises the steps of:
(A) providing a i' , ~ ~y effective arnount of a therapeutic wouod healingof r,..l.~l, l One (I A-D) selected from the group consisting of (LA) (a) pyruvate selected from the g~up consisting of pyruvic acid, 1' 'ly acceptable salts of pyruvic acid, and r~ctures thereof;
(b) an ~ ..1 ,1. and (c) a mixture of saturated and I ' fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular .. ,.1..... ~ and of mArnTnAli~ cells;
a.B) (a) pyruvate selected from the group consisting of pyruvic acid, ~ly acceptable salts of pyruvic acid, and mi~ttures thereof;
(b) lactate selected from the group consisting of lactic acid, - lly acceptable salts of lactic acid, and mi~tures thereof; and (c) a mi~cture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular .. 1.... ~.. c and ~ of m~Ammqliqn cells;

WO g6103149 PCTIUS95/08551 21 ql 603 (LC) (a) an ~ , and (b) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular ~ and - Of m~ql~ cells;

~ D) (a) lactate selected from the group cODsisting of lactic acid, lly acceptable salts of lactic acid, and mixtures thereof;
(b) an ' ~ and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular .. , . ,1. -. ~ and of ' cells; and (B) providing a l,l - ." - . ;. ~11y acceptable carrier; and (C) admi~ing the therapeutic wound healing ~ from step ~A) and the r3 ~ lly acceptable carrier from step (B) to form a therapeutic l,~
1~ '~""'1"'-' ;""

Throughout this ~rrl;~qfinn various r ' 1' '' have been referenced.
The disclosures in these 1~.3.1;~ are , ' herein by reference in order to ~re fully describe the state of the aTt.
The present invention is f~ther illustrated by the following examples which are not intended to limit tbe effective scope of tbe claims. AU p_rts and p.~ UlGg~ in the examples and throughout the ~ - ;.". and claims are by weight of the final ~ ,n- ,.. . unless otherwise specified.
E. E~mples Of The Therapeutic Wound lEIea~ng C
Of r ~ ~ Ore (I~-D) Sf:lrdy 1 This study ,i.. - ~ ~ a comparison of the viability of U937 monocytic cells after exposure of the cells to various - ;..~,8 ~. and ~. .,i.: l;..

WO g6/03l49 2 19 16 () 3 r~"~ l of . ,~ ,. This study also ~ a ~ nC~n of the levels of hyd}ogen peroxide produced by U937 monocytic cells and .~ epiderrnal 1 ~D
after exposure of the cells to various 1 and ~ Of The results of this study are illustrated in Eigures 1 1 and examples 1-26 below.
s r ~ ~ epiderrnal 1 ~ ~ ~D and monocytes were employed to exarr~ine the ability of various ' to reduce levels of hydrogen pero~cide in these cells. Hydrogen peroxide was measured after the cells were exposed to ultraviolet light in the wavelength range from 290 to 320 nm ~UV-B) or to the ~~ ~ compound 12-0 ~ . ,' ' ' 13-acetak CIPA). Sodium pyruvate was tested at various ..,.... ,;.,u.. to determine the effect of:
of this antioxidant on the hydrogen perw;ide production by epidem~al cells and monocytes. !\f L"" - ---" pyruvate, calcium pymvate, zinc pyruvate, and .~. ~1,: - ;,. ~
of sodium pyruvate with ascorbic acid, lactic acid, and Vitamin E were then tested to deterrnine the effect of these salts and ~ of ~ on the hydrogen peroxide production by epidermal cells and monocytes.

~' ' epide~ llV~ ~D were isolakd by Il~D~Lvll of epithelial sheets and grown in rnodified basal MCDB 153 medium ~ . u 1 with epidermal growth factor, bovine pituitary extract, and Ly.1 ",."1;~,, Cells weremaintained in a hurnidified incubator with 5% carbon dioxide at 37~C. K~ D
were seeded in 60 mm culture dishes at a cell density of 3 x 105 cells per dish and the cultures were exposed to I M.E.D. dose of ul~ ;ol-l B light (100 mJ/cm2) or treated with 100 ng/rnl of TPA.
U937 monocytic cells are a cultured cell line grown in RPMI media with 10~/0 fetal calf serurn Cells were maintained in a 60 rnm culture dish at 5% carbon dioxide at 37CC. at a seeding density not exceeding 1 x lo6 cells per dish.

Sodiurn pyruvate, lactic acid, ascorbic acid, and Vitarnin E were dissolved in distilled water, with sufficient surfactant. The c~--- : ~l;~ of the sodium pyruvate solutions prepared were I mM, 10 mM, 50 mM, 100 mM, and WOg6/03149 21 91 6 03 r~ S 1~ 1 ~

200 mM. The C1. . . ,-~ of the lactic acid solulions prcparcd were 1.0%, 0.1%, and 0.05%. The ~ of the ascorbic acid soluùons preparcd were 1.0%, 0.1~/O~ 0.05~/0~ and 0.025~/0. The ~ of the vitamin E soluùons prepared wcre 1 U, 10 U, 50 U~ and 100 U. The test solutions wcre adjusted to a pH value of 7.4 with 1.0N sodium hydroxide solutio~ and then sterile filtered. The appropriate of test solution or ' of tcst solutions was added to the ceDs ' 1~ prior to exposure of the cells to ultraYiolet light-B or TPA [lOOnglml].
Stock solutions were prepared so that the vehicle did not constitute rnore than IC/o of the total volume of the culture media.
~ ~~ " ' hydrogen peroxide production by m.n~mA1;An epidermal }ClalJIIOU~LUD and U937 monocytes was mcasured using ~ ...n....r~ - diacetate (DCFH-D~ Molecular Probes, Eugene, Ore.). DCFEI-DA is a non-polar non-fluorescent compound that readily diffuses into cells where it is hydrolyzed to the polar non-fluorescent derivaùve DCFH which then bccomes trapped within the cells~ In the prcsence of; ~ A I hydrogen perwcide, DCFlI is oxidized to the highly fluoroecent compound DCF. Hence, ceDular n - .. r ~ intensity is directly ~1uyw ~ull~1 to the level of; .~ . hydrogen peroxide produced. Cellular n....,~ . intensity can be monitored by fluorimetrY and by flow cytometry.
~ ' '' epidermal hcl~L~u~ ~D and U937 cultured monocytes (1 x 106 per dish) wcre incubated at 37CC. with 5 uM of DCFEI-DA Production of hydrogen peroxide was rneasured using a Coulter Profile analytical flow cy[ometer.
Linear and log intensity of green n.. r-.. -- ~ data was collected For each analysis, a quantity of 10,000 to 20,000 eYents was ~ Optical aligmnent for the instrument was performed d~uly Cnr~ffi 1r~ntc of variation for forward angle light scatter and integrated green 11 -, G- e were generally less than two. Each analysis was repeated three tirnes and the '1" - ~:~- ,-- of n .... ... ~ was e~rcssed in terms of F ~ ..... 1~ (finol, 10-15 mohs) of DCF ~idized per ceD, which is a direct measure of the; , ........ 11~ hydrogen peroxide produced. AltematiYely, in the saturated and ' fat~y acid ex mples in examples 27-52~ fluorimetry was used to assess the DCF oxidation per ceD.

