CA2218539A1

CA2218539A1 - Immunostimulating-wound healing compositions and methods for preparing and using same

Info

Publication number: CA2218539A1
Application number: CA002218539A
Authority: CA
Inventors: Alain Martin
Original assignee: Individual
Current assignee: Warner Lambert Co LLC
Priority date: 1995-05-22
Filing date: 1996-04-26
Publication date: 1996-11-28
Also published as: US5658957A; EP0828514A1; ZA964054B; NZ307055A; WO1996037230A1

Abstract

This invention pertains to therapeutic immunostimulating-wound healing compositions. The compositions comprise a therapeutically effective amount of an immunostimulating 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 immunostimulating-wound healing compositions may be utilized in a wide variety of pharmaceutical products. This invention also relates to methods for preparing and using the therapeutic immunostimulating-wound healing compositions and the pharmaceutical products in which the compositions may be used.

Description

WO 96t37230 PCI'IUS96/05901 IMMUNOSTIMU1ATING WOUND HEALING COMPOSl~IONS
AND METHODS FOR PREPARING AND USING SAME

BACKGROUND OF THE INVENTION

1. Field of the Invention This invention ~ ~i"s to therapeutic immlmoshmlll~hng-wound healing compositions for enhancing wound repair in the outer and inner layers of the skin.
More particularly, the immnnoshmlll~hng wound healing compositions comprise an immunostimlll~hng agent and a t~.c/~ulic wound healing composition and/or its metabolites. This invention also pertains to m~tho1c for ~l~al;llg and using theimmunoshmlll~hng-wound healing compositions and the ph~rm~cellhcal products in which the therapeutic compositions may be used.

A pleft;ll~;d embo~1im~nt of the th~ c.lLic wound healing cully~o~ilion of this invention comprises (a) pyruvate sclcct~<l from the group con~;chng of pyruvic acid, ph~rm~cellhcally acceptable salts of pyruvic acid, and ~luies thereof, (b) an antioxidant, and (c) a ~ lulc; of salul~led and lmC~t~AI~d fatty acids wherein the fatty acids are those fatty acids requ*ed for the repa* of cellular nl~llbl~les and resuscitation of l "~ n cells. In another aspect of this invention the wound healing composition comrrice,c (a) pyruvate st~lecte(l from the group concichng of pyruvic acid, ph~rm~enhically acceptable salts of pyruvic acid, and llli~Lules thereof; and (b) an zlnhoxi-l~nt CA 022l8539 l997- lO- l7 WO 96/37230 PCTlUS~S (i5 2. Description of the Background Wound Healing Wounds are intemal or eYt~n~1 bodily injuries or lesions caused by physical S means, such as m~c-h~ni~1 Gll~mir~l viral, b~ct~i~l or thermal means, which disrupt the normal cv..l;...l;ly of structures. Such bodily injuries include contll~;ong, wounds in which the skin is unbroken, incisions, wounds in which the skin is broken by a cutting insl ulll~lll, and l~cto~tion~, wounds in which the skin is broken by a dull or blunt in~lluul.~,..L. Wounds may be caused by accidents or by surgical procedures.
Patients who suffer major wounds could benefit from an enhancement of the wound healing process.

Wound healing consists of a series of processes whereby injured tissue is repaired, speGi~li7e~l tissue is regenerated, and new tissue is ~ ~l Wound healing con~i~t~ of three major phases: a) an infl~mm~ti-~n phase (0-3 days), b) a cellular proliferation phase (3-12 days), and (c) a rem-d~ling phase (3 days-6 months).

During the infl~mm~tion phase, platelet ag~,.~alion and clotting form a matrix which traps plasrna proteins and blood cells to induce the influx of various types of cells. During the cellular proliferation phase, new connective or gr~nl]l~tion tissue and blood vessels are formed. During the r~mod~ling phase, gr~nnl~ic)n tissue isrepl~cecl by a network of collagen and elastin fibers leading to the fonn~t-on 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 cytotoxicity, the ~up~c;s~ion of ;on~ and the shm~ tion of cellular viability and proliferation. Wounds require low levels of oxygen in the initial stages of healing to ~iu~pl'eSS oxidative darnage and higher levels of oxygen in the later stages of healing to pl.,.ll~te collagen formation by fibroblasts.

WO 96/37230 PCT/USgli ~5 M~mmAliAn cells are cQntinllously l -l.os~l to ac~iv~lGd oxygen species such as ~u~.uAide (~2-)~ hydrogen peroxide (H202), hydroxyl radical (OH-), and singlet oxygen (102). In vivo, these reactive oxygen ;~ e are generated by cells in response to aerobic metabolism, catabolism of drugs and other xenobiotics, ultraviolet and x-ray r~ tion, and the ~ei~haluly burst of phagocytic cells (such as white blood cells) to kill invading b~ct~i~ such as those introduced through wounds. Hydrogen peroxide, for ~Y~mple7 is produced during ~ aliol~ of most living org~qnicme especially by ~IlGssGd and injured cells.

These active oxygen species can injure cells. An important example of such damage is lipid peroxi~1~tion which involves the oxidative degradation of lm~
lipids. Lipid peroxidation is highly ~letnm~nt~l to mc.~,~,e structure and function and can cause nu~ uus cytopathological effects. Cells defend against lipid per)xi~1~ti-)n by producing radical scavengers such as ~ul,c.~Aide ~liemlltAee c~t~l~ee and peroxi~l~ee. Injured cells have a decreased ability to produce radical scavengers.
Excess hydrogen peroxide can react with DNA to cause backbone breakage, produce mutations, and alter and liberate bases. Hydrogen peroxide can also react with pyrimirlines to open the 5, 6-double bond, which reaction inhibits the ability of pyrimidines to hydrogen bond to compl~ y bases, ~ en-ler et al. (1971). Such oxidative biochemical injury can result in the loss of cellular ~llGI.. ~ e integrity, reduced enzyme activity, changes in transport kinetics, changes in ~--G~ e lipidcont~nt, and leakage of potassium ions, amino acids, and other cellular m~t.-ri~l Antioxidants have been shown to inhibit damage associated with active oxygen species. For example, pyruvate and other ,41pha-ketoacids have been reported to react rapidly and stoichiometrically with hydrogen peroxide to protect cells from cytolytic effects, O'Donnell-Tormey et al., J. Exp. Med., 165, pp. 500-514 (1987).

~ 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 k~Atr)~ei.e, -,~e,.~ tely, which consist of applying to the affected area a topical composition comprising from about 1% to about 20% of a lower aliphatic compound co..~;..;.lg WO 96/37230 PCT/US9~'~5~01 from two to six carbon atoms selected from the group con~ictin~ of Alpha-hydroxyacids, Alpha-ket-~cirlc and esters thereof, and 3-hydroxybutryic acid in a pharm~cenhc~lly acceptable carrier. The ~1iph~tic compounds include pyruvic acid and lactic acid.
s United States Patents Nos. 4,105,783 and 4,197,316, both issued to Yu et al., disclose a method and composition, respectively, for treating dry skin which con~ictc of applying to the affected area a topical composition compricing from about 1% to about 20% of a compound selected from the group concictln~ of amides and ~ 11 salts of ~Ipha-hydroxyacids, ~-hydroxyacids, and ~Ipha-ketoacids in a pharrn~ce -tic~11y acceptable carrier. The compounds include the amides and a~ ll salts of pyruvic acid and lactic acid.

United States Patent No. 4,234,599, issued to Van Scott et al., discloses a method for treating actinic and nc~n~ctinic skin keratoses which consists of applying to the affected area a topical composition comprising an errci~iLive amount of acompound selected from the group con.cicting of Alpha-hydroxyacids"~-hydroxyacids, and ,4lpha-ketoacids in a ph~ cellt~c~11y acceptable carrier. The acidic compounds include pyruvic acid and lactic acid.
United States Patent No. 4,294,852, issued to Wil<1n~llPr et al., discloses a composition for treating skin which comprises the Alpha-hydroxyacids,,~-hydroxyacids, and Alpha-keto~ci-lc ~licclose-l above by Van Scott et al. in combination with C3-C8 aliphatic alcohols.
United States Patent No. 4,663,166, issued to Veech, discloses an electrolyte solution which comprises a ~Lule of ~lactate and pyruvate in a ratio from 20:1 to 1: 1, respectively, or a ~ Lul c; of D-~¢-hydroxybutyrate and ~ceto~cet~te, in a ratio from 6:1 to 0.5:1, respectively.
Sodium pyruvate has been reported to reduce the number of erosions, ulcers, and hemorrhages on the gastric mucosa in guinea pigs and rats caused by , WO 96/37230 PCT/U5961'~5901 acetylsalicylic acid. The ~n~lgesic and ~lti~ylGLic ~r~c.Lies of acetylsalicylic acid were not i-.-~>ailGd by sodium pyruvate, P lc~l..,.,.."" ~r7nçimittelforscllllng~ 33, pp. 410-415 and 415-416 (1983).

Pyruvate has been reported to exert a positive inotropic effect in st~lnned myocardum, which is a prolonged venhicular dysfunction following brief periods of coronary artery occll~ n~ which does not produce irreversible damage, Mentzer et al., Ann. Surg., 209, pp. 629-633 (1989).

Pyruvate has been reported to produce a relative stabili7~hon of left venh icular pressure and work parameter and to reduce the size of infarctions. Py. UV~lG
improves l~ ~Lion of spontaneous beating of the heart and lG~L~-ation of normal rates and pressure development, Bunger et al., J. Mol. Cell. Cardiol., 18, pp. 423-438 (1986), Mochizuki et al., J. Physiol. (Paris), 76, pp. 805-812 (1980), Regitz et al., Cardiovasc. Res., 15, pp. 652-658 (1981), Giannelli et al., Ann. Thorac. Surg., 21, pp. 386-396 (1976).

Sodium pyruvate has been reported to act as an antagonist to cyanide intoxication (~LGsul--ably through the formation of a cyanohydrin) and to protect against the lethal effects of sodium sulfide and to retard the onset and development of functional, morphological, and biochemical measures of acrylamide neuropathy of axons,Schwartz etal.,Toxicol. Appl. Ph~rm~cQl.,50,pp. 437-442(1979),Sabri etal.,Brain Res., 483, pp. 1-11 (1989).

A chemotherapeutic cure of advanced L1210 leukemia has been reported using sodium pyruvate to restore abnormally deformed red blood cells to nonn~l The deformed red blood cells prevented adequate drug delivery to tumor cells, Cohen,Cancer Chemother. Ph~rm~col., 5, pp. 175-179 (1981).

Primary cultures of hGLGl~ Lu~ic tracheal hransplant exposed in vivo to 7, 12-dimethyl-benz(a)anthracene were reported to be s~lcceecfully ..~ ;..ed in enrichment m~r~ rn suppl~rn~onte~l with sodium pyruvate along with cultures of interleukin-2 WO 96137230 PCT/US9G~'05301 stiml~lat~l peripheral blood Iymphocytes, and p1~m~cytomas and hybri~lom~e, pig embryos, and human blastocysts, Shacter, J. lmmllnol Meth~e, 99, pp. 259-270 (1987), Marchok et al., C~ancer Res., 37, pp. 1811-1821 (1977), Davis, J. Reprod. Fertil. Suppl., 33, pp. 115-124 (1985), O~au.~,tu et al., No To ~hinkei, 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, and4,645,764, all issued to Stanko, ~ieclose mt-thorls for plcv~llLhlg the Accl~mlllAtit n of fat in the liver of a ~ lAl due to the ingestion of alcohol, for controlling weight in a III~-IIIIIIAl, for inhibiting body fat while increasing protein concentration in a Ill~ 1, and for controlling the deposition of body fat in a living being, respectively. The methods comprise admini.et~ring to the m~mm~l a therapeutic m~Lul~;; of pyruvate and dihydroxyacetone, and optionally riboflavin. United States Patent No. 4,548,937,issued to Stanko, discloses a method for controlling the weight gain of a ll~.. lllnAl which comprises a~lminiet~ring to the 11l;3.11lllAl a therapeutically effective amount of pyruvate, and optionally riboflavin. United States Patent No. 4,812,479, issued to Stanko, ~lieclc)s~e a method for controlling the weight gain of a Illi.llllllAl which comprises atlminietering to the " ".. "" ~Al a therapeutically effective amount of dihydroxyacetone, and optionally riboflavin and pyruvate.

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

-CA 02218~39 1997-10-17 WO 96/37230 PCI~/USS16/03~01 United States Patent No. 4,416,982, issued to Tauda et al., ~ os~s a method for decomposing hydrogen peroxide by reacting the hydrogen peroxide with a phenol or aniline derivative in the presence of perolri~ e S United States Patent No. 4,696,917, issued to Lindstrom et al., ~ cl~ses an eye irrigation solution which cqmrri~es Eagle's ~;.. ;.~.. F~nh~l Me~ lm with Earle's salts, chondroitin sulfate, a buffer solution, 2~ r~iapl.~eth~n- 1, and a ~yluv~le.
The irrigation solution may optionally contain ascorbic acid and Alpha-tocopherol.
United States Patent No. 4,725,586, issued to Lindstrom et al., discloses an irrig~ti~n solution which comprises a balanced salt sol-lti- n, chondroitin sulfate, a buffer solution, 2-l~ oethanol, sodium bicarbonate or d~,A~I~.se, a pyruvate, a sodium phosphate buffer system, and cystine. The irrigation solution may optionally contain ascorbic acid and gamma-tocopherol.

United States Patent No. 3,887,702 issued to Baldwin, discloses a composition for treating fingemails and toenails which consists essçnti~lly of soybean oil or sunflower oil in combination with Vitamin E.