WO96/03149 2 1 9 1 603 P~

The viability of the U937 rnonocytic cells after exposure of the cells to various . ;",;.i-.s~ for 24 hours was measured. The viability of the cells was deterrnmed by exposing the cells to the dye propidiun iodide. Permeable cell ...... ,1.. , .. ~ which absorbed the dye were not considered viable. The viabiiity of the cells was .=, ' as the percentage of cells that excluded propidium iodide.
Figure 1 depicts in bar graph foImat tbe viability of U937 monocytic cells afterexposure of the cells to no antioxidant (E~arnple 1, control), to sodicun pyruvate (E~ample 2), to ascorbic acid ~Exarnple 3), to lactic acid ~arnple 4), and to Vitamin E ~Example 5). Figure 2 depicts in bar graph format the viability of U937 monocytic cells after exposure of the cells to various ~ of !~perifir~l'~/, the viability of U937 monocytic cells was measured after exposure to no antioxidant (Example 6, control), to ascorbic acid and lactic acid ~ample 7), toascorbic acid and Vitarnin E ~Example 8), to sodium pyruvate and ascorbic acid (Exarnple 9), to sodiurn pyruvate and lactic acid (Example 10), to sodium pyruvate and Vitamin E (Exarnple 11), to lactic acid and Vitamin E (E~ample 12), and to sodnun pyruvate, ascorbic acid, and lactic acid (Example 13).

Figure I shows that ascorbic acid is cytotoxic to rnonocytes at as low as 0.25%. Figure 2 shows that the ~;ylulu~ y of ascorbic acid was reversed by the addition of 10 mM of sodiurn pyruvate. Figures I and 2 show that the viability rate of 15% to 20% of the cells when treated with ascorbic acid was increased to 95% to 98% upon addition of sodium pyruvate. Lactic acid and Vitamin E did not reverse the ~iyu~twu~,;ly of ascorbic acid.

Sodium pyruvate was then tested at various to determine the effect of ~ .... e~ ,. i ;.. - of this antioxidant on the hydrogen peroxide production by epidermal cells and monocytes. I'" ' epidermal l~ ~o.,~;. and monocytes were exposed to (a) I M E.D. dose of ultraviolet light-B and (b) 100 nglml of 12-O-t~,h~,.,~u..,~l~Lu,1.ol-13-acetate CIPA) in the presence of sodium pyruvate at the following c-, . ~ 200 mM, 100 mM, 50 mM, 10 mM, I rnM.

WO 96103149 2 1 ~ 1 6 0 3 The optirnurn L.~ SA~ 11 of sodiurn pyruvate to reduce the hydrogen peroxide production by epiderrnal cells and ~nocytes was found to be 10 n~M.
C-~ . of sodium pymvate of 50 mM and above were cytoto~ic to botn epiderrnal ht~ "i, and ~nocytes.

r~ 1 pyruvate, calcium pyruvate, zmc pyruvate, ascorbic acid, Iactic acid, and Vitamin E, and ~ of sodium pyruvate with ascorbic acid, lactic acid, and Vitamin E were then tested to deterrnine the effect of these salts and of - ' on the hydrogen pero~cide production by epiderrnal cells and ~nocytes. The following test solutions were prepared.

(a) sodium pyruvate [10 rnMl;
(b) zinc salt [10 n~
(c) mAgn.~ m salt [10 n~Il;
(d) calcium salt [10 n~q;
(e) sodiurn pyruvate [10 mMl and ascorbic acid [0.025%];
(f) sodiurn pyruvate [10 n~ll and lactic acid tO.05%];
(g) sodiurn pyruvate [10 mMl, lactic acid, [0.05%], and ascorbic acid [0.025%];
(h) lactic acid [1.0%, 0.1%, and 0.05%];
(i) ascorbic acid [1.0%, 0.1%, 0.05%, and 0.025%];
(j) Vitarnin E [I U, 10 U, 50 U, and 100 U]; and (k) vehicle solvent controls.

There was no significant difference among the zinc, ,, and calcium salts of pyruvic acid on the hydrogen pero~;ide production by epidc~mal cells and monocytes. The zinc and calciurn salts of pyruvic acid induced .1 IF~ .n.S;,-;.,.. of ~cl~l~illu~ or ~.lv~.lP..,.Ice, the sodium salt was used in subsequent tests.

~ W0 96/03149 2 1 9 1 6 0 3 P~ S ( The optimum c- ...e~ of lactic acid to reduce the hydrogen pero cide production by epidermal cells and monocytes was found to be 0.05%. Theoptimum ~ of ascorbic acid was found to be 0.025~/c. The higher of both of these W___r ~ were found to be cytotoxic to both types of cells. The optimum ~A~- - ' '.:'AI;~'' of Vitamin E was found to be 50 U.

Figure 3 depicts in bar graph format the levels of hydrogen peroxide produced by U937 monocytic cells afier exposure of the cells to no antio~idant ~ample 14 control) to sodium pyruvate (Example 15) to ascorbic acid (Example 16) to lactic acid (Example 17) and to Vitamin E (Example 18). Sodium pyruvate and Vitamin E D;~lifi~allily rednced the hydrogen perocide production by monocytes.

Figure 4 depicts in bar graph format the levels of hydrogen peroxide produced by U937 monocy~ic cells after exposure of the cells to vafious ~ .,1. -1;
of r- -- Specifically the levels of hydrogen peroxide prcduced by U937 ~nocytic cells were measured after exposure to no antioxidant (Example 19 control) tc~ ascorbic acid and lactic acid (Example 20) to ascorbic acid and Vitamin E
(Example 21) to sodium pyruvate and ascorbic acid (Example 22) to sodium pyruvate and lactic acid (Example 23) to sodium pyruvate and Vitamin E (Example 24) to lactic acid and Vitamin E (Example 25) and to sodium pyruvate ascorbic acid and lactic acid (Example 26). The r~....T...-I;...~ of lactic acid (0.05%) and Vitamin E
(50 U) S;~,.-r. a lay reduced the hydrogen peroxide production by monocytes.

The . .. ~ alterations in epiderrc~i ~claLillu~ D were observed in control cultures and in cultures exposed to . iol t B. Ceils in the layer closest to the demmis are basal l~clat;llo~.~a. These ceils proliferate and migrate into the spinous and granular layers of the epidermis where the cells begin to ~1-rr " ' The d;rrclcllu 1~)ll pattem results in cells ~ ..Lf~ and foming cornified envelopes at the uppermost portion of the epidemis the statum comeum. The ~ r~ ll of ;..J~ ., is controlled by the levels of calcnn 1 ~ and other elements in the medium. Cells in culture systems promoting .~ ~. .nllll-l;l .. appear as an epidem~al WO96/03149 2 1 9 1 603 ~ r~

sheet forming ~ ,...., or tight junctions with each other. KclahLIv~ that become ~' or float rn the media were considered responding to a CytOtO~iC
event.

The following ,.. ~ gi.. l alterations in tbe m~ l' epidem~al aLi~v~ ~ were observed for the following contrl cultures:

10 mM Sodium Pvruvate: Tight junctions of cells were formed and tbe ~ u~
rate of tbe cells was higher than the rate of the contrvl cells.
0.025% Ascorbic Acid: Cells were floating in a cyt~toxic response to ascorbic acid.

0.025% Ascorbic acid and 10 mM Sodium Pvruvate: F,,e,w tight junctions of cells were observed and cells appeared simllar to the cells in the sodium pyruvate culture.
0.05% Lactic Acid: Cells appeared ~L~lly altered as an epidermal sheet and as flat granular cells.

0.05% Lactic Acid and 10 mM Sodium PvTuvate: Cells formed an epidem~al sheet butappeared smaller than the cell in the lactic acid culture.

50 U Vit~nin E: Cells appeared ~e same as ~e cells in ~e control culb~e.

50 U Vitamin E and 10 mM Sodium Pvruvate: Cel'is increased in number and changedin appearance resernbling the cells in the sodium pyruvate culture.

The following ~ g; 1 alterations in the m~rnm~ epidermal hclaL~IO~;~., were observed for the ~ g cultures e~posed to ultraviolet ligVht-B, 100 mJoules, for 24 hours:
10 mM Sodium Pvruvate: Cells proliferated more rapidiy than the cells in the contrl culture.

WO96/03149 2 1 ~ 1 6 03 0.025% Ascorbic Acid: Cells were and floating in a cytoto ic response to ascorbic acid greater than the CytOtOXiC response of the ~ E cells without . ;ol~ . B light exposure.

0.05~/O Lactic Acid: Cells for ned an epidermal sheet and were rnore granular than cells in the cont~ol culture without uluav;vl~,~ B light exposure.

50 U Vitamin E: Cell growth was inlubited but cells appeared sirnilar to cells in the control culture without ullldvioL ~ B light exposure.
50 U Vitarnin E and 10 rnM Sodium Pvruvate: Cells appeared sirnilar to cells in the control culture and 1 ~ r ' ~ to a greater extent than cells in the cont~ol cultures without IJIl~dVi~ B light exposure.

~.. ~ g;. 1 alteraions in the U937 rnonocyic cell line were also observed for control cultures and cultures exposed to ult~aviolet light-B 100 rnJoules for 24 hours. The foilowing .~.. 1.. lc and .. 1 .. -~.. of at the set out below ~;~l~;rn ~lly iniubited the levels of hydrogen peroxide produced by U937 monocytic cells:

Sodiurn pyruvate at 10 mM and 50 mM;
Vitarnin E at 50 U and 100 U; and Lactic acid at 0.05% and Vitamin E at 50 U.

E~amples Of l'he Therapeutic Wound nealing rl of r ~, One (I.A-D) Stndy 2 This study d a o l17 p~c ~n ofthe levels of hydrogen peroxide produced by U937 monocytic cells and epidermal hc.~Lillu. ~. after exposure of the Wo 96/û3149 2 l 9 1 6 G 3 P~

cclls to va~ious ~.. ,.1, -1;.. of .. ,.:1.. ~;.1~., with and without a mixture of saturated and, ' fatty acids. The results of this study are iilustrated in Figures 5-7 and examples 27-52 below.

~ ' ' epidermal hcla~ ,D and U937 monocytic cells and the test solutions of sodium pyruvate, lactic acid, ascorbic acid, and Vitamin E were prepared as descnbe above for Examples 1-26. T " ' hydr~gen peroxide production by the ' epidermal 1 .~ ~C~D and U937 ~nocytes was also measured as descnbed above.
A mixture of fatty acids derived from chicken fat was preparcd for addition to the cultured cells by mixing 0.1% of the chicken fat with the culture media.
At the t~ , of the culture rnedia, 37 ~C., the chicken fat was misable. This chicken fat m~ure was added to cultures of cells prior to exposure of tbe cells to ultraviolet-B light or TPA treatment.

As set out in examples 1-26, mammalian epidermal ' ~i, and monocytes were exposed to (a) I ~E.D dose of ultraviolet light-B and (b) 100 ng/ml of 12-O-Ie~L~,yl,- ' ' 13-acetate in tbe presence of various . ' and c.. l . l ;.. c of ~ with and without a mixture of saturated and unsaturated fatLy acids [0.1%, 0.5~/0, and 1.0% chicken fat].

Figure 5 depicts in bar graph forrnat the levels of hydrogen peroxide produced by U937 monocytic cells after exposure of the cells to various _~
of -~ with and without a mixture of saturated and ~ ' fatty acids.
Specifically, the levels of hydrogen peroxide produced by U937 monocytic cells were measured after exposure to lactic acid and Vitamin E without fatly acids (Example 27) and with fat~y acids (Example 28), to ascorbic acid and lactic acid without fatty acids (Example 29) and with fatty acids (E~ample 30), and to ascorbic acid and Vitarnin E
without fatty acids (Example 31) and with fatty acids ~xample 32). The ability of the c - .1.;" l;.. ~ of lactic acid and Vitamin E, ascorbic acid and lactic acid, and ascorbic acid and Vitarnin E to reduce the hydrogen peroxide production by ~nocytes was W096/03149 21 9 i 603 P_l/o..,.";

increased in the presence of fany acids. The ~st effective ~....,1.: ;:..,. to reduce the hydrogen peroxide production of rnonocytes was lactic acid (0.05%) and Vitamin E(50 E) in the presence of a rni,ctu~ of saturated and I ' fany acids (0.5%).

Figure 6 depicts in bar graph format the levels of hydrogen permide produced by epidermal I ' ~ ~;. after exposure of the cells to various with and without a rnixture of saturated and I ' fatty acids. Specifically, the levels of hydrogen peroxide produced by epidermal I ,~ were rneasured after exposure to no antioxidant without fatly acids (Exampie 33, control) and with fatty acids (Ex~unple 34), to sodium pyruvate without fatty acids (Exarnple 35) and with fatty acids (~ arnple 36), to ascorbic acid without fatty acids (Example 37) and with fatty acids (~ ample 38), to lactic acid without fatty acids (Example 39) and with fatty acids (Example 40), and to Vitamin E without fatty acids (Exarnple 41) and with fatty acids ~unple 42). The ability of sodiurn pyruvate and Vitamin E to reduce the hydrogen peroxide production by epidermal ~claL;IlOc~ ~D was increased in the prcsence of fatly acids. The rnost effective c"" l , ~ to reduce the hydrogen pero~cide production of epidermal ~clalhlu~ were sodium pyruvate in ' ' ' with a rnixture saturated and ' fatty acids and Vitamin E in u"l- ";.." with a rnixture of saturated and, ' fatty acids.
ZO
Figure 7 depicts in bar graph format the levels of hydrogen peroxide p~oduced by epidermal keratinocytes after exposure of the cells to various c~ . I ", ~- ;", ,~
of ' with and without a mixture of saturated and unsaturated fany acids.
Specifically, the levels of hydrogen peroxide produced by epidermal kcla~v~ weremeasured after exposure to no antioxidant without fatty acids (Example 43, control) and with fatty acids (E~ample 44), to sodium pyruvatc and ascorbic acid without fatty acids ~ ample 45) and with fatty acids (Example 46), to sodium pyruvate and lactic acid without fatty acids (Example 47) and with fatty acids (E~ample 48), to sodium pyruvate and Vitamin E without fatty acids (Example 49) and with fany acids (Example 50), and to ascorbic acid and Vitamin E without fatty acids (Example 51) and with fatty acids (Exarnple 52). Tne ability of all ' of ' to reduce the hydrogen peroxide production by epidem~al Lclahllo~ ~ t.,i. was increased in WO96/03149 21 q 1 603 r~

the presence of fatty acids. In orda of potency, the ~st effeciive ~ to reduce the hydrogen peroxide production of epidermal }Cl~llillU~ D were sodium pyruvate and Vit= E, sodmm pyruvate and lactic acid, and Vitamin E, each in ~,.. 1.:.. -:;.. witb a mixture of saturated and ' fatty acids (0.5~/0).