United States Patent No. 4,847,069, issued to Bissett et al., discloses a photuplv~ctive composition comprising (a) a sorbohydrox~mic acid, (b) an anti-infl~.. ~ol~ agent selected from steroidal anti-infl;~ ly agents and a natural anti-infl~.. ~1.,ly agent, and (c) a topical carrier. Fatty acids may be present as an emollient. United States Patent No. 4,847,071, issued to Bissett et al., ~ closes a photopn~ctive composition comprising (a) a tocopherol or tocopherol ester radical scavenger, (b) an anti-infl~ c,ly agent sçlected from steroidal anti-inflil.. ,.. ,.l.. y agents and a natural anti-infl~.. ~tc-ly agent, and (c) a topical carrier. United States Patent No. 4,847,072, issued to Bissett et al., discloses a topical composition comprising not more than 25% tocopherol sorbate in a topical carrier.

United States Patent No. 4,533,637, issued to Yamane et al., discloses a culture ...~lli...., which comprises a carbon source, a nucleic acid source precursor, amino acids, vit~minc, minerals, a lipophilic nutrient, and serum albumin, and cyc~ Ytnn~ The lipophilic substances include lm~ l~t~ fatty acids and lipophilicvitamins such as Vitamin A, D, and E. Ascorbic acid may also be present.

United Kingdom patent application no. 2,196,348A, to Kovar et al."1i~clos~s S a synthetic culture m~ m which comr~i~çs inorganic salts, monos~r,rh~rides, amino acids, vit~min~ burr~ g agents, and optiona11y sodium pyruvate adding magnesiurnhydroxide or m~gn~ m oxide to the ~ ion The oil phase may include chiclrt n fat.

United States Patent No. 4,284,630, issued to Yu et al., ~ closes a method for stabilizing a water-in-oil emlll~ion which cnmpri~es adding magnesium hydroxide or m~gn~eillm oxide to the ~mnl~ic)n The oil phase may include chicken fat.

Pl~ uaLion HTM has been reported to increase the rate of wound healing in artificially created rectal ulcers. The active ingredients in P~ lion HTM are skin ICi~ ly factor and shark liver oil, Subl~lyam 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 peroxide production, enhance growth, and protect the systems against the toxicity of reactive oxygen ;~ tes The unsaturated fatty acids and saturated fatty acids contained within c.hick~n fat enhanced membrane repair and reduced cytotoxicity. The antioxidants glllt~thione and thioglycollate reduced the injury inclllce-l by oxygen radical species, Martin, Ph.D. thesis, (1987-89).
United States Patent No. 4,615,697, issued to Robinson, discloses a controlled release ~ nt composition comprising a treating agent and a bioadhesive agent comprising a water-swellable but water-insoluble, fibrous cross-linked carboxy-functional polymer.

European patent application no. 0410696A1, to Kellaway et al., discloses a mucoadhesive delivery system comprising a treating agent and a polyacrylic acid cross--WO 96t37230 PCT/US~G~5~01 linked with from about 1% to about 20% by weight of a polyhydroxy cw~ ulld such as a sugar, cyclitol, or lower polyhydric alcohol.

SI~Y OF THE INVENTION

This invention pertains to t~e~d~ciuLic immlmoshmlll~hn~wound healing compositions for treating psoriasis and other keratolytic disorders for enh~ncing wound healing in the inner and outer layers of the skin. The compositions of this invention comprise therapeutically effective amount of an immnnostimlll~ting agent and a wound healing composition. A ~rer~l.ed embodiment of the wound healing composition of this invention comprises (a) pyruvate s~lected from the group coneiehng of pyruvic acid, ph~rm~ceutically acceptable salts of pyruvic acid, and n~ ulcs thereof; (b) an antioxidant; and (c) a llli~lulc of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular m~ l,l~les and resuscitation of ~ n cells. The therapeutic immllnostimlll~ting-wound healing compositions of this invention may be utilized in a wide variety of ph~rm~eutical products. This invention also relates to methods for preparing and using the immunostim--l~ting-wound healing therapeutic compositions and the ph~rm~ceuticalproducts in which the therapeutic compositions may be used.

This invention further comprises ~llgmPnt~l therapeutic immunostim.-l~ting-wound healing compositions comprising immlmo~timlll~tin~ agents and a therapeutic wound healing composition in combination with one or more additional m~ m~nte This invention also relates to methods for preparing and using the ~ m~ont~A
therapeutic imrnunostim-ll~ting-wound healing compositions and the pharmaceutical products in which the ~llgm~nted compositions may be used.

CA 02218~39 1997-10-17 WO 96/37230 PCI~/US96/05901 BRIEF DESCR~PTION OF THE DRAWINGS

Figure 1 depicts in bar graph format the viability of U937 monocytic cells following exposure of the cells to various ~ntioxi~l~nt~ (Examples 1-5).
Figure 2 depicts in bar graph format the viability of U937 monocytic cells following exposure of the cells to various combin~tion~ of ~nti-)xi~l~nt~ (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 antioxidants (Examples 14-18).

Figure 4 depicts in bar graph folmat the levels of hydrogen peroxide produced by U937 monocytic cells following exposure of the cells to various combinations of antioxidants (Examples 19-26).

Figure 5 depicts in bar graph format the levels of hydrogen peroxide produced by U937 monocytic cells following exposure of the cells to various combinations of antioxidants with and without a mixhure of s~hlr~t~l and unsahlrated fatty acids (Examples 27-32).

Figure 6 depicts in bar graph forrnat the levels of hydrogen peroxide produced by epiderrnal keratinocytes following exposure of the cells to various antioxidants with and without a llfi~ e of saLul~t~d and nn~ ted fatty acids (Examples 33-42).

Figure 7 depicts in bar graph format the levels of hydrogen peroxide produced by epiderrnal keratinocytes following exposure of the cells to various combin~tic-ni of antioxidants with and without a mixh~re of sahurated and unsahlrated fan~ acids (Examples 43-52).

CA 02218~39 1997-10-17 WO 96137230 PCTIUS96/OS90l Figure 8 depicts in bar graph foImat a ~.~...".~,y analysis of the levels of hydrogen peroxide produced by epidennal keratinocytes following exposure of the cells to the individual cum~o.lents of the wound healing composition, to various combinations of the wound healing composition, and to the wound healing composition.
S
Figures 9A-9D are photographs of wounded mice after 4 days of ~ with:
no composition (Fig. 9A, control); a petrolatum base fonn~ tion Co~ Iive yeast cell derivative, shark oil, and a ~ C of sodium pyruvate, vitamin E, and chicken fat (Fig. 9B); a petrolatum base forrn~ ion cu..l~;..;..g live yeast cell derivative and shark 10oil (Fig. 9C); and Preparation HTM (Fig. 9D).

Figure 10 is a photograph of a wounded mouse after 4 days of Lle~l.. .~-.1 with a petrolatum base formnl~ n only (Example D).

15Figures 1 lA-1 lD are photographs of wounded mice after 3 days of tre~ nt with: no composition (Fig. 1 IA); BetafectinTM with Pl~ alion H~M with the woundhealing composition (Fig. llB); Betafectin~U (Fig. llC); live yeast cell derivative containing the wound healing composition (Fig. 1 ID).

20Figure 12 is a photograph of a wounded mouse after 3 days of treatment with neosporin cont~ining the wound healing composition (D).

Figures 13A-13D are photographs of the histological results of wounded mice after 3 days of lle~ .t with: no composition (Fig. 1 lA); Betafectin~U with Pl~lion 25HrM with the wound healing composition (Fig. 1 lB); Betafectin~ (Fig. l l C); live yeast cell d~livalive cu.~ i.,;.lg the wound healing composition (Fig. 1 lD).

Figure 14 is a photograph of the histological results of a wounded mouse after 3 days oftre~tm~nt with neosporin co~ ;";,~g the woundhealing composition (D).

WO 96137230 PCrlUS96/0~01 DETAILED DESCRIPTION OF T~i INVENTION

This invention pertains to th~..,.pc~.l;c immnnos~imlll~ting_wound healing compositions which c-~mpri~e an jmmllnos~mlll~hng agent and a wound healing composition and/or its metabolites. In one embodiment the wound healing c."ll~o~iLio-l c.,~ lises (a) pyruvate selecte/l from the group con~i~ting of pyruvic acid, ph~rm~ce~lt-c~lly acceptable salts of pyruvic acid, and ~LUIGS thereof; (b) an antioxidant; and (c) a ll~Lu~ of salulaLGd and unsaLuldled fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular lllc.l-bl~les and resll~cit~tion of .. ,.~ n cells.

Applicant has discovered therapeutic wound healing compositions for preventing and reducing injury to m~mm~ n cells and increasing the resuscitation rate of injured ".,..~"~ n cells. Cells treated with the therapeutic wound healing compositions of this invention show decreased levels of hydrogen peroxide production, increased resistance to cytotoxic agents, increased rates of proliferation, and increased viability.
Cellular cultures co~ il.g the therapeutic wound healing compositions showed ~onh~nced dirrGl~ tion and proliferation over control cultures and rapidly formed ;ltt:~'h~ ; or tight junctions between the cells to form an epidermal sheet. Wounded ... ,.. ~l.c treated with the therapeutic wound healing compositions show significantly improved wound closing and healing over ullLIGalGd ..-i1...,..~1.~ and ~--i1....-.~1.~ treated with conventional healing compositions. The wound healing compositions may be used alone or in combination with other m~AicO...~ More specifically, the therapeuticwound healing compositions of this invention are used in combination with therapeutic amounts of antikeratolytic agents for treating psoriasis and other keratolytic skin disorders. The antikeratolytic-wound healing compositions of this invention may be further combined with afl(liti~)n~l medic~m~nt~ for enhanced therapeutic efficacy.

The therapeutic wound healing compositions of this invention are Embodiment One. There are several aspects of Embodiment One of therapeutic wound healing compositions in this invention. In a first aspect, a.A), the therapeutic wound healing composition comprises (a) pyruvate selected from the group con~ ting of pymvic acid, ph~rrn~ce~lhrAlly acceptable salts of pyruvic acid, and ~~ s thereof, (b) an ~nhoxi-l~nt and (c) a ~ u e of s~ A andnn.~ l~ fatty acids whc.~ the fatty acids are those fatty acids required for the repair of cellular ~ lalles and resuscitation of .,.i....,..Ali~n cells. In a second aspect, (I.B), the therapeutic wound S healing composition compri~Çs (a) ~yluval~ s~lecte~l from the group con~ichng of pyruvic acid, ph~rm~cellt1cally acceptable salts of pyruvic acid, and ~ S thereof, (b) lactate selected from the group con~i~hng of lactic acid, ph~rm~ce~tir.~lly acceptable salts of lactic acid, and ~lulcis thereof, and (c) a IlJiAlul~ of saLulated and unsaLulated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and r~sllecit~hon of ,.. ~ lll.Ali~n cells. In a third aspect, (I.C), the therapeutic wound healing composition comprises (a) an Antio~cil1~nt and (b) a llfi~Lule of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of .. ~.. ~li~n cells. In a fourth aspect, (I.D), the therapeutic wound healing composition comprises (a) lactate selected from the group con~i~hng of lactic acid, ph~rm~ce~ltic~lly acceptable salts of lactic acid, and ll~ s thereof, (b) an antioxidant, and (c) a ~-~lw~ of saluldL~d and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular lll~-L,bl~les and resuscitation of l..,,.".~.Ali~n cells.
The therapeutic wound healing compositions of this invention are further combined with a therapeutically effective amount of an immllnostimlll~ting agent (X) to form immnnostimlll~ting-wound healing compositions (I.A-D + X). The immlmostimlll~tlng-wound healing composit~on.~ may be used alone or in combination with other medics.. r.. ~.~i This invention also pertains to methods for preparing and using the immunostim~ ting-wound healing compositions and the ph~rm~re~ltical products in which the therapeutic compositions may be used.

The therapeutic immnnos~mlll~hng-wound healing compositions of this invention may be further combined with one or more ~ On~l me~ for treating wounds to form ~llgm~.nt~l1 immlmostimlll~tlng-wound healing compositions.
This invention also relates to methods for preparing and using the ~llgmt-.nt~(1 WO 96/37230 PCI~/US~ 5!101 thc,~culic immllnoshmlll~hn~-wound healing compositions and the ph~Tm~celltic~
products in which the ~llgmPntç~l compositions may be used.

The term "injured cell" as used herein means a cell that has any activity disrupted for any reason. For example, an injured cell may be a cell that has injured .. G,~ es or damaged DNA, RNA, and ribosom~ for example, a cell which has (a) injured l--G -lI,l~les so that transport through the I~ anes is ~ hed r~sllltinE in an increase in toxins and normal cellular wastes inside the cell and a decrease in llUll;C~ and other components necç~Ty for cellular repair inside the cell, (b) an increase in concentration of oxygen radicals inside the cell because of the decreased ability of the cell to produce antioxidants and enzymes, or (c) darnaged DNA, RNA, and ribosomes which must be repaired or replaced before normal cellular functions can be resllmt-A The term "resuscitation" of injured l..~.,....~li~n cells as used herein means the reversal of cytotoxicity, the stabilization of the cellular ~-lGll-~-~le, an increase in the proliferation rate of the cell, and/or the norm~li7~tion of cellular functions such as the secretion of growth factors, h(~rml-n~s, and the like. The term "cytotoxicity" as used herein means a con~1itic-n 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 plUlll~tGS cellular proliferation.
The term "prodrug", as used herein, refers to compounds which undergo biotran~rc,~ dtion prior to exhibiting their ph~rm~cological effects. The chemical modification of drugs to overcome ph~rm~ceutical problems has also been termed "drug l~t~nh~ti-)n " Drug l~tenh~tion is the ch~ c~l m-ltlific~hon of a biologically active co~ oulld to form a new compound which upon in vivo enzymatic attack willliberate the parent compound. The chemical alterations of the parent compound are such that the change in physicochemical properties will affect the absorption, distribution and enzymatic metabolism. The ~l~finitic)n of drug l~tenh~hon has also been el~t~n(led to include nonenzymatic regeneration of the parent compound.
Regen~r~hc)n takes place as a consequence of hydrolytic, dissociative, and otherrç~chon~ not necessarily enzyme mP~ t~1 The terms prodrugs, l~t~nh~hcl drugs, and bic,l~ ,ible derivatives are used interchangeably. By inference, l~tenh~hQn implies WO 96/37230 PCT/US!~6~'~5~01 a time lag t?l~nf-nt or time Cu~ Ol~ t involved in re~G~ -g the bioactive parentmolecule in vivo. The term prodrug is general in that it inc~ es lA~ ;At~l drug dcliv~livcs as well as those substances which are ~l,vcllcd after ~Amini~tration to the actual snhstAnce which combines with lcc~lol~. The term prodrug is a generic term - S for agents which undergo biotrAn~r~ AI;~n prior to exhibiting their phArrnAcolcgical actions. In the case where the ~ ed drug is not the active agent, but rather is biotransformed to the active agent, the term "prodrug" also inclll-les compounds which may not necessarily undergo biotran~ru---~AI;on to the ~-lmini~t~red drug but may undergo biotran~r~l...At;on to the active agent which exhibits the desired pharmacological effect.