Because of the ~,.ylu uiu-,;i~ of cells towards ascorbic acid descrrbed above, the ascorbic acid ~A,...I, -:,.. without sodium pyruvate were not considaed , different from the control test solmion.

Summarg Allalysis Of The Data E'rom Studies 1 and 2 Hurnan epidermal I y~D were isolated by LI~LJalu~uull of epithelial sheets and grown in modified base MCDB 153 medium rrl ' with epidermal growth factor and bovine pituitaTy extract. Cells were seeded in culture dishes at a density of 3 x 105/dish. Prior to exposure to W B light ~lOOmJ/crn2) or treatrnent with lOOng/ml TPA, the cultures were treated with the appropriate ;-- of wound healing , T..~ . production of hydrogan peroxide was measured using DCFH-DA, a nonpolar compound tbat readiiy diffusos into cells, hydrolyzad to a nonpolar derivative. In the presence of : " ' hydrogen peroxide, DC~:H is oxidized to a highly fluoroscent compound DCF. Thus,cellular '' ~ i intensity is directdy pT~rnrrinn~l to levels of hydrogen peroxide produced and can be monitored by flow cytometTy. Hydrogen peroxide is cytotoxic,therefore low~ levels of hydrogen peroxide production is desirable for cellular viability.

In all cases, the tbree component wound healing ,,-- q - ~;.,.. suTpassed the predicted outcomes, clearly ri.. ~ '~i synergy.

W096/03149 2 1 ~ 1 ~03 .

Results 1 - Control 250 250 0 2 - Fatty Acids 250 230 -20 (0.5~/0) 3 - Sodimn Pymvate 250 490 +240 (lOmM) 4 - Vitamin E 250 400 +150 (50 units) S - PyTuvate & 250 430 +180 Fatty Acids 6 - Vitamin E & 250 200 -50 Fatty Acids 7 - Pyruvate & 250 290 +40 Vitamin E

8 - Pyruvate & 250 120 -130 Vitarnin E & Fatty Acids Column I shows the different treatment groups.
Column 2 shows the production of H202 in control cdls (finol/cell).
Colurnn 3 shows the production of H202 after treatment with wound healing r Colurnn 4 shows the difference rn production of H2~2 fiom control after the treatment.

All ~ were assessed against the controls, which produced 250 H202 i~nol/cell. The positive numbers represent H2~2 production in excess of tbecontrol and the negative numbers represent H202 production below the control. These results are set out in Figure 8.

9 2l ~ i 603 r~l,u~

(' ' " of Single Ingredient E~ects Fatty Acids (-2û) & Vitamin E (+150) & Pyruvate (+240) +370 Is The Predickd Three Component Effect -130 Is The Wound healing ~ Acblal Effect 500 Is The Difference Between Predicted Effect minus Actual effect (Synergy) (' 'of Paired and Single Ingredients 10Pymvak & Fatty Acids (+ 1~0) ~ vitamin E (+ 150) +330 Is The Predicted Predicted Three Component E~ect -13û Is The Wound healing ~ l Actual Effect 460 Is The Difference between Predicted Effect minus Actual Effect (Synergy) Vitamin E & Fatty Acids (-50) & Pyruvate (+240) + 190 Is The Predicted Three Component Effect -130 Is The Wound healing ~ ;.." Actual Effect 20320 Is The Differcnce between Predicted Effect minus Actual Effect (Synergy) Pyruvak & Vitamin E (+40) ~ E~atty Acids (-20) 25+20 Is The Predicted Three Cr~mr~n~nt Effect -130 Is The Wound healing ~ Actual Effect 150 Is The Difference between Predicted Effect minus Actual Effect (Synergy) In all cases, the three component wound healing ~ ~1 ;' "' surpassed the predicted outcomes clearly ~ . unpredickd synergy.

WO 96/03149 1 ~_1/L_,5,.
E~amples Of The Therapeutic Wound Healing ~' A ' of r hc One (I.~-D) Study 3 This study .' a .u~ . of the wound healing abilities of the therapeutic wound healing ~~.,,~1..,, ~;...-~ of the present invention versus .lltiullal wound healing ~u~ .v~ The results of this study are illustrated in examples A-D.
The wound healing ..u~ ,v~ of Examples A-D were prepared having the ~s set out in Table A.

Examples Ingredient A B C D
Prep-H~M
sodium pyruvate -- 2% - -vitamin E - 1% -- --chicken fat - 2% - -shark liver oil 3% 3% 3%
petrolatum in 64% 66.5% 68%
mineral oil arnounts 22.53Q/o 25.03% 26.8%
paraffin totaling 5% 5~/' 5o/
emulsifer 1000/o 0.2% 0.2% 0.2%
These CullllJull~llto are present in Preparation HTM

Wound healing ~ ;.." A was commercially available Preparation HnU Wound healing s~ B was a petrolatum base r~ ul.lliull containing live yeast cell derivative, shark oil, and a mixture of sodium pyruvate, REClIFIED SHEET (RULE 91) ISA/EP
_ . _ ... . . . . .. ... ... .......... ............

wo 96/03149 vitamin E, and chicken fat Wound healing r.~ C was a petrolatum base A ~ containrng live yeast cell derivative and shark oil. Wound healing ';"" D was a petrolatum base fnmAIlh~ n only.

Wound healing studi_s were ca~ied out using hairless mice (SKR-I, 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 6 mice for evaluation at either day 3 or day 7 for a total number of 60 animals in the study. The mice were -Ih..~ l with ether and a midline 3 cm full thickness 1.".~ AI incision was made with a number 10 scalpel blade. Incisions were closed using steel clips at I cm intervals. F~ A-D set out above were applied in a . . 1. " ": i blinded study to the wounds on day 0 at 2 hours following woundnng and reapplied at 24 hour intervals during the 7 days of the study. The wounds were examined daily and scored on a basis of 0-5 for closure on each day of the study, with a score of 5 IC~ D.~llLillg the wound best healed.

The animals were sacrif ced on day 3 and day 7 using cervical .. The dorsal skin including the rncision was dissected without the ~ ' tissue. The skin was placed in neutral buffered fonnalin and ~ - ~lu .lly sectioned and stained with hematoxylin and eosin. The wounds were examined llli~,luD.,~ ,ally and lc;~ ,D.,..Ialiv~; tissue sections were pl~ ~u~ hfJ

On each day of the experiment, the score and r_nk order of the fonmll~ion~ for closure of wounds and speed of healing were as follows:
B (5) >> D (4) >> C (2) >1= E, Control (2) > A (I) P huLu~Dl~f/llD of the wounded mice on day 4 are set out in Figures 9A - 9D and 10.

Figures 9A-9Dandl0showthatFullllulAliull B,whichwasapetrolatum base r.",....l- ...., conta;ning live yeast cell derivative, shark oil, and a mixture of sodium pymvate, vitamin E, and chicken fat, was a D;Lldri.,~udly better wound healing RECTIFIED SHEET (RU!E gl) ISA/EP

WO 96l03l4g 21 9 1 6 G 3 agent than the other r."....,l~ These results are supported by the subjechive grading of the wound closures and the speed of healing on each day (1-7) of the experiment as well as on the objective hjrr~l/)gAr~1 1 ' ..11;11-';~.. of tissue sechions to measure the extent of ~ y cell infiltrate within the wound and the extent of ~l ;h ~ ." at the wound edges. The final result was that less scar tissue was present at day 7 on the mice treated with Ful~uul.~liull B.

FullllulaLiull D, which was a white petrolahlm ~( ' only, was judged to be D;~llir.,.ll.lly more effective to promote healing than either Formulation C, which was a petrolahJm base r..",. ~-~;.... containing shark liver oil and live yeast cell derivahive~ or FullllulaLiul~ A, which was Preparation HTM. The superior ability of Fullllulaliun D over r. ~ 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 Fulllluk~liun B apparently offsets the depletion of the live yeast cell derivative.

rulllluLlLivll c, which was a petrolatum base r.. ~ " containing live yeast cell derivahive and shark oil~ was judged ,,~ . to the control (untreated wound) in speed of wound closure and extent of healing. Formulahon A, which was Preparahion H~A, appeared to be the least effective healing r-----"~-~;--., by both subjechive grading of wound healing and by objective i of tissue sechions.
The superior ability of Formulation D and FulllluLlLiull C over FUI~IIUI~IL;UI~ A to irnprove healing may be due to their ability to act as an occlusive wound dressing that prevents Ll - .p..l~ .., Al water loss and thus promotes healing and wound closure. The poor ability of FUI~IIUI.~L;UII A to improve healing may be due to the potential~.yLulu~i~,;Ly of ph~,l,yllll~ nitrate present in Preparahion HlM as a preservahive.

These results show that the wound healing ~ of the present invention which comprise a mixhrre of sodium pyruvate, vitamin E, and chicken fat increase the l~lvl;r~laliun and 1~ rate of I~IAIIIIII l' cells. The wound RECTIFIED SHEET (RULE 91 ISA/EP

wo 96/03149 2 l 9 1 6 3 3 ~ / L ~

heahng ~ mediate low levels of o~cygen in the initial stages of healing to suppress oxidative dilmage and higher levels of oxygen in the later stages of healing to promote collagen formation.

~ O ' ~Vound Heal~ng 1~ .
~ - Two (LA-D t X) Applicant has discovered therapeutic antifungal-wound healing 4~ (IA-D ~ X) which comprise a first antifungal agent ~) and the wound healing c~ of r .1 u 1~ . . s One (IA-D). Preferably, the wound healing ~~" 'l"'- ';"" ~A) comprises (a) pyruvate, (b) an ~ ' t, and (c) a mi~cture of saturated and I ' fatty acids. and a wound healing ~ y~ The first antifungal agent is lactic acid or sorbic acid. Antifungal agents can treat fungal infections in a patient but do not promote the wound healing process. Wound healing f~ can increase the rate of injured ."~ - cells and the y~"l;~ rate of new m~mm~ cells to replace dead cells but do not treat fungal infections. Applicants have found that the ~ .. of an antifungal agent and a wound healing ~ results in a therapeutic ~~ _ I wound healing ~ which reduces the duration and severitv of fungal infections The therapeutic antifungal-wound healing ~ul~ - - may fmther comprise a second antifungal agent and an anti- n y agent.

~ a preferred c ~ " t, the therapeutic ~n~ifimg?l wound healing Z5 ~ y~ of the prese~t invention may be i3~;~L~cd to a patient in the form of a vaginal douche or a vaginal such as a vaOs~al insert or ~uy~u~ y.
Vaginal ....-:~l.., ;, .. ~ such as ReplensTM, relieve the discomfort of vaginal dryness.
Vaginal . . . ~ provide relief from the discomfort of vaginal dryness by providing continuous hydration to the vaginal tissue. Vaginal 1. ~ typicaLy contain purified water, glycerin mineral oil, POIYI~1L1JUY~ Carbomer 934P, L~l' , ' palmoil glyceridel and sorbic acid. Ill this . ~l .o~ , the l,;o~ll~ , properties of the vaginal douche or are employed to deLver the antifungal agent and the ~ WO 96/03149 2 1 9 1 6 0 3 r~

wound healing r~ to the vaginal walls to provide long lasting lubrication, antifungal treatrnent, and wound healing agents to facilitate healing for pr~
women.

The, ' ~ of the first antifungal agent and the wound healing rA...~ ;.. ofthepresentinventionprovidesap~ l c"".l~ useful for treating fungal infections and having an enhanced ability to prevent and reduce inJury to .. --,, 1 --, cells and fmther increase the rate of injured . ~
cells. The tissue damage associated with fungal infections is believed to be caused by the production of cellular produced active o:cygen species. Cr~h;~ rm of the first antifungal agent and the wound healing ~ rnay suppress such reactive oxygen-linked tissue injury.

The first antifungal agent in the ~nhfimg ~ ud healing of the present invention may be selected from the group consisting of lactic acid and sorhic acid. Preferably, the first antifungal agent is lactic acid. More preferably, the first antifungal agent is a mixture of lactic acid and sorhic acid. Lactic acid (2-L~yLw~L I acid) promotes the growth of lactooacill~ and inhibits the growth of pathogens. Sorbic acid (2,4-l~ ..-:- acid) is a natoral antifungal agent which ~Is Cand~da aloicans. Sorbic acid and lactic acid are soluble in water.

The first antifungal agent of the present invention may be used in many distinct physical forms well known in the 1.1.--...--...l:. -~ art to provide an initial dosage of the first antifimgal agent and/or a further time-release form of the first antifungal agent. Without being limited thereto, such physical forms include free forms and, , ' ' forms, and rn~tures thereof.

The amormt of first antifungal agent used in the present invention may vary depending upon the therapeutic dosage Ir .. ---l-l or permitted for the par~icular first antifungal agent. In general, the amount of first antifungal agent present is the ordinary dosage required to obtain the desired result. Such dosages are ~nown to the skilled practitioner in the medical arts and are not a part of the present invention.

WO96/03149 2 1 9 1 6 ~3 In a preferred, ' ' t, the first antifungal agent in the ~ n"rl . ' healing .~ q -- u - is present in an amount from about 0.05% to about 10%, preferably from about 0.1% to about 5C/o~ and more preferably from about 0.2% to about 4C/o, by weight.
s The therapeutic antifungal-wound healing o~q ~ of the present invention may further comprise a second antifungal agent. The second antifungal agent may be selected from a wide variety of water-soluble and ~. ~ ' ' '- drugs and their acid addition or metallic salts. Both organic and inorgamc salts may be used provided the second antifungal agent rnaintains its ,.. ~.1.. ,.,.~: value. The second antifungal agent may be selected from a wide range of therapeutic agents and mixtures of therapeutic agents which may be ~ ' ' in sustained release or prolonged action form N~ i..g Illustrative specific examples of antifungal agents include the foUowing ",~ 1;- .. : ".. ~ .. 1~ l.. U;",_,.. l~ l;.~.. -,.il~ t~c.. n~7.~1~ povidon~
iodine, and l,.. s,~,", ,.. 1~ Preferred second antifungal agents to be employed may be selected from the group consisting of ~f~wll~vle and ~ Jlr The second antifimgal agent of the present invention may be used in rnany distinct physical forms well known in the l~ art to provide an initial dosage of the second antifungal agent and/or a further time-release folm of tbe second antifungal agent~ Wlthout berng lirnited tbereto, such physical f = include free forrns and c r ~ ' ~ folms, and mi~h~es thereof.