The term "metabolite", as used herein, refers to any substance produced by metabolism or by a metabolic process. "Metabolism", as used herein, refers to the various chemical reactions involved in the tran~r~lll~lion of molecules or chemical compounds occurring in tissue and the cells therein.

I. Wound Healin~ ComPositions A. Embodiment One (I.A-D) The cells which may be treated with the thcl~ ic wound healing compositions in the present invention are ~..A.~".AliAn cells. ~lthollgh applicant will describe the present therapeutic wound healing compositi~n~ as useful for treating ~A~ AliAn epidermal keratinocytes and ...A...---AliAn monocytes, applicant contemrlates that the therapeutic wound healing compositi~n~ may be used to protect or resuscitate all I~Allll~lAliAn cells. Keratinocytes are representative of normal ~ lAliAn cells and are the fastest proliferating cells in the body. The correlation between the reaction of keratinocytes to injury and therapy and that of l~",.."~ n cells in general is very high.
Monocytes are representative of speci~li7e~ ."~ n cells such as the white blood cells in the immune system and the organ cells in liver, kidney, heart, and brain. The n cells may be treated in vivo and in vitro.

WO 96137230 PCTIUS3.~ 5301 Epidermal keratinocytes are the spec~ l epithelial cells of the epidermis which synth~ei7e keratin, a scleroprotein which is the principal conetihl~nt of epi~l~rmie, hair, nails, homy tissue, and the organic matrix of the enamel of teeth.
M~mm~ n epidermal k~hin~cytes c~neh~h~7t~ about 95% of the epidem~al cells and together with melanocytes fomm the binary system of the epi~l~rmie In its various sllccçeeive stages, epi~l~rm~l k~hnocytes are also known as basal cells, prickle cells, and granular cells.

Monocytes are mnnon~lcle~r phagocytic leukocytes which undergo lc~ al~ l y bursting and are involved in reactive oxygen m~li~ted damage within the epitl~Tnie Leukocytes are white blood cells or corpuscles which may be cl~eeifie 1 into two main groups: granular leukocytes (granulocytes) which are leukocytes with abundant granules in the cytoplasm and nongranular leukocytes (nongranulocytes) which are leukocytes without specific granules in the cytoplasm and which include the lymphocytes andmonocytes. Phagocyte cells are cells which ingest microorg~nieme or other cells and foreign particles. Monocytes are also known as large mononuclear leukocytes, andhyaline or transitional leukocytes.

Epidermal keratinocytic cells and monocytic cells have multiple oxygen generating mech~nieme and the degree to which each type of mech~niim functions differs in each type of cell. In monocytes, for example, the lC~il~ y bursting process is more pronounced ~an in epidermal keratinocytes. Hence, the componentsin the therapeutic wound healing compositions of the present invention may vary depending upon the types of cells involved in the contlihon being treated.
As set out above, in a first aspect of Embodiment One (I.A), the therapeutic wound healing composition for treating .. ~.. ~li~n cells, preferably epiderrnal keratinocytes, comprises (a) pyruvate selected from the group consisting of pyruvic acid, ph~rm~ elltically acceptable salts of pyruvic acid, and I~ Lul~S thereof, (b) an antio~ nt, and (c) a l~ e of ~alulaled and unsalulaled fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of ~ n cells. In a second aspect of Embodiment One (I.B), the CA 02218~39 1997-10-17 WO 96~37230 PCIlUS9C/O~igOl dlerapeutic wound healing C~mpOSitil~n for treating ~ n cells, preferably epidermal keratinocytes, comrri~es (a) pyruvate selectecl from the group c~ n~i~ting of pyruvic acid, ph~rm~çelltiç~1ly acceptable salts of pyruvic acid, and ~ LUIGS thereof, (b) lactate selected from t-h-e group con~i~hng of lactic acid, ph~rm~celltiç~lly - 5 acceptable salts of lactic acid, and llfi~lul~s thereof, and (c) a llli~UlG of satula~ed and unsalulatGd fatty acids wherein the fatty acids are those fatty acids le~luil~id for the repair of cellular ~llGl,lII~Ies and resns~ tirn of ",5l."lll~ n cells. In a third aspect of Embodiment One (I.C), the thcl~c.llic wound healing composition for treating n cells, preferably epidermal keratinocytes, comprises (a) an antioxidant and (b) a ~ Lulc of saturated and unsalul~lGd fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of ll, ..ll,l.~li~n cells. In a fourth aspect of Embodiment One (T.D), the therapeutic wound healing composition for treating "lil."~ n cells, preferably monocytes, comprises (a) lactate selected from the group con~i~ting of lactic acid, ph~rm~ceutically acceptable salts of lactic acid, and Illi~UlGS thereof, (b) an antioxidant, and (c) a mi~LUlG of sa~ul~LGd and unsa~ula~ed fatty acids wherein the fatty acids are those fatty acids yuil~d for the repair of cellular lllGlllLanes and resuscitation of l".~ n cells.

Pyruvic acid (2-oxopropanoic acid,~lpha-ketopropionic acid, CH3COCOOH) or pyruvate is a filn~ .nt~l int~ tt~. in protein and carbohydrate metabolism and in the citric acid cycle. The citric acid cycle (tricarboxylic acid cycle, Kreb's cycle) is the major reaction sequence which executes the reduction of oxygen to generate adenosine triphosphate (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 thereafter utilized in â variety of biological processes and is a precursor in the biosynthesis of many fatty acids and sterols. The two major sources of acetyl coenzyme A are derived from the metabolism of glucose and fatty acids. Glycolysis consists of a series of tran~llllalions wherein each glucose molecule is transformed in the cellular cytoplasm into two molecules of pyruvic acid.
Pyruvic acid may then enter the mitocholll1ri~ where it is oxi-li7~cl by coenzyme A in ~ the presence of enzymes and cofactors to acetyl coenzyme A. Acetyl coenzyme A can then enter the citric acid cycle.

WO 96/37230 PCI~/US96105901 In mllecle, pyruvic acid (derived from glycogen) can be reduced to lactic acid during anerobic metabolism which can occur during exercise. Lactic acid is reoxi~li7ed and partially retransformed to glycogen during rest. Pyluv~tt; can also act as an -n~ioxi~i~nt to neutr-1i7~ oxygen r~licAle in the cell and can be used in the S multifunction oxidase system to reverse ~;y~,~icity.

The pyruvate in the present invention may be srl~cl~cl from the group c~neieting of pyruvic acid, ph~rm-Acellhcally acceptable salts of pyruvic acid, prodrugs of pyluvic acid, and l~ es thereof. In general, the ph~A~nAce~ cAlly ~ccepPble salts of pyruvic acid may be alkali salts and Alk-Aline ear~ salts. Preferably, the pyluvate is selected from the group cone-ie-ting of pyruvic acid, lithium ~yluv~le, sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate, mAng~nese pymvate, methyl pyruvate, Alpha-ketoglutaric acid, and l~ Lult;s thereof.
More preferably, the pyruvate is selected from the group of salts coneie-ting of sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate, m~ng~nese pyruvate, and the like, and llfi~ules thereof. Most preferably, the py~uvate is sodium pyruvate.

The amount of pyruvate present in the therapeutic wound healing compositions of the present invention is a therapeutically effective amount. A
therapeutically effective amount of pyruvate is that amount of pyruvate neccssAry for the inventive composition to plc~,ent and reduce injury to Il~AllllnAliAn cells or increase the resuscitation rate of injured ",A.~ Ali-An cells. The exact amount of pyruvate is a matter of plG~l-,.,ce subject to such factors as the type of concli~-. n being treated as well as the other ingredients in the colll~o:~iLion. In a pr~ ,d embo~liment~ pyruvate is present in the therapeutic wound healing composition 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 composition.

L-Lactic acid ((S)-2-hydlu~y~l~anoic acid, (+) Alpha-hydroxypropionic acid, C~H3CHOHCOOH) or lactate occurs in small quantities in the blood and muscle fluid of ~A~"~"Ale Lactic acid concentration increases in muscle and blood after WO 96137230 PCT/US96~'~5gOl vigorous activity. Lactate is a culll~onent in the cellular fee~lhaclr m~ch~niQm and inhibits the natural 1'CQ~Pi1~1Y bursting process of cells thereby su~pl~s~illg the production of oxygen radicals.

S The lactate in the present invention rnay be se1ecte~1 frorn the group con.QiQhn~ of lactic acid, ph~rm~ce11hc~11y acceptable salts of lactic acid, prodrugs of lactic acid, and ~ ules thereof. In general, the ph~rm~ce~1hcally acceptable salts of lactic acid rnay be alkali salts and ~lk~line earth salts. Preferably, the lactate is selecte~l from the group conQ;~ting of lactic acid, lithiurn lactate, sodium lactate, potassium lactate, rnagnesiurn lactate, calcillm lactate, zinc lactate, m~ng~nese lactate, and the like, and nli~lules thereof. More preferably, the lactate is selected from the group consisting of lactic acid, sodiurn lactate, potassium lactate, magnesium lactate, calcium lactate, zinc lactate, m~ng~nese lactate, and ~lul~s thereof. Most preferably, the lactate is lactic acid.
The ~ll(~unt of lactate present in the therapeutic wound healing compositions of the present invention is a therapeutically effective amount. A ther~pe11hc~11y effective allluulll of lactate is that amount of lactate necessary for the inventive composition to ~I~VCIIt and reduce injury to ",i...",.~ n cells or increase the r.~,s11Q~it~hnn rate of injured ".,.~ n cells. For an ingestible composition, a therapeutically effective amount of lactate is that amount necessary to suppress the ~s~ilal~,l y bursting process of white blood cells to protect and resuscitate the n cells. In general, a tkc.a~c.llically effective amount of lactate in an ingestible composition is from about 5 to about 10 times the amount of lactate norrnally found in serum. The exact amount of lactate is a matter of pl~r~.~.lce subject to such factors as the type of con~ on being treated as well as the other ingredients in the composition. In a ~lert;lled embo~1im~nt~ lactate is present in the therapeutic wound healing composition 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%, byweight of the therapeutic wound healing composition.

WO 96137230 PCT/U~ 5~01 AntioXi~nt~ are substances which inhibit ~xirl~tion or Su~lGss reactions p~ olGd by oxygen orperoxides. ~nhnxi~nt~, çspec~ y lipid-soluble ~ntir~ nte, can be absorbed into the cellular llwll~ e to neutralize oxygen r~lic~l~ and thereby protect the "-G"-bl~u,e. The ~nti~xi~l~nt~ useful in the present invention rnay be S select~l from the group con~i~tin~E~ of all forms of Vitamin A (retinol), all forms of Vitamin2 (3, 4-didehydlulGIillol), all forms of carotene such as ~lpha-carotene, ,~-carotene (beta"~-carotene), gamma-carotene, delta-carotene, all forms of Vitarnin C
(D-ascorbic acid, ~ascorbic acid), all forms of tocopherol such as Vitamin E (Alpha-tocopherol, 3 ,4-dihydro-2,5, 7, 8-tetramethyl-2-(4, 8, 1 2-trimethyltri-decyl)-2H- 1-benzopyran-6-ol), ,~-tocopherol, gamma-tocopherol, delta-tocopherol, tocoquinone, tocotrienol, and Vitamin E esters which readily undergo hydrolysis to Vitamin E such as Vitarnin E acetate and Vitarnin E succinate, and ph~ ceutically acceptable Vitarnin E salts such as Vitamin E phosph~te, prodrugs of Vitamin A, carotene, Vitarnin C, and Vitamin E, ph~rm~ceutically acceptable salts of Vitamin A, carotene, Vitamin C, and Vitamin E, and the like, and llli~ s thereof. Preferably, the antioxidant is selected from the group of lipid-soluble antioxidants consisting of Vitarnin A, ~-carotene, Vitarnin E, Vitamin E acetate, and ll~lules thereof. More preferably, the antioxidant is Vitarnin E or Vitamin E acetate. Most preferably, the ~nhoxi-l~nt is Vitamin E acetate.
The amount of antioxidant present in the therapeutic wound healing compositions of the present invention is a therapeutically t;rr~.;live amount. Atherapeutically effective amount of antioxidant is that amount of antioxidant necessary for the inventive composition to plev~.lt and reduce injury to ~ "~ n cells or increase the resuscitation rate of injured "~,.. "",~ n cells. The exact amount of antioxidant is a matter of ~-t;r~ nce subject to such factors as the type of condition being treated as well as the other ingredients in the composition. In a ~fert;.l~,d embodiment, the antioxidant is present in the therapeutic wound healing composition in an amount from about 0.1% to about 40%, preferably from about 0.2% to about 30%, and more preferably from about 0.5% to about 20%, by weight of the therapeutic wound healing composition.