The amount of second alltifungal agent used in the present inveDtion may vary depending upon the therapeutic dosage ~ r .. ~ or permitted for the particular second antifungal agent In general, the arnount of second antifimgal agent present is the ordinary dosage required to obtain the desired result. Such dosages are ~-nown to the skilled ~ in the rnedical arts and are not a patt of the present rnvention In a preferred ,..,1.~1 ". ,1~ the second antifungal agent in the antifungal-wound healing ~;.. l.. - l: .,, is present in an arnount from about 0 05% to about 10%, preferably f~om about 01% to about 5%, and more preferably from about 0.2% to about 4%, by weight.

WO 96/03149 2 19 16 0 3 r~

The therapeutic ~ healing c ~ of the present invention may further cornprise an anti- r'a y agent. The anti - n y agent may be selected f~n a wide variety of water-soluble and water-insoluble drugs and their acid addition or metallic salts. Botn organic and inorganic salts may be used provided the anti- n ' y agent rnaintains its ' value. The anti-- n y agent may be selected from a wide range of therapeutic agents a~nd rni~tures of therapeutic agents which may be _' ' in sustained release or prolonged action form. ~ iDustrative specific ex~unples of non-steroidal anti-...1~ .,.. ~ ~ly agents include the foDowing ~-- ]~ ibuprofen, naprwcen, sulindac, diflunisal, piro~icam, ".. L.. ,.. l~, ;.. etodolac, ' ' sodiurn, f~snnprob~n calcium, ketoprofen, mefenarnic acid, ' ketorolac 1",.. l1~-.. ;,.. 9 diclofenac, and evening primrose oil (containing about 72% linoleic a~cid and about 9% gamma-Gnolenic acid). Nonlirniting illuslrative specific e~amples of salicylate anti-~ n ' y agents include the following ' a~..,.yl~ .yl;c acid, " _.l.":.. _ salsalate, diflunisal, ~L~ b~l;.,~Lc acid, and choline ,, l~i~li~,y' N~ .-..flu.g illustrativo specific examples of steroidal anti n y agents include the following ".. li~-.. ,.. ~ flunisolide, I,;-.. ;.. nl~ ;-.... ,l;.,r acetonide,1, l ,.,. :1 " - ~G, )j b. l~ ir ~ hy.l.....
cortisone, .l~ , methyl ~JIC ' ~ ' ~i, and The amount of anti- n ' y agent used in the present invention may vary depending upon the therapeutic dosage Ir .. l ~ or pem~itted for the particular anti~ n- ~ ~ y agent. In general, the amount of anti- n y agent present is the ordinary dosage required to obtain thc desired result. Such dosages are known to the skilled l in the medical arts and are not a part of the present invention. In a preferred ~ ' ~ the ~ n ' y agent in the antifungal-wound heaGng ~ -~ l -:-;-- is present in an amount from about 0.05% to about 10%, prefeirably from about 0.1% to about 5%, and ~re proferably from about 0.2% to about 4%, by weight.

w0 96/03149 2 1 9 1 6 0 3 B. Methods Por M~g The AntifungAI-Wound EIealing C
Of r ~ - Two lLA-D I X) The present invention extends to methods for making the therapeutic ' wound healing c..."~ ;.. ,; (IA-D + X). ln general, a therapeutic ~ ' wound healing ~ ~ is made by fo~ning an admixture of the wound healing ~ - - of r,.~l.o.3. .. ,l One (IA-D) and an antifungal agent. In a first aspect of r ~ " Two ~[A + X), an /III~ lb vound healing therapeutic ~ ' " is made by forming an admixture of an antifungal agent and a wound healing ""~ comprising (a) a pyruvate, (b) an ~ntif~ifl~nt and (c) a mi~cture of saturated and, ' fatty acids. In a second aspect of r-~i ' Two (LB
+ X), an antifung~l w on-~3 healing therapeutic c~ is made by for ning an admi~ture of an antifungal agent and a wound healing ~ comprising (a) a pyruvate, (b) a lactate, and (c) a rnixture of saturated and ~ ' fatty acids. InathirdaspectofFl.,' .~1;1~... Two([.C+~),an a,l,ir~ ~ w ~ 3 heaUngtherapeutic ~,~- 1 -- l ." is made by formmg an admixture of an antifimgal agent and a woundhealing cc~ - c~ coruprising (a) an ~ntic~Yifl~lt and (b) a mixture of saturated and ' fattv acids. In a fourth aspect of r.l~ Two (LD + 7~), an ~ ~ 1 wound healing thefapeutic ~ is rnade by forming an admixture of an antifungal agent and a wound healing c~ " comprising (a) a lactate, (b) an icn~if3~nt and (c) a r~ixtme of saturated and unsaturated fatty acids.

In a preferred rl. l ~ll . .I the invention is directed to a method for preparing a therapeutic a.Lrh.. bal wound healing ~ IA + X) which comprises the steps of admixing the following ingredients:
(A) a IP rl :i..~lly effective amount of an antifiungal agent; and (B) a wound healing c~ ;.. . which comprises:
(a) pyruvate selected from the group consisting of pyruvic acid, ~ ;l Ally acceptable salts of pyruvic acid, and mi,ctures thereof;
(b) an ~ ;l - and WO96/03149 2 1 9 1 6 0 3 F~

(c) a mixture of saturated and ' fatty acids whcrei~ the fatty acids are those fatly acids required for the repair of cellular ' and ,. of . ~ --. cells.

5C. Methods For r ~, The A ~ O ' Woulld Healing C
Of r ~ Two a~-D t X) The present invention extends to methods for employing the therapeutic Ol.~ir.. b.~l.. JU.Id healing ~.. q.. -:~ (IA-D + X). In general, a therapeutic ;-is employed by contacting the therapeutic ~ with a wound. In a preferred ~.,.~1.. ,.':.. 1 the invention is directed to a method for healing a infected wound in a mammal with an antifungal-wound healing ~ ~ (IA + X) which comprises the steps o~
(A) providing a therapeutic antifungal-wound healing ~ which comprises:
(1) a 1~ lly effective atnount of an antifungal agent; and (2) a wound healing c..- l OC:~ -- \ which compIises:
(a) pyruvate selected from the group consisting of pyruvic acid, ~ 1Y acceptable salts of pyruvic acid, and mixtures thereof;
(b) an A, .1;. .. ;.1 1 and (c) a mixture of saturated and ~ C~ l fatty acids wherein the fatty acids are those fatty acids reqmred for the repair of cellular metnbranes and 1-- of m~mmAl~ cells; and (B) contacting the ~Lir~l .. ' healing c-~ ) with the infected wound.