CA 02218~39 1997-10-17 WO 96/37230 PCT/US~ '(i!;!)01 The ll~u~Lulc of salulated and unsah~rated fatty acids in the present invention are those fatty acids required for the repair of ~ n cellular clll~lalles and the pr~lleti~-n of new cells. Fatty acids are carboxylic acid compounds found in animal and vegetable fat and oil. Fatty acids are cl~eeifie~l as lipids and are composed of S chains of alkyl groups cc.. l;~;.. ;.. g from 4 to 22 carbon atoms and 0-3 double bonds and charact~,l.,ed by a t~rmin~l carboxyl group, -COOH. Fatty acids may be s~hlr~t~l or unsalulàlcd and may be solid, semisolid, or liquid. The most C~ saLulaLcd fatty acids are butyric acid (C4), lauric acid (C12), p~lmitic acid (C16), and stearic acid (Clg). Unsalu~alGd fatty acids are usually derived from vegetables and consist of alkyl chains co~ ;.. ;.. g from 16 to 22 carbon atoms and 0-3 double bonds with the characteristic termin~l carboxyl group. The most cc,llllllol unsahurated fatty acids are oleic acid, linoleic acid, and linolenic acid (all C18 acids).

In general, the Illi~Lu,G of sahurated and unsatulatcd fatty acids required for the repair of ~ n cellular In~lllblalles in the present invention may be derivedfrom animal and vegetable fats and waxes, prodrugs of salulaLed and lmc~ r~l fatty acids useful in the present invention, and llli~Lul~s thereof. For example, the fatty acids in the therapeutic wound healing composition may be in the form of mono-, di-, or trigylcerides, or free fatty acids, or ~ Lulcs thereof, which are readily available for the repair of injured cells. Cells produce the chemical components and the energy Ic~luiled 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 plcr~llcd animal fats and waxes have a fatty acid composition similar to that of human fat and the fat contained in human breast milk. The ~lcrellcd animal fats and waxes may be selected from the group cnniiehng of human fat, ç~licl~en fat, cow fat (defined herein as a bovine ~1Omestic animal regardless of sex or age), sheep fat~ horse fat, pig fat, and whale fat. The more plcrcllGd animal fats and waxes rnay be s~.lectecl from the group con~ ting of human fat and chicken fat. The most ~IGr~,llGd animal fat is human fat. Mixtures of other fats and waxes, such as vegetable waxes (especiallysunflower oil), marine oils (especially shark liver oil), and synthetic waxes and oils, - which have a fatty acid composition similar to that of animal fats and waxes, and preferably to that of human fats and waxes, may also be employed.

CA 02218~39 1997-10-17 WO 96/37230 PCT/US96105!101 In a l,.Gr.,..~d embo lim~nt~ the ~lule of s~hlrate~l and lm.c,~l...,.tecl fattyacids has a composition similar to that of human fat and c~mpri.ces the following fatty acids: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, myristoleic acid, p~lmihc acid, palmit~l~ic acid, stearic, oleic acid, linoleic acid, S linolenic acid, arachidic acid, and gadoleic acid. Preferably, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic, oleic acid, linoleic acid, linc~lenic acid, arachidic acid, and gadoleic acid are present in the l~lule in about the following percentages by weight, respectively (carbon chain number and number of nnc~hlrahon~c are shown parenthetically, respectively): 0.2%-0.4% (C4), 0.1% (C6), 0.3%-0.8% (C8), 2.2%-3.5%
(C10) 0 9%-5 5% (C12), 2.8%-8.5% (C14), 0.1%-0.6% (C14:1)' 23-2%-24-6% (C16)~
1 8%-3 0% (Cl6 1)~ 6-9%-9.9% (C18), 36.0%-36.5% (C18 1), 20%-20.6% (C18 2), 7-8% (C18:3)~ 1-1%-4-9% (C20), and 3.3%-6.4% (C20 1).

In another ~ Ç~.. Gd embo~lim~-nt, the ~ 1G of saLul~led and unsatulated fatty acids is typically chicken fat comprising the following fatty acids: lauric acid, myristic acid, myristoleic acid, pent~lec~nnic acid, palrnitic acid, palmitoleic acid, ~~galic acid, .~ aloleic acid, stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, and gadoleic acid. Preferably, lauric acid, myristic acid, myristoleic acid, pl?nt~1ec~noic acid, palmitic acid, palmitoleic acid, rnargaric acid, .~ uoleic acid, stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, and gadoleic acid are present in the n.i~ in about the following percentages by weight, respectively:
0 1% (C12), 0-8% (C14), 0-2% (C14 1), 0-1% (Cls), 25-3% (C16), 7-2% (C16 1), 0-1%
(C17), 0-1% (C17:1)~ 6-5% (Cl8)~ 37-7% (C18 1), 20-6% (C18 2), 0.8% (C18 3), 0.2%
(C20), and 0.3% (C20 1), all percent~gçs +/- 10%

In another ~cr~ ,d embodiment, the mixture of s~lu.dled and unsaturated fatty acids comprises lecithin. Lecithin (phosphatidylcholine) is a phosphatide found in all living orf~nicm.c (plants and ~nim~l.c) and is a significant con.ctit~lent of nervous tissue and brain substance. Lecithin is a l~f"~ e of the diglycerides of stearic, p~ itic~ and oleic acids, linked to the choline ester of phosphoric acid. The product of co~ ;e is pre~lomin~ntly soybean lecithin obtained as a by-product in the CA 022l8539 l997- lO- l7 WO 96/37230 PCT/U5!16J~901 m~n~lf~r.~lring of soybean oil. Soybean lecithin c~nt~in~ palmitic acid 11.7%, stearic 4 0%, p~lmitoleic 8.6%, oleic 9.8%, linoleic 55.0%, linolc.nic 4.0%, C20 to C22 acids (includes arachidonic) 5.5%. T eçithin may be l~res~ d by the formula:

I

CHOCOR
I

CH2O-P(O)2-OCH2CH2N (CH3)3 wherein R is selected from the group con~ieting of stearic, p~lmitic~ and oleic acid.

The above fatty acids and pe.cc.ll~ges thereof present in the fatty acid ll~~ are given as an example. The exact type of fatty acid present in the fatty acid and the exact amount of fatty acid employed in the fatty acid lffi~Lule 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 experiment~tiQn .
The amount of fatty acids present in the therapeutic wound healing compositions of the present invention is a thc.apc~Lically effective amount. A
therapeutically effective amount of fatty acids is that amount of fatty acids necessary for the inventive composition to ~revent and reduce injury to ...i1....n~ n cells or increase the resuscitation rate of injured ~ n cells. The exact amount of fatty acids employed is subject to such factors as the type and distribution of fatty acids employed in the ll~ ule, the type of con-lihon being treated, and the other ingredients in the composition. In a plc;r~lled embo-1im~.nt, the fatty acids are present in the therapeutic wound healing composition 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 composition.

WO 96/37230 PCTtUS96~ 01 In accord with the present invention, the therapeutic wound healing compositi~n.C of Embodiment One (I.A-D) for treating .. ~.. Ali~n cells may be s~lectecl from the group con~icting of:

S (I.A)(a) pyruvate selecte(l ~om the group c n~icting of pyruvic acid, ph~nn~çe~ltlc~lly acceptable salts of pyruvic acid, and llfi~lu.cs thereof;
(b) an antioxidant; and (c) a ~ ul~ of saLu-~ted and llnc~ ccl fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular ll.~.llI).~les and rçs lech~tlon of " ,,", . ., .Ali~n cells;

(I.B)(a) pyruvate selected from the group concichng of pyruvic acid, ph~nn~ce~ltlcally acceptable salts of pyruvic acid, and ~Lu.cs thereof;
(b) lactate selected ~om the group concicting of lactic acid, ph~n~eutically acceptable salts of lactic acid, and lliLX~UlCS thereof; and (c) a ,.fi~u,e of saLu-attd and nnc~ tçd fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of ",i,.""~Ali~n cells;

~.C) (a) an antioxidant; and (b) a .-~ ~Lule of satu-aLcd and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular Illc~ b~ es and resuscitation of l,.i3.",..~ n cells;

(I.D) (a) lactate s~lect~d from the group corl~ichng of lactic acid, pharm~cel-tically acceptable salts of lactic acid, and ,~L~u,es thereof;
(b) an antioxidant; and (c) a Llfi~Lulc of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular ,nc,llb,a~les and r~s~Ccit~hion of ",i~ n cells.

Preferably, the wound healing compositions of F.mho lim~nt One (I) for treating ,,"..",,~ n cells, preferably epidermal ke~tinc-cytes, may be select~l from the group con~i~hng of:

S ~LA) (a) pyruvate sçle~ilecl from the group con~i~ting of pyruvic acid, ph~rm~celllicc~lly acceptable salts of pyruvic acid, and ll~ s thereof;
(b) an ~ntil~Xi~1~nt and (c) a llfi~ , of sdlwdl~d and unsdluldlcd fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular ll~c.l~ dlles and resuscitation of ",i.. "",~ n cells, ~.B) (a) pyruvate selected from the group con~i~ting of pyruvic acid, ph~n~celltically acceptable salts of pyruvic acid, and ~Lules thereof;
(b) lactate selectecl from the group concicting of lactic acid, pharm~ce ltically acceptable salts of lactic acid, and ll~Lul~s thereof; and (c) a llfi~Lulc of salul~tcd and unsa~ulaled fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular lllcll~l~les and resuscitation of m~ n cells, and (I.C) (a) an antioxidant; and (b) a Illi~ C of sa~ tcd and uns~Luldlcd fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular lllcllll~ es and resuscitation of ~ n cells.

More preferably, the wound healing compositions of Embo~limt~nt One (I) for treating "~;-""~ n cells, preferably epidermal keratinocytes, may be selected from the group consisting of:

(I.A) (a) pyluvate selected from the group con~ ting of pyruvic acid, ph~rm~ce~ltically acceptable salts of pyruvic acid, and llfi~Lulcs thereof;
(b) an antioxidant; and WO 96137230 PCT/U59~ 01 (c) a l~ e of ~ cl and unsalula~d fatty acids wherein the fat~y acids are those fat~ acids ~ ,d for ~e repair of cellular ~ ~es and resllccit~t-on of ~ n cells; and WO 96/37230 PCT/US~'05~Ul (I.C) (a) an ~ntioxi~nt and (b) a ~ Lulc of s~ ~l and unsalulaled fatty acids where*l the fatty acids are those fatty acids rc~luilcd for the repa* of cellular m~ es and reellecitAtion of ~ n cells.
~ S
More preferably, the wound healing composihone of Embod*nent One (I) for treating ~ AliAn cells, preferably epidermal kerAtinocytes, may be selected from the group coneieting of:

(I.A) (a) pyruvate selectecl from the group conei.eting of pyruvic acid, pharm~çe~ltically acceptable salts of pyruvic acid, and llfi~ s thereof;
(b) an antioxidant; and (c) a l~luue of s~ and unsaturated fatty acids whe.eill the fatty acids are those fatty acids lc~luilcd for the repair of cellular IllcnLlailes and resuscitation of ~ "",Ali~n cells; and (I.B) (a) pyruvate selected from the group conei.eting of pyruvic acid, pharm~cenhcally acceptable salts of pyruvic acid, and I~ LulcS thereof;
(b) lactate s~lectecl from the group concicting of lactic acid, pharmAce ltically acceptable salts of lactic acid, and llli~LLulcs thereof; and (c) a ll i~Lulc of saLulaLcd and unsaLulaLcd fatty acids wherein the fatty acids are those fatty acids lc~luilcd for the repa* of cellular Ill~ Ll~les and resuscitation of . . .;1 . ", . .Ali~n cells.

Most preferably, the wound healing compositions of Embotlim~nt One (I) for treating ,~ ."",Ali~n cells, preferably epidermal keratinocytes, comprise:

(I.A) (a) pyruvate selecte~l from the group coneieting of pyruvic acid, pharmAce~-tic~lly acceptable salts of pyruvic acid, and I~ LuucS thereof;
(b) an antioxidant; and CA 02218~39 1997-10-17 WO 96/37230 PCT/US~G~'~5~01 (c) a Illi~lUlG of s ~ ;d and lm~ ~ fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular ~ es and resuscitation of . "~ . ". . .~ n cells.

Most preferably, the wound healing compositions of Embodiment One (I) for treating ~ n ce11s, preferably monocytes, comprise:

(I.D) (a) lactate s~lecte~l from the group con~ichng of lactic acid, ph~rm~ceutically acceptable salts of lactic acid, and llf~LulcS thereof;
(b) an ~nti~xi~l~nt and (c) a ~lfi~lulG of s~Lul~lGd and uns~lulaled fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of ."~n....~ n cells.

Throughout this tli~c1osllre~ applicant will suggest various theories or mt-ch~niirn~ by which applicant believes the c~ onents in the therapeutic wound healing compositions and the antiviral agent function together in an unexpected synergistic manner to prevent and reduce injury to .. i.. ~li~n cells, increase the resuscitation rate of injured ~Il;1llllll~1i~n cells, and reduce viral titers. While applicant may offer various meçll~nicm~ 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 int~ndecl to limit the effective scope of the claims.

In the first aspect of Embodiment One (I.A), applicant believes that pyruvate can be transported inside a cell where it can act as an ~ntioxi~l~nt to neutralize oxygen radicals in the cell. Pyruvate can also be used inside the cell in the citric acid cycle to provide energy to increase cellular viability, and as a precursor in the synthesis of important biomolecules to promote cellular proliferation. In ~d-liti~ n, pyruvate can be used in the multifunction oxidase system to reverse cytotoxicity. Antioxidants, especially lipid-soluble antioxidants, can be absorbed into the cell lllwllbl~le to neutralize oxygen radicals and thereby protect the membrane. The s~tnr~t~cl and unsalulaLGd fatty acids in the present invention are those fatty acids lG~luilGd for the CA 02218~39 1997-10-17 WO 96t37230 PCTtUS~/OJ901 resuscitation of ~ n cells and are readily available for the repa* of injured cells and the proliferation of new cells. Cells injured by oxygen radicals need to produce unsalulàted fatty acids to repair cellular ,.l~ es. However, the proclnr,tion ofunsalulaled fatty acids by cells requires oxygen. Thus, the injured cell needs high S levels of oxygen to produce nn~tll~t~ fatty acids and at the same time needs to reduce the level of oxygen within the cell to reduce oxidative injury. By providing the cell with the unsaturated fatty acids needed for repair, the need of the cell for unsalulàl~d fatty acids is reduced and the need for high oxygen levels is also reduced.