D. A ~ A ~ Wound Healing r-of r ~ ' Two~A-D~X~
In another aspect of F--' '' Two, the therapeutic antifimgal-wound healing ~(I A-D + X) of the presc,nt invention mAy be further combined WO96/03149 2 1~ 1603 r~l,u.

with ~ uDefol for treating wounds (M) to form augrnented antifungal-wound healing ~ D + X + M). In this ~- .L~I .. 1 the .A.,. ~ ; . of the ... ' healing r~ q ~ of the present invention and the useful for treating wounds provides an augmented .~r _ 1 wound healing ~, - q ~- -i - having an enhanced ability to increase the p 1 r and IG~
rate of ' cells. For example; the therapeutic ~. .q...- ;.. c of the presentinvention may be used in ~..,. '. -:i ... with " useful for treating woundssuch as ' _ agents (BetafectinTM), antiviral agerlts, r~ G~ ulyL~, agerlts, _--'h ' r y agents, otber antifungal agents, tretinoiu, sunscreer. agents, ,1 .. . L,lr,y.. ~1 agents, topical ;~ r agents, ~-~ih rt~iAl agents, b;U~]L~D;~;
agents, respiratoTy bursting irhnbitors (lactic acid, adenosine), inlubitors of y -l ~1 ,1i, synthesis (ibuprofen, aspirin, ' ' , ~ f ~ acid, retinoic acid, padimate O, meclomen, u~.yb~,l~u~ steroidal anti-;~.n- ~ .,y agents (c~ ..;.k including synthetic analogs), 1: 1: 1, -1 agents (neosporin ointment, silvadine), antiseptic agents, anesthetic agents (pramoxine Ly ~ lidocaine, benzocaine), cell nutrient media, bum relief . 1: -~ sun bum . ~ :.. c, acne ~ ~u 1:., ~,insectbiteandsting,.. l: I; ,c woundcleansers,wounddressings,scar reducing agents (vitamin E), and the l~ce, and mi~ctures thereof, to further enhance the ~r ' and ~~ LU-- rate of ln~nnm~ .n ce~ls. Preferably, the .. u~ , useful for treating wounds is selected from the g~up consisting of i""" ,~1;,",,1_l;,,~.
agents, antiviTal agents, D~ ;lr.~lyLil~ agents, anti~ ~ y agents, antifungal agents, tretinoin, sunscreen agents, ' I g ~' agents, topical "";;1,:~1.,., r agents, . dAl agents, I~;uo~h~ , agents, respiratory bursting inhlbitors, ir~ubitors of ,gl 1; synthesis, A l a ~ Vl ~;_1 agents, cell nut~ient media, scar reducing agents, ~5 and mixtures thereof. More preferably, the ~ l useful for treating wounds is selected from the group wnsisting of ' e agents, antiviral agents, I~ agents, anti~ ~ y agents, antifungal agents, acne treating agents, sunscreen agents, .1 .., ~ 1 agents, ~ agents, ~ -t~l agents, . agents, and mixtures thereo~
In a preferred lll,l,lJ.ii .. .: the invention is directed to an augmented antifungal-wound healing c~- q~ u..,. (LA + X + M) which comprises:

~ Wo 96/03149 2 1 9 l 6 0 3 r~ os (A) a therapeutic antifungal-wound hoal~ng ~ which comprisos:
(l) a Ih. ~l .1;. lly effective amount of an antifungal agent; and (2) a wound healing ~ which cornprises:
(a) pyruvate selected from the group consisting of pyruvic acid, ~ ' "y acceptable salts of pyruvic acid, and mixtures thereof;
(b) an ' , and (c) a mixture of saturated and I ' fatly acids wherein the fatty acids are those fatty acids required fo} the repair of cellular membranes and of ll-~ cells; ard a~) a ~ - '1 useful for treating wounds.

The present invention oxtends to methods for making the augmented antifungal-wound healing c....~ In general, the augmented rA~ are rnade by admixing the therapeutic ~ wound healing ~ ;.... with the ~ .1;. .. l useful for treating wounds to prepare the augrnented . ~ ~ ' wourldhealing ~AII.y...~

The present invention also extends to methods for ernploying the augmentod a~ bl v~ound healing r.~ In general, an augmented ~ ~ ' wound heaiing c. " ~ ;"" is employed by contacting the f, " "l ~ with a wound. In a preferred ~. "I.~lh . ,. l. the invention is directed to a method for healing an infected wound in a mammal with arl augmented antifungal-wound healing u- ~ a.A + X + M) which comprises the steps of:
(A) providing a therapeutic augmented ~ vound healing which comprises:
(I) a Ih -l" ..u. 71,~, effective amount of an antifungal agent;
(2) a wound healing ~ which comprises:
(a) pyruvate selected from the group consisting of pyruvic acid, 1~} - ..1 ~ lly acceptable salts of pyruvic acid, and mixtures thereof;
(b) an ,.~ ;.l .. l, and WO 96/03149 2 1 9 1 6 0 3 F.~

(c) a mixture of satus;sd and, ' fat y acids wherein the fatty acids are dhose fatly acids required for the repair of cellular 'and of .~ -- cells; arld (3) providing a .., 1: - . useful for treatirlg wounds; and ~3) contacting the augrnented -r _ 1 wound healing c~ with dhe infected wound.

The types of wounds which may be healed using dhe .u.tir~l25..1 ~round healing ~ . . and the augmented r ~ 1,, Jll~ld healing ~ of the present invention are those which result from an infected injury which causes epidem~al damage.

Methods for healing a wound comprise topically a-~ d :g dle of the present invention direcdy to a wound site to increase dhe healing rate of the wo~Lnd. The ~ is mairtaiIIed in contact with the wound for a period of time sufficient to increase the 1~ r. ~1;- ~, and . ~ l ;- ." rate of the cells.

E.r. ' Of The A '-- ~ ' Wolmd l~ealing ~' , Of E ~ ~ Turo a.A-D + X) and (LA-D + X + M) Once prepared, the inventive therapeutic an~fungal-wound healing ~ ~ ~ y ~ and augrnented ~ r g 1 ~ healing ~ may be stored for fulure use or may be formulated in effective amounts with ~' - ,., ~ ~. 11 y acceptable carriers such as pl.., .,. - ~, S; ~1 appliances and topical vehicles (oral and non-oral) to prepare a wide variety of ~' ' c..~ -...- The pl ~"-,"1;.~lly acceptable carriers which may be employed and the methods used to prepare the y~ have been descrbed above in connection widh dhe r.. . ,l- ;.. ~ of the wound healing ~A~ .ll .. - ;.. ~ of r--~ ' One ([A-D).
lil a preferred . .. l.~l; . dhe invention is directed to an antifungal-wound healing pl - ,.,- r .1;. Al ~....,1..-: .. which comprises:

WO 96~03149 2 1 9 1 6 ~ 3 r Im~. ~

(A) a therapeutic ~nrifimg-~ .~.1 healing ~ q ~ A + X) which comprises:
(I) a i' , lly effective amount of an antifungal agent; and (2) a wound healing ~ which comprises:
(a) pyruvate selected from the group consisting of pyruvic acid, 4 i ' - "~, acceptable salts of pyruvic acid, and mixtures thereof;
(b) an A~ and (c) a mixLure of saturated and I ' fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular l -- ''1/-A- - ~ and ,~ ;"" of ".- "". ~ cells; and (B) a pl . ", ~ ;. All y acceptable carrier selected _om the group consisting ofr~ appliances, b;Oadl~ and occlusive vehicles.

In another preferred ~,1.~l,~ the invention is directed to a method for preparing a ~' ~ 1 1.......... ,.. l.. - 1;.. for increasing the ~ ;f .A~;~.. and rate of '~ cells, which comprises the steps of:
(A) providing a i r ' ~ ~by effective amount of an antifungal-wound healing ~'""1"'- ';"" (IA + X) which comprises:
(I) an antifungal agent; and (2) a wound healing ~1,. . q .. .- ~
(a) pyruvate selected from the group consisting of pyruvic acid, ~,1 - .., - ~..1;. ~lly acceptable salts of pymvic acid, and mixtures thereof;
(b) an A~ 1, and (c) a mixture of saturated and ' fatty acids wherein the fatty acids are those fatty acids required fo} the repair of cellular ". ,~l A,. ~ and 1;.... of mAmmaliAn cells;
(B) provi&g a pl - ,~ y acceptable calrier; and (C) admixing the antifungal-wound healing .,~ q~ from step (A) and the r - 1" acceptable carrier from step ~3) to fomm a j' 1

Claims

I claim

1. A therapeutic antifungal-wound healing composition which comprises a therapeutically effective amount of a first antifungal agent selected from the group consisting of lactic acid and sorbic acid and a wound healing composition, wherein the wound healing composition 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 resuscitation of injured mammalian cells.