The combination of pyruvate inside the cell and an ~nti~xitl~nt in the cellular membrane functions in an unexpected synergistic manner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved by use of either type of component alone. The presence of ll~lul' ,S of saturated and unsaturated fatty acids in the therapeutic wound healing composition significantly enhances the ability of pyruvate and the antioxidant to inhibit reactive oxygen production. By stabilizing the cellular membrane, unsat~aled fatty acids also improve membrane function andenhance pyruvate transport into the cell. Hence, the three components in the therapeutic wound healing composition of the first aspect of Embodiment One (I.A) function together in an unexpected synergistic manner to ~ lt and reduce injury to I.. ~.. ~li~n cells and increase the res~l~çit~tiQn rate of injured ll.,.. -----~li~n cells.

~ the second aspect of Embodiment One (I.B), lactate is employed instead of an antioxidant. Antioxidants react with, and neutralize, oxygen radicals after the radicals are already formed. T ~r~te~ on the other hand, is a component in the cellular fee~lb?cl~ mrr.h~ni~m and inhibits the ~ ilatOIy bursting process to suppress the production of active oxygen species. The combination of pyruvate to neutralize active oxygen species and lactate to ~u~p-css the Ic~lJilal~ly bursting process functions in a synergistic manner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved by use of either type of component alone. The presence of Illi~lUI~,S of salulaled and unsalulated fatty acids in the therapeutic wound healing composition significantly enhances the ability of pyruvate and lactate to inhibit reactive oxygen production. Hence, the three components in the therapeutic wound healing CC~ OSiL~On in the second aspect of F.mho~lim~nt One (I.B) fim~hon together in asynergistic manner to protect and res leÇ.it~te ,~"..~",,~ n cells.

In the third aspect of Embodiment One a.C), the presence of ~ S
S of saturated and unsaturated fatty acids in the the~ lic wound healing composition in this embolim-qnt eignific~ntly P.nh~nces the ability of the ~nho~ nt to inhibit reactive oxygen production. The combination of an ~nhnxi-l~nt to neutralize active oxygen species and fatty acids to rebuild cellular llleml,ldlles and reduce the need of the cell for oxygen functions in a synergistic manner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved by either type of component alone. Hence, the components in the therapeutic wound healing composition in thethird aspect of Embodiment One a.C) function together in a synergistic manner toprotect and resuscitate ~ n~ n cells.

In the fourth aspect of Embodiment One a.D), lactate is employed because the le~ y bursting process is more pronounced in monocytes than in epidermal keratinocytes. The combination of lactate to suppress the lG~ildlCJI~ybursting process and an antioxidant to neutralize active oxygen species functions in a synergistic manner to reduce hydrogen peroxide pro~ chrn in the cell to levels lower than can be achieved by either component alone. The presence of nli~Lu.c;s of saturated and uns~ ed fatty acids in the therapeutic wound healing composition in this embodiment cignific~ntly enhances the ability of lactate and the ~nhioxi~1~nt to inhibit reactive oxygen production. Hence, the three components in the therapeutic wound healing composition in the fourth aspect of Embodiment One a.D) function together in an unexpected synergistic manner to protect and resllecit~te l~ n cells.

Accordingly, the combination of ingredients set out in the above embo-l;...r..l.. functions together in an enhanced manner to prevent and reduce injury to ~ ",~ n cells and increase the r~slleçit~tion rate of injured l"~"""~ n cells.
The therapeutic effect of the combination of the components in each of the aboveembo~;.. ~ is m~rk~-lly greater than that expected by the mere ~ lih-n of the individual therapeutic components. Hence, applicant's th~.a~c.lLic wound healingcompositions for treating ...;.."~ n cells have the ability to decrease intrac~ r levels of hydrogen peroxide prc~lct-on, increase cellular resi~t~nce to ~;yl~ ic agents, increase rates of cellular proliferation, and increase cellular viability.
s B. Methods For Making The Therapeutic Wound Hesling Compositions Of Embodiment One (I.A-D) The present invention extends to methods for making the therapeutic wound healing compositions of Embodiment One (I.A-D). In general, a therapeutic wound healing composition is made by forrning an a~-~ u,c of the components of the composition. In a first aspect of Embodiment One (I.A), a therapeutic wound healing composition is made by forming an ~lhll;xllllc of (a) pyruvate selected from the group consisting of pyruvic acid, ph~rm~ce~lti~lly acceptable salts of pyruvic acid, and mi~urcs thereof, (b) an antioxidant, and (c) a ~ of sd~uldled and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of ~ .,l"l.~ n cells. In a second aspect ofEmbodiment One (I.B), a therapeutic wound healing composition is made by formingan adl",~lulc of (a) pyruvate selected from the group con~i~ting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and llliX.~UlCS thereof, (b) lactate selected from the group consisting of lactic acid, ph~ ce~ltic~lly acceptable salts of lactic acid, and ~ s thereof, and (c) a ~u-c of sa~ula~cd and unsalula~cd fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular mclllbl~les and resuscitation of m~ n cells. In a third aspect of Embodiment One (I.C), a therapeutic wound healing composition is made by forming an ad,."~lu,c of (a) an antioxidant and (b) a Illi~UlC of saturated and unsa~ulalcd fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular mc~..b.alles and resuscitation of l"~ n cells. In a fourth aspect of Embodiment One (I.D), a therapeutic wound healing composition is made by forming an ~-l.. ,;xl.~lc of (a) lactate selected from the group con~i~tinP of lactic acid, ph~rm~ce~l~cally acceptable salts of lactic acid, and ll~ GS thereof, (b) an antioxidant, and (c) a .",~lu,c of si~ te~1 and WO 96/37230 PCT/US9G~'~5~01 unsaturated fatty acids wherein the fatty acids are those fatty acids ~ci~luil~d for the repair of cellular .~.~....h~ es and resllerit~h- n of l~ n cells.

For some applic~hone~ the ~ .e may be formed in a solvent such as water, and a s~ ,t~nt may be added if required. If neceee~.~y, the pH of the solvent is adjusted to a range from about 3.5 to about 8.0, and preferably from about 4.5 to about 7.5, and more preferably about 6.0 to about 7.4. The ~.1...;~1...~ is then sterile filtered. Other ingredients may also be incorporated into the therapeutic wound healing composition as dictated by the nature of the desired composition as well known by those having ordinary skill in the art. The l]ltim~te therapeutic wound healing compositions are readily prepared using methods generally known in the ph~rm~r.eutical arts.

In a ~lGrt;--ed embodiment, the invention is directed to a method for ~ ~il-g a therapeutic wound healing composition (I.A) for preventing and reducing injury to ~. ..",~ n cells, and increasing the r~ellecit~h-n rate of injured "~i1."".~ n cells, which comprises the steps of ~lmixin~ the following ingredients:
(a) pyruvate s~lecte-l from the group conei.ehn~ of pyruvic acid, ph~rm~ellhcally acceptable salts of pyruvic acid, and llf~lules thereof;
(b) an antioxidant; and (c) a llfu~LLLIe of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resllecit~hcn of injured ~ ",.~ n cells.

C. Methods For li.m~ ~ing The Ther~pel-tie Wound Healing Compositions of F.~boAi~ent One (I.A-D) The present invention extends to m-oth~ e for employing the therapeutic wound healing compositions of Embodiment One a) in vivo and in vitro. In general, a therapeutic wound healing composition is employed by contacting the therapeutic composition with l~ n~ n cells.

WO 96/37230 PCT/US~)G~ Cl In a first aspect of Ell,l)o~ One (I.A), the invention is directed to a m~thofl for preventing and re(l~lcing injury to ~ n cells, and increasing the resllccit~hon rate of injured ~ n cells, which co~ c the steps of (A) providing a therapeutic wound healing composition which comprises (a) pyruvate S s~lect~l from the group concichng of pyruvic acid, ph~rm~cellhc~lly acceptable salts of pyruvic acid, and llfixLulGs thereof, (b) an antioxidant, and (c) a llfi~lulG of s~tn~t~
and unsdLuldLGd fatty acids wherein the fatty acids are those fatty acids required for the resllccit~h~n of injured "",.~""~ n cells, and (B) cont~chng the therapeutic wound healing composition with the ~ n cells.
In a second aspect of Embodiment One (I.B), the invention is dil~ Gd to a method for preventing and reducing injury to ",i.."",~ n cells, and increasing the resuscitation rate of injured .. i.. ~li~n cells, which comprises the steps of (A) providing a therapeutic wound healing composition which comprises (a) pyruvate selectecl from the group conci.cting of pyruvic acid, ph~rm~ce~ltically acceptable salts of pyruvic acid, and, I~Lul~s thereof, (b) lactate select~l from the group conci.cting of lactic acid, pharm~ce~lhiç~lly acceptable salts of lactic acid, and llfir.Lul~,s thereof, and (c) a ~ UlG of saturated and unsa~ ~d fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured ~ n cells, and (B) contacting the therapeutic wound healing composition with the .. ~ n cells.

In a third aspect of Embodiment One ~.C), the invention is directed to a method for preventing and redncing injury to ,..~..""~ n cells, and increasing the resuscitation rate of injured . - Ii1 ~ .... I~li~n cells, which comprises the steps of (A) providing a therapeutic wound healing composition which comprises (a) an antioxidant, and (b) a llfi~LulG of saturated and uns~LuldLGd fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured ",:.."",~ n cells, and (B) cont~cting the therapeutic wound healing composition with the m~mm~ n cells.

In a fourth aspect of Embodiment One (I.D), the invention is directed to a m~thod for plGvGllLillg and red~ ing injury to ~ ."",~ n cells, and increasing the resuscitation rate of injured .. "" .. ~ n cells, which comprises the steps of (A) WO 96/37230 PCI~/US96/05901 providing a therapeutic wound healing composition which comrri~çs (a) lactate s~lecte~l from the group con~i~hng of lactic acid, ph~rm~celltic~lly acceptable salts of lactic acid, and ~ ,s thereof, (b) an ~nti.~xi-l~nt and (c) a l~ tUl~, of .~tlr~t~l and~m.c~hlr~tel1 fatty acids wherein the fatty acids are those fatty acids required for the S r~ cit~tiQn of injured ~ "l,~ n cells, and (B) contacting the therapeutic wound healing composition with the ~ n cells.

In a ~lcrcllcd emb~imtont the invention is dhe~;lcd to a method for healing a wound in a ",~."",~1 which comrriees the steps of:
(A) providing a therapeutic wound healing composition (I.A) which comprises:
(a) pyruvate selecte(l from the group con~i~ting of pyruvic acid, ph~rrn~ce ltically acceptable salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant, and (c) a l~ Lulc of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured ".~."".~ n cells;
and (B) contacting the therapeutic wound healing composition with the wound.

The types of wounds which may be healed using the wound healing compositions of Embodiment One (I.A-D) of the present invention are those which result from an injury which causes epidermal damage such as incisions, wounds inwhich the skin is broken by a cutting in~L~ lcllt, and lacerations, wounds in which the skin is broken by a dull or blunt insL~ ,.lt. The therapeutic compositions may also be used to treat various ~lerrn~tological disorders such as hype,kcl~lusis, photo-aging, burns, donor site wounds from skin transplants, ulcers (cutaneous, decubitus, venous stasis, and diabetic), psoriasis, skin rashes, and sunburn photoreactive processes. The topical therapeutic compositions may also be used orally in the form of a mouth wash or spray to protect and accelerate the healing of injured oral tissue such as mouth sores and burns. The topical therapeutic compositions may further be used in ophth~lmological preparations to treat wounds such as those which result from corneal ulcers, radialkc~ t~llly~ corneal transplants, epikeratophakia and other surgically in~ ce~l wounds in the eye. The topical therapeutic compositions may in addition be CA 02218~39 1997-10-17 WO 96t37230 PCT/US3 '1~53~1 used in anorectal creams and ~uE)~o~ ;rs to treat such c~n~litione as pruritus and, proctitis, anal fissures, and h~m~-rrhoi~le In a p~cr~ Gd embo~lim.qnt, the thG~ ic compositions are used to treat wounds such as incisions and lacerations.

S The wound healing c~mrositione of Embo lim--nt One a.A-D) of the present invention may be utilized in topical products, ingestible products, and tissue culture m~ m to protect ".~ "~ n cells and increase the rçsleçit~rion rate of injured l~...l,ll,~li~n cells. For example, the therapeutic wound healing compositions may be used in topical skin care products to protect and increase the resneçit~h~n rate of skin tissue such as in the trç~tm~nt of various ~l~rm~tc-logical disorders such as hyperkeratosis, photo-aging, and sunburn photoreactive processes. Injury to skin can occur for a variety of reasons. Injury often occurs to individuals who wash their hands often, to individuals who are exposed to stressful c"vilu~ l con~litione (overexposure to sun or chemicals), or to the elderly or individuals with an underlining disease. The addition of the wound healing compositions of the present invention to a lotion provides a source of antioxidants to the skin which would protect the skin from the harmful effects of W light, cht-mic~le, and severe drying. The wound healing compositions can be used for the following indications: a) Moietnri7ing and protecting; b) Healing dry cr~ç1~çcl skin; c) Treating irritated skin such as diaper rash;
d) We~ling severe dry skin due to other ~liiÇ~e~s (venous ~ rm~tltie); e) Treating psoriasis and other hyperprolirela~ive ~lieÇ~çs; f) Protecting skin from W lightdamage (antioxidant skin repl~ç~m~nt); g) Treating seborrheic c~n~litione; and h) Treating shaving wounds in an after shave lotion.