2. The composition according to claim 1, wherein the first antifungal agent is a mixture of lactic acid and sorbic acid.

3. The composition according to claim 1, further comprising a second antifungal agent selected from the group consisting of miconazole, clotrimazole,tioconazole, terconazole, povidone-iodine, and butoconazole.

4. The composition according to claim 1, wherein the pyruvate is selected from the group consisting of pyruvic acid, lithium pyruvate, sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate, methyl pyruvate, ~-ketoglutaric acid, pharmaceutically acceptable salts of pyruvic acid, prodrugs of pyruvic acid, and mixtures thereof.

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

6. The composition according to claim 1, wherein the antioxidant is selected from the group consisting of all forms of Vitamin A including retinol and 3,4-didehydoretinol, all forms of carotene including ~-carotene, .beta.-carotene, gamma-carotene, and delta-carotene, all forms of Vitamin C including D-ascorbic acid and L-ascorbic acid, all forms of Vitamin E including .alpha.-tocopherol, .beta.-tocopherol, gamma-tocopherol, delta-tocopherol, tocoquinone, tocotrienol, Vitamin E esters which readily undergo hydrolysis to Vitamin E including Vitamin E acetate and Vitamin E succinate, and pharmaceutically acceptable Vitamin E salts such as Vitamin E phosphate, prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E, pharmaceutically acceptable salts of Vitamin A, carotene, Vitamin C, and Vitamin E, and mixtures thereof.

7. The composition according to claim 6, wherein the antioxidant is Vitamin E acetate.

8. The composition according to claim 1, wherein the mixture of saturated and unsaturated fatty acids is selected from the group consisting of animal and vegetable fats and waxes.

9. The composition according to claim 8, wherein the mixture of saturated and unsaturated fatty acids is selected from the group consisting of human fat, chicken fat, cow fat, sheep fat, horse fat, pig fat, and whale fat.

10. The composition according to claim 9, 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 gadoleic acid.

11. The composition according to claim 1, wherein the first antifungal agent is present in the therapeutic wound healing composition in an amount from about 0.05% to about 10%, by weight of the therapeutic wound healing composition.

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

13. The composition according to claim 1, wherein the antioxidant is present in the therapeutic wound healing composition in an amount from about 0.1%
to about 40%, by weight of the therapeutic wound healing composition.

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

15. The composition according to claim 3, wherein the second antifungal agent is present in the therapeutic wound healing composition in an amount from about 0.05% to about 10%, by weight of the therapeutic wound healing composition.

16. The composition according to claim 1, further comprising an anti-inflammatory agent.

17. The composition according to claim 16, wherein the anti-inflammatory agent is selected from the group consisting of ibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin, etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine, diclofenac, evening primrose oil, acetylsalicylic acid, mesalamine, salsalate, diflunisal, salicylsalicylic acid, choline magnesium trisalicylate, flunisolide, triamcinoline, triamcinoline, acetonide, beclomethasone diproprionate, betamethasone diproprionate, hydrocortisone, cortisone, dexamethasone, predinisone, methyl prednisolone, and prednisolone.

18. The composition according to claim 16, wherein the anto-inflammatory agent is present in the therapeutic wound healing composition in an amount from about 0.05% to about 10%, by weight of the therapeutic wound healingcomposition.

19. A method for treating a fungal wound in a mammal with an healing-wound healing which comprises the steps of:
(A) providing a therapeutic antifungal-wound healing composition which comprises:
(1) a therapeutically effective amount of a first antifungal agent selected fromthe group consisting of lactic acid and sorbic acid; and (2) 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; and (B) contacting the antifungal-wound healing composition with the fungal wound.

20. The composition according to claim 19, further comprising a second antifungal agent selected from the group consisting of miconazole, clotrimazole,tioconazole, terconazole, povidone-iodine. and butoconazole.

21. The composition according to claim 19, further comprising an anti-inflammatory agent.

22. A method for preparing a therapeutic antifungal-wound healing composition which comprises the steps of admixing the following ingredients:
(A) a therapeutically effective amount of a first antifungal agent selected fromthe group consisting of lactic acid and sorbic acid; and (B) 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.

23. The method according to claim 22, further comprising a second antifungal agent selected from the group consisting of miconazole, clotrimazole,tioconazole, terconazole, povidone-iodine, and butoconazole.

24. The method according to claim 22, further comprising an anti-inflammatory agent.

25. An augmented antifungal-wound healing composition which comprises:
(A) a therapeutic antifungal-wound healing composition which comprises:
(1) a therapeutically effective amount of a first antifungal agent selected fromthe group consisting of lactic acid and sorbic acid; and (2) 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; and (B) a medicament useful for treating wounds.

26. The composition according to claim 25, further comprising a second antifungal agent selected from the group consisting of miconazole, clotrimazole,tioconazole, terconazole, povidone-iodine, and butoconazole.

27. The composition according to claim 25, further comprising an anti-inflammatory agent.

28. The augmented antifungal-wound healing composition according to claim 25, wherein the medicament useful for treating wounds is selected from thegroup consisting of immunostimulating agents, antiviral agents, antikeratolytic agents, anti-inflammatory agents, other antifungal agents, acne treating agents, sunscreen agents, dermatological agents, antihistamine agents, antibacterial agents, bioadhesive agents, respiratory bursting inhibitors, inhibitors of protaglandin synthesis, antimicrobial agents, antiseptic agents, anesthetic agents, cell nutrient media, burn relief medications, sun burn medications, insect bite and sting medications, wound cleansers, wound dressings, scar reducing agents, and mixtures thereof.

29. A method for treating a fungal wound in a mammal with an augmented antifungal-wound healing composition which comprises the steps of:
(A) providing a therapeutic augmented antifungal-wound healing composition which comprises:
(1) a therapeutically effective amount of a first antifungal agent selected fromthe group consisting of lactic acid and sorbic acid;
(2) 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; and (3) providing a medicament useful for treating wounds; and (B) contacting the augmented antifungal-wound healing composition with the inflammed wound.

30. The method according to claim 29, further comprising a second antifungal. agent selected from the group consisting of miconazole, clotromazole, tioconazole, terconazole, povidone-iodine, and butoconazole.

31. The method according to claim 29, further comprising an anti-inflammatory agent.

32. An antifungal-wound healing pharmaceutical composition which comprises:
(A) a therapeutic antifungal-wound healing composition which comprises:
(1) a therapeutically effective amount of a first antifungal agent selected fromthe group consisting of lactic acid and sorbic acid; and (2) 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; and (B) a pharmaceutically acceptable carrier selected from the group consisting of pharmaceutically appliances, bioadhesives, and occlusive vehicles.

33. The composition according to claim 32, further comprising a second antifungal agent selected from the group consisting of miconazole, clotrimazole,tioconazole, terconazole, povidone-iodine, and butoconazole.

34. The composition according to claim 3 further comprising an anti-inflammatory agent.