The topical therapeutic wound healing compositions may also be used orally in the form of a mouth wash or spray to protect and accelerate the healing of injured oral tissue such as mouth sores and burns. The topical therapeutic woundhealing compositions may further be used in ophth~lm- logical preparations such as eye care products to neutralize hydrogen peroxide used in the cleaning of contact lenses.
The topical therapeutic wound healing compositions may in addition be used in anorectal creams and suppositories to treat such contlitiQne as pruritus ani, proctitis, anal fissures, and hemorrhoids. Initially as white blood cells enter a wound site, the WO 96/37230 PCT/USgl ~Ol cells release oxygen r~-lic~lc, fitepleting the ~ntin~ ntc at the wound site, thus ~ p~;.;l.g the heal*ng process. Incorporating the wourld heal*ng compositions of the present invention into a wound heal*ng form~ tion would facilitate healing by providing the site with usable ~ntioxitl~ntc, and a source of fatty acids needed for ~G~ e repa*. The wound heal*ng compositions can be used for the following in-lic~tiQnc a) ~e~ling of cuts and scrapes; b) Burns (heals bums with less scaring and scabbing); c) Decubitus ulcers; d) Bed sores, ~1GS:jUre ulcers; e) Fissures, ~em~-rrhoi-lc;
f) Use in combination with immnn~shmlll~tQrc (cimlll~t~l healing in heal*ng deficient people); g) Post surgical wounds; h) R~n-l~ges; i) Diabetic ulcers; j) Venous lllcr~ti-~n;
and k) Use in combination with wound cle~ncing agents.

The therapeutic wound healing compositions may also be used in ingestible products to protect and increase the resuscitation rate of erosions, s~m~rh ulcers, and hrm- rrh~ges in the gastric mucosa. Other ingestible therapeutic products include: stroke medications; ~lo;~ e disease medications; arthritis medications;ulcer medications; cancer m-oAir~tiQne (cytotoxic agents); heart mr~lic~tion to *mprove regional ventricular function and restore normal heart rate and P1'GS~UIG functions; lung medication to repair injured tissue; liver m~lie~tion to ~u~)plCSS lipogenesis of alcoholic origin and prevent hepatic steatosis; kidney medication to suppress urinary calculi (kidney stones); ~letnxi~cation medication to antagonize heavy metal pnieoning, cyanide poisoning, sodium sulfide poisoning, other types of poieoning ; and reduce and neutralize the production of oxygen radicals which produces injury to tissue, to protect and further enhance the resllecit~h~n rate of the injured ,l~.l",~ n cells. The therapeutic wound healing compositions may be used in ingestible products to treat infl~.. ~tnly ~liee~ees such as hep~tit1e, g~etntie, colitis, esoph~gitie~ arthritis, and pancreatitis.

The therapeutic wound healing compositions of the present invention may also be used in tissue culture media and organ transplant media to prevent and reduce injury to ~ n cells and increase the resllecit~ti- n rate of injured n cells. Tissue cultures and transplant organs encounter reactive oxygen species generated in the culture media by the injured cells. Organs particularly WO 96137230 PCT/US95'~301 susceptible to oxidative ~mage during transport and transplantation due to repe~i~ n injury following i.echemi~ are corne~e, livers, hearts, and kidneys. The ~ .c~1tir.
wound healing compositions may be useful to abrogate reperfusion injury to such transplant organs.

In a specific embo~lim~ont, the invention is directed to a method for preserving ~ n cells in a culture ...P~ which comprises the steps of:
(A) providing a therapeutic wound healing composition selected from tbe group of con.~ in~ of:
a.A) (a) pyruvate selecte~l from the group concietin~ of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mi~Lules thereof;
(b) an antioxid~nt; and (c) a ll~Lwc; of s~Lu-~Lc;d and unsatul~led fatty acids wherein the 15fatty acids are those fatty acids required for the repair of cellular membranes and rçs-1ecit~tion of ~,..... ~1i~n cells;

a.B) (a) py-ruvate selectecl from the group coneietln~ of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and Il~i~lwes thereof;
20(b) lactate selected from the group con.cieting of lactic acid, ph~rm~ce11tically acceptable salts of lactic acid, and llfi~ s thereof; and (c) a ~ Lw~ of saturated and unsaLu-aled fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular rr.~ es and resuscitation of m~mm~ n cells;
(I.C) (a) an antioxidant; and (b) a llli~LWe of saturated and unsaturated fatty acids where*n the fatty acids are those fatty acids required for the repa* of cellular membranes and ~ resuscitation of ..... ;.. ~ n cells;
(I.D) (a) lactate se1ected from the group con~ietin~ of lactic acid, ph~ centically acceptable salts of lactic acid, and llf~Lulc;s thereof, WO 96/37230 PCT/US9C~OJ~Ol (b) an ~nhoxitl~nt and (c) a ~tu-ci of s~ f~1 and nn~ d fatty acids wherein the fatty acids are those fatty acids .c;quil~;d for the repair of cellular Illel~.~,es and rÇsllccit~tion of ~ n cells; and S (b) an antioxi~l~nt and (c) a ~u~Lul~ of s~hlr~trcl and unsalu ated fatty acids wherein the fatty acids are those fatty acids l~-luiled for the re~sllccit~t1l:?n of injured Ill.. "~ n cells, (B) providing m;1.. ~1i~n cells in a culh~re mrAitlm; and (C) cont~cting the therapeutic wound healing composition from step (A) with the ll~ n cells in the culhure mf~ lm from step (B).

D. Formulations Of The Therapeutic Wound Healing Com~o~ ns Of Embodiment One (I.A-D) Once pr~lfd, the inventive therapeutic wound healing compositions of Embodiment One (I.A-D) may be stored for fuhure use or may be f~rrn~ tf d in effective a-lloun~ with ph~rm~cellhc.~lly acceptable carriers to prepare a wide variety of ph~rm~r.elltlcal compositions. Examples of ph~ r,eutically acceptable carriers are pharm~cel-tical appliances, topical vehicles (non-oral and oral), and ingestible vehicles.

Examples of ph~ c.elltlr,~l appliances are sutures, staples, gauze, bandages, burn dreeeing.e, artificial skins, liposome or micell formulations, microcapsules, aqueous vehicles for so~king gauze dressings, and the like, and ll~.~Lul~;S
thereof. Non~ral topical compositions employ non-oral topical vehicles, such as creams, gels formulations, foams, o;..l...~ ; and sprays, salves, and films, which are inten-le-l to be applied to the skin or body cavity and are not intrn-led to be taken by ~ 30 mouth. Oral topical compositions employ oral vehicles, such as ~--~uLhwashes, rinses, oral sprays, suspensions, and dental gels, which are intt-nd~ to be taken by mouth but are not intto.n-led to be ingested. Ingestible compositions employ ingestible or partly , WO 96137230 PCT/u~ '0~301 ingestible vehicles such as collr~ ;u..Ary bulking agents which include hard and soft col-rG~Lionery such as lozenges, tablets, toffees, nougats, ~lspen~ic ne, chewy c~n~lie and chewing gums.

S In one form of the invention, the thc~a~ Lic wound healing composition is incol~ulaled into a ph~rm~cel1hc~1 appliance which may be in the form of sutures, staples, gauze, b~n-l~g~e, burn drçeeing~e~ artificia1 skins, liposome or micellffirmlll~horle, microcapsules, aqueous vehicles for so~kinp~ gauze dreseing~e~ and the like, and ll.iALulGs thereof. A variety of tr~lihon~l ingredients may optionally be included in the pharm~ce~1hc~l composition in effective amounts such as buffers,preservatives, tonicity adjusting agents, antioxidants, polymers for adjusting viscosity or for use as extenders, and excipients, and the like. Specific illustrative examples of such tr~liti~n~l ingredients include acetate and borate buffers; Lhilll.,.osal, sorbic acid, methyl and propyl paraben and chlorobutanol ~resGI vaLives; sodium chloride and sugars to adjust the tonicity; and excipients such as .. ~ l, lactose and sucrose. Other conventiQn~l ph~rm~ce~ltic~1 additives known to those having ordinary skill in the ph~rm~cel1tical arts may also be used in the ph~rrn~cellhcal composition.

In accordance with this invention, therapeutically effective amounts of the therapeutic wound healing composihone of the present invention may be employed in the pharmaceutical appliance. These amounts are readily determined by those skilled in the art without the need for undue ~A~cL;...~nt~hon The exact amount of the therapeutic wound healing composition employed is subject to such factors as the type and concentration of the therapeutic wound healing composition and the type of pharrn~ce~lhcal appliance employed. Thus, the amount of therapeutic wound healing composition may be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the need forundueexpGl;.. .~ t~;on Ina ~ rc;ll ~dembo~limPnt,theph~rm~ceuticalcompositionwill comprise the therapeutic wound healing composition in an amount from about 0;1% to about 5%, by weight of the ph~rm~ce~1hcal composition. In a more ~lerGllGd embodiment, the ph~rm~se1ltical composition will comprise the therapeutic wound healing composition in an amount from about 0.1% to about 3%, by weight of the -ph~rrn~r,alhr~l coll,~o~ilion. In a most ~ ,f~ Gd embo lim.ont, the ph~rm~r,ellhr,~l composition will compri~e the t~le.apc.llic wound healing composition in an amount from about 0.1% to about 1%, by weight of the ph~rrn~r,ellhc~l composition.

The present invention extends to m~th~ls for making the ph~rm~rellhr,~l c~mpositi-n~ In general, a ph~rm~rellhr~l cnmro~ih(n is made by c~?nt~r,hn~ a th~la~ ~ically effective amount of a th~a~ ic wound healing co~ o;,ilion with a ph~rrn~cellhr,~l appliance and the other ingredients of the final desired ph~rrn~ce~lhc~l composition. The therapeutic wound healing composition may be in a solvent and may be absorbed onto a ph~ ceuhcal appliance.

Other ingredients will usually be incol~u.~lGd into the composition as ~ t~tecl by the nature of the desired composition as well known by those having oldillaly skill in the art. The nlhm~te ph~rrn~rellhcal compositions are readilyprepared using methods generally known in the ph~rm~ce~lhcal arts.

In another form of the invention, the thel~Gulic wound healing composition is incol~ldlGd into a non-oral topical vehicle which may be in the form of a crearn, gel, foam, ointrn~nt, spray, and the like. Typical non-toxic non-oral topical vehicles known in the ph~rm~ceutical arts may be used in the present invention. The ef~lled non-oral topical vehicles are water and ph~r}n~ce~hc~lly acceptable water-miscible organic solvents such as ethyl alcohol, isopropyl alcohol, propylene glycol, glycerin, and the like, and ll~lul~,s of these solvents. Water-alcohol l~ lu es are particularly plc;rt;lled and are generally employed 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 10:1, respectively.

The non-oral topical therapeutic wound healing compositions may also contain convrntiQn~l additives employed in those products. Convt-ntion~l additives include hum~ct~nt~, emollients, lubricants, stabilizers, dyes, and ~elru~lles, providing the additives do not hl~ rclc with the therapeutic properties of the therapeutic wound healing composition.

-Suitable hllm~ct~ntc useful in the non-oral topical therapeutic wound healing compositions include glycerin, propylene glycol, polyethylene glycol, soll,i~l, fructose, and the like, and ~ Lul~s thereof. ~lTm~ct~nti, when employed, may be present in ~ll.~ ls from about 10% to about 20%, by weight of the topical therapeutic - 5 wound healing composition.

The coloring agents (colors, colorants) useful in the non-oral topical therapeutic wound healing composition are used in ~ lLs effective to produce thedesired color. These coloring agents include pigTnt-nt~ which may be incorporated in amounts up to about 6% by weight of the non-oral topical therapeutic wound healing composition. A prcrt;ll~d pigment, ~ ... dioxide, may be incorporated in a~loullt~
up to about 2%, and preferably less than about 1%, by weight of the non-oral topical therapeutic wound healing composition. The coloring agents may also include natural food colors and dyes suitable for food, drug and co~metic applic~tiQn~ These coloring agents are known as F.D.& C. dyes and lakes. The materials acceptable for the foregoing uses are preferably water-soluble. lllustrative nonlimi~ing examples include the indigoid dye known as F.D.& C. Blue No.2, which is the ~ o~lium salt of 5,5-indigotin(1i~ll1fonic acid. Similarly, the dye known as F.D.& C. Green No.l comprises a triphenylm~th~ne dye and is the monosodium salt of 4-[4-(N-ethyl-~-sulru~ e.~ylarnino) diphenylmethylene]-[l-(N-ethyl-N-P-sulrl~niumbenzyl)-delta-2,5-cyclohexadieneimine]. A full recitation of all F.D.& C. coloring agents and their corresponding chemical structures may be found in the Kirk-Othmer Encyclopedia of Chemical Technology, 3rd Fclition, in volume 5 at pages 857-884, which text is incorporated herein by reference.
In accordance with this invention, therapeutically effective amounts of the therapeutic wound healing compo~ition~ of the present invention may be ~lmixe~l with a non-oral topical vehicle to form a topical therapeutic wound healing composition. These amounts are readily ~let~rminecl by those skilled in the art without the need for undue experim~nt~tion In a ~rerc;llcd emboclim~nt~ the non-oral topical - therapeutic wound healing compositions will compri~e the therapeutic wound healing composition in an amount from about 0.1% to about 10% and a non-oral topical CA 02218~39 1997-10-17 WO 96137230 PCI'/US~ 5~01 vehicle in a quantity sufficient to bring the total amount of composition to 100%, by weight of the non-oral topical therapeutic wound healing composition. In a more plGr~Lled embodiment, the non-oral topical therapeutic wound healing compositions will comprise the therapeutic wound healing composition in an amount from about 0.1% to about 5%, and in a most ~-G~lGd emb~lim~-nt, the non-oral topical therapeutic wound healing compositions will compri.~e the thela~ lic wound healing composition in an amount from about 0.1% to about 2%, and a non-oral topical vehicle in a quantity sufficient to bring the total amount of composition to 100%, by weight of the non-oral topical therapeutic wound healing composition.
The present invention extends to m~thculs for preparing the non-oral topical therapeutic wound healing compositions. In such a method, the non-oral topical therapeutic wound healing composition is p~ ~Gd by ~Amixing a therapeutically errG-;LivG amount of the therapeutic wound healing composition of the present invention and a non-oral topical vehicle. The final compositions are readily prepared using standard methods and apparatus generally known by those skilled in the ph~rm~ceutic~l arts. The apparatus useful in accordance with the present invention compri~es mixing apparatus well known in the ph~ ceuhcal arts, and therefore the s~l~?c~on of thespecific apparatus will be apparent to the artisan.
In another form of the invention, the therapeutic wound healing composition is incorporated into an oral topical vehicle which may be in the form of a mouthwash, rinse, oral spray, suep~n~ion, dental gel, and the like. Typical non-toxic oral vehicles known in the ph~rm~celltical arts may be used in the present invention.
The plerGIl~d oral vehicles are water, eth~nol and water-ethanol llfi~lwGs. The water-ethanol l~ LwGs are generally employed in a weight ratio from about 1:1 to about20:1, preferably from about 3:1 to about 20:1, and most preferably from about 3:1 to about 10:1, respectively. The pH value of the oral vehicle is generally from about 4 to about 7, and preferably from about 5 to about 6.5. An oral topical vehicle having a pH value below about 4 is generally irrit~ting to the oral cavity and an oral vehicle having a pH value greater than about 7 generally results in an unpleasant mouth feel.

CA 02218~39 1997-10-17 WO 96137230 PCT/US96/115~Cl The oral topical the~peuhc wound healing composihon~ may also contain conventional additives noTm~lly employed in those products. Conv~nhl n~ladditives include a fluorine providing compound, a sweettoning agent, a nav-"..,g agent, a coloring agent~ a hllm~ct~nt, a buffer, and an .-nmll~ifier, providing the additives do S not in~.relc with the therapeutic p~ ,Llies of the thc.d~ Lic wound healing composition.

The coloring agents and l~ c~ , and the a~ unl~ of these additives to be employed, set out above as useful in the non-oral topical therapeutic wound healing composition may be used in the oral topical therapeutic wound healing compositlon.

Fluorine providing compounds may be fully or slightly water soluble and are characterized by their ability to release fluoride ions or fluoride co..l;~;..;..E~ ions in water and by their lack of reaction with other components in the composition. Typical fluorine providing compounds are inorganic fluoride salts such as water-soluble alkali metal, alkaline earth metal, and heavy metal salts, for example, sodium fluoride, pot~sillm fluoride, ~.. ,.. ;.. fluoride, cuprous fluoride, zinc fluoride, stannic fluoride, stannous fluoride, barium fluoride, sodium fluorosilicate, ~mmnnillm fluorosilicate, sodium fluorozirconate, sodium monofluorophosphate, ~h.. ~ .;.. i-- mono-and di-fluorophosphates and fluorinated sodium calcium pyrophosphate. Alkali metal fluorides, tin fluoride and monofluorophosph~t~s, such as sodium and stannous fluoride, sodium monofluorophosphate and ll~lul~s thereof, are ~rcrc.lcd.

The amount of fluorine providing compound present in the present oral topical therapeutic wound healing composition is dependent upon the type of fluorine providing compound employed, the solubility of the fluorine compound, and the nature of the final oral therapeutic wound healing composition. The amount of fluorine providing compound used must be a nontoxic amount. In general, the fluorine providing compound when used will be present in an amount up to about 1%, - preferably from about 0.001% to about 0.1%, and most preferably from about 0.001%

WO 96137230 PCI~/US9~1~5~01 to about 0.05%, by weight of the oral topical th~ ;c wound healing comFoeitinn When ~wcc~ g agents (~we~ ) are used, those ~weG~ rfi well known in the art, inc~ ing both natural and artificial swect~.,c.~, rnay be employed.
S The sweetening agent used may be select~ from a wide range of m~t.-ri~le inrln-ling water-soluble :j~eet~ -g agents, water-soluble artificial ~WCt;l~ .;..g agents, water-soluble sweG~ ;--g agents derived from naturally occurring water-soluble ~wecl~ -g agents, dipeptide based ~wecl~ .;..g agents, and protein based sweek-ning agents, including llP~A~ ,s thereof. Without being limited to particular ~ t~,..;..g agents, reprcsc.llalive calegolies and examples incl~lcle:
(a) water-soluble sweetening agents such as monosac-~h~ri-1~e, ~lie~cçh~rides and polysaccharides such as xylose, ribose, glucose (dextrose), m~nnose g~l~ctose, fructose (levulose), sucrose (sugar), m~ltl~se, invert sugar (a ~lulc of fructose and glucose derived from sucrose), partially hydrolyzed starch, corn syrup solids, dihydrochalcones, monellin, steviosides, and glycyrrhizin, and ~ LulcS thereof;
(b) water-soluble artificial sweet~nçre such as soluble s~ch~rin salts, i.e., sodium or calcium saccharin salts, cyclamate salts, the solillm, ~.. ;.. -- or calciurnsaltof3,4-dihydro-6-methyl-1,2,3-o~thi~7ine4-one-2,2-dioxide, thepotassium salt of 3,4-dihydro-6-methyl-1 ,2,3~x~thi~7ine-4-one-2,2-dioxide (A~,e-~lf~m~-K), the free acid form of saccharin, and the like;
(c) dipeptide based sweeteners, such as L-aspartic acid derived ~wc~Lellers, such as L-aspartyl-L-phenyl~l~nine methyl ester (A~L~ulle) and m~teri~le described in United States Patent No. 3,492,131, L-Alpha-aspartyl-N-(2,2,4,4-t~ 11.yl-3-thietanyl)-D-alanin-amide hydrate (Alitame), methyl esters of L-aspartyl-~phenylglycerineandL-aspartyl-L-2,5-dihydrophenyl-glycine, L-aspartyl-2,5-dihydro-L-phenyl~l~nine; L-aspartyl-L-(1-cyclr-hPYtsn)-alanine, and the like;
(d) water-so1uble sweeteners derived from naturally occurring water-soluble ~wecl.,llers, such as chlorinated dcliv~Livcs of ordinary sugar (sucrose), e.g., chlorodeoxysugar derivatives such as derivatives of chlorodeoxysucrose or chlorodeoxygalactosucrose, known, for example, under the product d~-eign~tion ofSucralose; examples of chlorodeoxysucrose and chlorodeoxygalacto-sucrose derivatives include but are not limited to: 1-chloro-1'-deoxysucrose; 4-chloro4-deoxy-Alpha-D-CA 02218~39 1997-10-17 galacto-pyranosyl-Alpha-D-f~u-;~r~.A~ e, or 4-chloro-4~eoxygalactosucrose; 4-chloro-4-deoxy-Alpha-D-galacto-pyranosyl-1-chloro-1-deoxy-J3-D-fructo-filr-Anosi~le or 4,1'-dichloro-4,1'-dideoxy~lArtosnrrose; 1',6'-dichloro-1',6'-dideoxysucrose; 4-chloro-4-deoxy-Alpha-D-galacto-pyranosyl-1,6-dichloro-1,6-dideoxy-13-D-fructo-filr~n~si~l~ or 4,1 ',6'-trichloro-4, 1 ',6'-trideoxygalacto-sucrose; 4,6-dichloro4,6-dideoxy-Alpha-D-galacto-pyranosyl-6-chloro-6-deoxy-~-D-fruck,ru.A.~osi-le, or 4,6,6'-trichloro-4,6,6'-trideoxyg~lAr,t~s~lcrose; 6,1 ',6'-trichloro-6, 1 ',6'-trideoxysucrose; 4,6-dichloro-4,6-dideoxy-Alpha-D-galacto-pyranosyl-1,6-dichloro-1,6-di~eoxy-~3-D-fru.;l~,rulA..osi~le or 4,6,1 ',6'-tetrachloro-4,6, 1 ',6'-tetradeoxygalacto-sucrose; and 4,6,1 ',6'-tetrachloro-4,6,1',6'-tetradeoxy-sucrose; and (e) protein based ~wGGlGIlers such as th~llmA-~ccous danielli CTh I and Il).

In general, an effective arnount of sweetening agent is utilized to provide the level of sweetness desired in the particular oral topical therapeutic wound healing composition, and this amount will vary with the sweetener selected and the final oral therapeutic product desired. The amount of :~wcc~ cl nonnAlly present is in the range from about 0.0025% to about 90%, by weight of the oral topical therapeutic woundhealing composition, depending upon the swtGlGller used. The exact range of a ~
for each type of sweetener is well known in the art and is not the subject of the present invention.

The flavoring agents (flavors, n~vu~ ) which may be used include those flavors known to the skilled artisan, such as natural and artificial flavors.
Suitable flavoring agents include mints, such as peppc.,llil.t, citrus flavors such as orange and lemon, artificial vanilla, cinnAmon, various fruit flavors, both individual and mixed, and the like.

~ The amount of flavoring agent employed in the oral topical therapeutic wound healing composition is normAlly a matter of plGr~l~,.lce subject to such factors as the type of final oral therapeutic wound healing composition, the individual flavor employed, and the strength of flavor desired. Thus, the amount of flavoring may be CA 02218~39 1997-10-17 WO 96137230 PCT/US9G/05, C 1 varied in order to obtain the result desired in the final product and such v~ri~ti~?ne are within the capabilities of those skilled in the art without the need for undue experim~nt~tion. The flavoring agents, when used, are g~-.n~.r~lly utilized in amounts that may, for example, range in ~ ~ from about 0.05% to about 6%, by weight of the oral topical therapeutic wound healing composition.

Suitable buffer solutions useful in the non-oral topical therapeutic wound healing compositions include citric acid-sodium citrate solntlc n, phosphoric acid-sodium phosphate solution, and acetic acid-sodium acetate solution in ~ll~)U~ up to about 1%, and preferably from about 0.05% to about 0.5% by weight of the oral topical therapeutic wound healing composition.

In accordance with this invention, therapeutically effective amounts of the therapeutic wound healing compositions of the present invention may be admixed with an oral topical vehicle to form a topical therapeutic wound healing composition.
These amounts are readily determined by those skilled in the art without the need for undue expclilll~ ion. In a pl~;rcllcd embo~iim~nt~ the oral topical therapeutic wound healing compositions will comprise the therapeutic wound healing composition in an amount from about 0.1% to about 10% and a oral topical vehicle in a quantity sufficient to bring the total amount of composition to 100%, by weight of the oral topical therapeutic wound healing composition. In a more plcr~llcd embo~im~nt, the oral topical therapeutic wound healing compositions will compri~e the therapeutic wound healing composition in an amount from about 0.1% to about 5%, and in a most ~lcr~.lGd embo-limt-nt, the oral topical therapeutic wound healing compositions will comprise the therapeutic wound healing composition in an amount from about 0.1%
to about 2%, and a oral topical vehicle in a quantity sufflcient to bring the total amount of composition to 100%, by weight of the oral topical therapeutic wound healing composition.

The present invention extends to methods for ~,-c~ ,g the oral topical therapeutic wound healing compositions. ~ such a method, the oral topical therapeutic wound healing composition is prepared by ~cimixing a therapeutically t;rrcclivc amount WO 96137230 PCT/US9' l~,01 of the thc ~uLic wound healing composition of the present invention and an oral topical vehicle. The final compositions are readily l,l~dl~d using standard methQ lc and a~ lus generally known by those skilled in the ph~ ce~ltical arts. The a~dldlus useful in accordance with the present invention comprises mixing ap~alalus well known in the ph~rm~cellhc~l arts, and therefore the sçlectir~n of the specific d~dl~lUS will be a~pd~ to the artisan.

In a ~l~r~,llcd embodiment, an oral topical therapeutic wound healing composition is made by first dissolving coloring agents, sweetening agents, and similar additives in water. The therapeutic wound healing composition is then admixed with the aqueous solution. Then sufficient water or ethanol, or l~ Lulcs of water andethanol, are added to the solution with mixing until the final solution volume is reached. In a more ~r~ cd embodiment, the therapeutic wound healing composition is added to the solution as the final ingredient. The final oral topical therapeutic wound healing compositions are readily ~repared using m~tho-lq generally known in the ph~rm~e~tical arts.

The oral therapeutic wound healing composition may also be in the form of dental gel. As used herein, the term "gel" means a solid or semisolid colloid which cont~ine considerable qll~ntih~c of water. The colloid particles in a gel are linked together in a coherent ,l,e~hw~,lk which immobilizes the water contained inside the eshw~,lk.

The dental gel compositions of the present invention may contain the conventional additives set out above for oral topical therapeutic wound healing compositions such as mouthwashes, rinses, oral sprays, and suspensions and, in ~-ltlition, may contain additional additives such as a polishing agent, a ~lesen.citi7ing agent, and the like, providing the ~ i~ on~l additives do not h~tclrclc with the~ therapeutic properties of the therapeutic wound healing composition.
In a dental gel composition, the oral vehicle generally comprises water, typically in an ~IlllOUllt from about 10% to about 90%, by weight of the dental gel CA 02218~39 1997-10-17 composition. Polyethylene glycol, propylene glycol, glycerin, and lJ.iA~ S thereof may also be present in the vehicle as hnm~ct~nti or binders in ~o~ from about 18% to about 30%, by weight of the dental gel composition. Particularly prcr~ d oral vehicles comprise 11~iALU1C;S of water with polyethylene glycol or water with glycerin S and polypropylene glycol.

The dental gels of the present invention include a gelling agent (thicl~.ning agent) such as a natural or synthetic gum or gelatin. Gelling agents such as h~dr~Ayct~lyl cellulose, methyl celllllose glycerin, carboxypolymethylene, and gelatin and the like, and 1 IA~II1CS thereof may be used. The plGrcllcd gelling agent is hydroxyethyl celhllose Gelling agents may be used in amounts from about 0.5%
to about 5%, and preferably from about 0.5% to about 2%, by weight of the dental gel composltlon.

lS The dental gel compositions of the present invention may also include a polishing agent. In clear gels, a pnli~hing agent of colloidal silica and/or alkali metal ~luminosilicate complexes is pl~re-l~,d since these m~teri~l~ have refractive indices close to the refractive indices of the gelling systems commnnly used in dental gels.
In non-clear gels, a polishing agent of calcium carbonate or calcium dihydrate may be used. These polishing agents may be used in ~ l~ up to about 75%, and preferablyin ~~ ~ up to about 50%, by weight of the dental gel composition.

The dental gel may also contain a ~l~sen~iti7ing agent such as a combination of citric acid and sodium citrate. Citric acid may be used in an amount from about 0.1% to about 3%, and preferably from about 0.2% to about 1%, by weight, and sodium citrate may be used in an amount from about 0.3% to about 9%, and preferably from about 0.6% to about 3%, by weight of the dental gel composition.
In accordance with this invention, therapeutically effective amounts of the therapeutic wound healing compositions of the present invention may be ~1mix~1 into the dental gel compositions. These amounts are readily (let~rmined by thoseskilled in the art without the need for undue CA~C~ ;...~nt~tion. In a plcrcllcd WO 96/37230 PCT/U~96/05~01 embodiment, the dental gel compositions will comprise the therapeutic wound healing composition in an amount from about 0.1% to about 10% and an oral topical vehicle in a quantity sufficient to bring the total amount of composition to 100%, by weight of the dental gel composition. In a more pl~,rcllc;d embodiment, the dental gel S compo~eihone will comprise the therapeutic wound healing composition in an amount from about 0.1% to about 5%, and in a most ~-~,rcllcd embo limt-nt, the dental gel compo,eitionc will compriee the therapeutic wound healing composition in an amount from about 0.1% to about 2%, and an oral topical vehicle in a quantity sufficient to bring the total amount of composition to 100%, by weight of the dental gel 1 0 composition.

The present invention extends to methods for pl~alillg the therapeutic dental gel compositions. In such a method, the dental gel composition is prepared by admixing a therapeutically effective amount of the thcl~c.lLic wound healing composition of the present invention and an oral topical vehicle. The final compositions are readily p~ cd using methods generally known by those skilled inthe dental and ph~rm~cellhcal arts. The apparatus useful in accordance with the present invention comprises mixing apparatus well known in the ph~rm~r~ellhc~l arts, and therefore the selection of the specific apparatus will be apparent to the artisan.
In a plercl~d emb~lim~nt~ a thcla~ Lic dental gel composition is made by first dispersing a gelling agent in a h~ c~ L or water, or a llli~tUlC of both, then admixing to the dispersion an aqueous solution of the water-soluble additives such as the fluorine providing c~ ~oulld, ~w~lcllers and the like, then adding the polishing agent, and lastly admixing the flavoring agent and the therapeutic wound healingcomposition. The final gel IlliX.~UlC iS then tubed or otherwise p~c~gecl The liquids and solids in a gel product are proportioned to form a creamy or gelled mass which is extrudable from a pressurized container or from a collapsible tube. The final~ therapeutic wound healing compoeihone are readily prepared using methods generally known in the pharm~celltical arts.

WO 96137230 PCT/US~)G~ )01 In yet another form of the invention, the thPr~pellhc wound healing composition is incorporated into an ingestible vehicle. The ingestible vehicle may be a collre,;l;onlory bulking agent in the form of 1O7~nge$, tablets, toffees, nougats, sllep~on~ ne, chewy c~nclies, chewing gums, and the like. The ph~rm~celltic~lly S acceptable carriers may be ~ d from a wide range of m~t~ri~le inc.~ ling, but not limited to, ~lih~ente~ binders and adhesives, l~bric~nte flieint~grants~ coloring agents, bulking agents, flavoring agents, ~w~ .;..g agents and miect~ neQus m~t~ri~le such as buffers and adsorbents that may be needed in order to prepare a particular therapeutic confection.
The prep~r~tion of confectionery forrmll~tione is hiet ~ric~lly well known and has changed little through the years. Confectionery items have been cl~eeified as either "hard" confectionery or "soft" confectionery. The therapeutic wound healing compositions of the present invention can be incorporated into confectionery compositions by ~-lmixing the inventive composition into conventional hard and soft confections.

As used herein, the term confectionery m~t~ri~l means a product co~ g a bulking agent selected from a wide variety of m~t~ri~le such as sugar, com syrup, and in the case of sugarless bulking agents, sugar alcohols such as sorbitol and ...;...t~i~c.l and ~ s thereof. Confection~ry m~t~-ri~l may include such exemplary substances as lozenges, tablets, toffee, nougat, suspensions, chewy candy, chewing gum and the like. The bulking agent is present in a quantity sufficient to bring the total amount of composition to 100%. In general, the bulking agent will be present in amounts up to about 99.98%, preferably in ~ll >un~ up to about 99.9%, and more preferably in amounts up to about 99%, by weight of the ingestible therapeutic wound healing composition.

T o7~nges are flavored medicated dosage forms inten~lecl to be sucked and held in the mouth. Lozenges may be in the form of various shapes such as flat, circular, octagonal and biconvex forms. The lozenge bases are generally in two forms:
hard boiled candy lozenges and cc.~ cssed tablet lozenges.

CA 02218~39 1997-10-17 WO 96137230 PCT/US96/05~01 Hard boiled candy lc 7~ng~s may be processed and fc-rm~ te~l by c~,.lve~l;on~l means. In general, a hard boiled candy lozenge has a base composed of a m.,.lule of sugar and other carbohydrate bulking agents kept in an amorphous or glassy con~libon. This amorphous or glassy form is con~ red a solid syrup of sugars S generally having from about 0.5% to about 1.5% moisture. Such m~tPri~le noTm~lly contain up to about 92% corn syrup, up to about 55% sugar and from about 0.1% toabout 5% water, by weight of the final composition. The syrup component is generally d from corn syrups high in fructose, but may include other materials. Further ingredients such as flavoring agents, ~wc;~-.i..g agents, ~ci-llll~nte, coloring agents and the like may also be added.

Boiled candy lozenges may also be prepared from non-f-rm~nt~hle sugars such as sorbitol, m~nnitol, and hydrogenated corn syrup. Typical hydrogenated com syrups are Lycasin, a co-ll-llelcially available product m~nnf~ctured by Roquette Corporation, and Hystar, a cul.. llelcially available product m~nllf~ctllred by Lonza, Inc.
The candy lozenges may contain up to about 95% sorbitol, a llf~Lul~i of sorbitol and m~nnitol in a ratio from about 9.S:O.S up to about 7.5:2.5, and hydrogenated corn syrup up to about 55%, by weight of the solid syrup component.

Boiled candy lozenges may be routinely prepared by conventional methods such as those involving fire cookers, vacuum cookers, and scraped-surface cookers also referred to as high speed atmospheric cookers.

Fire cookers involve the tr~-lition~l m~tho~l of making a boiled candy lozenge base. In this m~thofl, the desired quantity of carbohydrate bulking agent is dissolved in water by heating the agent in a kettle until the bulking agent dissolves.
Additional bulking agent may then be added and cooking continued until a final temperature 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 atmospheric cooker uses a heat-exchanger surface which involves spreading a film of candy on a heat eYc~h~nge sllrf~c~ the candy is heated to 165~C. to 170~C. in a few ...;..~lçs. The candy is then rapidly cooled to 100~C. to WO 96t37230 PCTtUS!~ 5~01 120~C. and worked as a plastic-like mass enabling in~l~o.~ion of the additives, such as flavors, colorants and the like.

In vacuum cookers, the carboLy~le buL~Icing agent is boiled to 125~C
S to 132~C., vacuum is applied and ad~lihnn~l water is boiled offwithout exha h~hng When cooking is comrl~te the mass is a semi-solid and has a plastic-like conii.ct~nt~.y.
At this point, flavors, colorants, and other additives are ~timixecl in the mass by routine m~cl~nic~l mixing operations.

The ~ mixing required to ~.. ;r~.. 1y mix the flavoring agents, coloring agents and other additives during cu-lv~ n~l m~mlf~ctllring of boiled candy lozenges is ~1et~rmined by the time needed to obtain a ~ irc,..,. dishibution of the m~t~ri~lc Normally, mixing times of from 4 to 10 ...;....l~s have been found to be acceptable.
Once the boiled candy lozenge has been properly tempered, it may be cut into workable portions or formed into desired shapes. A variety of fcrming techniques may be utilized depending upon the shape and size of the final product desired. A general ~liccllcsion of the composition and p~ l ;on of hard confections may be found in H.A. Lieberman, ph~rm~ceutical Dosa~e Forms: Tablets, Volume I
(1980), Marcel Dekker, Inc., New York, N.Y. at pages 339 to 469, which flicclosllre is incorporated herein by reference.

The apparatus useful in accordance with the present invention comprises cooking and mixing apparatus well known in the confectionery m~mlf~ctllring arts, and therefore the selection of the specific apparatus will be ap~,..t to the artisan.

In contrast, compressed tablet confections contain particulate materials and are formed into structures under pressure. These confections generally contain sugars in an.ou,.L~ up to about 95~/O, by weight of the composition, and typical tablet excipients such as binders and lubricants as well as flavoring agents, coloring agents and the like.

In ~d-1ihon to hard confect-on~y m~t~li, the lozenges of the present invention may be made of soft cc,llrecl;cn~y m~t.-ri~l~ such as those cont~inçcl in nougat. The pl~ alion of soft confection~ such as nougat, involves collv~
mtotho~l~ such as the combination of t.,vo primary components, namely (1) a highboiling syrup such as a corn syrup, hydrogenated starch hydrolysate or the like, and (2) a relatively light l~ALulcd frappe, generally ~ d from egg albumin, gelatin, vegetable proteins, such as soy derived ~l~ ullds, sugarless milk derived compounds such as milk proteins, and 1 IAlUlCS thereof. The frappe is generally relatively light, and may, for example, range in density from about 0.5 to about 0.7 grams/cc.
The high boiling syrup, or "bob syrup" of the soft confectionery is relatively viscous and has a higher density than the frappe component, and frequently cont~in~ a substantial amount of carbohydrate bulking agent such as a hydrogenated starch hydrolysate. Conv~ntion~lly~ the final nougat composition is ~lc~al'cd by the addition of the "bob syrup" to the frappe under agitation, to form the basic nougat iAtulc. Further ingredients such as nav~ g agents, additional carbohydrate bulking agent, coloring agents, preservatives, medic....~ ..l,~, llliAlUICs thereof and the like may be added thcl~ancl also under ~git~tion A general discussion of the composition and preparation of nougat confections may be found in B.W. Minifie, Chocolate, Cocoa and ConfectionerY: Science and Technolos~y, 2nd edition, AVI Publishing Co., Inc., Westport, Conn. (1980), at pages 424-425, which disclosure is incorporated herein by reference.

The procedure for pl~illg the soft confectionery involves known procedures. In general, the frappe component is pr~alcd first and thereafter the syrup component is slowly added under ~git~tion at a temperature of at least about 65~C., and preferably at least about 100~C. The llliALulc of components is continl1ecl to be mixed to form a ullirullll llliAlUlC, after which the llliAlulc is cooled to a temperature below 80~C., at which point, the flavoring agent may be added. The llliAIu~e is further mixed for an additional period until it is ready to be removed and formed into suitable confectionery shapes.

WO 96/37230 PCT/US9~ '~5~Cl The ingeehble thcla~ Lic wound healing compositions may also be in the form of a ph~rm~ellti~ ~l suspension. ph~rm~ce~ltical susp~ncione of this invention may be p~ d by cc llv~ .l ;nn~l m~thotle long established in the art of ph~rm~celltic:ll compounding. Suspensions may contain adjunct m~teri~le employed in f~ rmlll~ting the S susp~ncione of the art. The suspensions of the present invention can comrnee (a) preservatives such as butylated hydroxyanisole (BHA), butylated hydl~)xytoluene (BHT), benzoic acid, ascorbic acid, methyl paraben, propyl paraben, tocopherols, and the like, and ..~,~Lules thereof. Pl~,s~..vaLivts are generally present in ~ ul~ up to about 1%, and preferably from about 0.05% to about 0.5%, by weight of the suspension;
(b) buffers such as citric acid-sodium citrate, phosphoric acid-sodium pho~eph~te, and acetic acid-sodium acetate in amounts up to about 1%, and preferably from about 0.05% to about 0.5%, by weight of the suspension;
(c) sllep~on~ling agents or thickeners such as c~lhllocice like methylcellulose, carrageenans like alginic acid and its derivatives, ~nth~n gums, gelatin, acacias, and microcrystalline cellulose in ;~"ou~L~ up to about 20%, and preferably from about 1% to about 15%, by weight of the suspension;
(d) antifoaming agents such as dimethyl polysiloxane in amounts up to about 0.2%, and preferably from about 0.01% to about 0.1%, by weight of the susp~n.eion;
(e) sweetening agents such as those sweeteners well known in the art, including both natural and artificial :,wciGLc;ners. Sweet~ning agents such as m- nos~c- h~rides, ~iie~cl~h~rides and polysaccharides such as xylose, ribose, glucose (dextrose), mannosej galactose, fructose (levulose), sucrose (sugar), maltose, invert sugar (a Illi~LLulc~ of fructose and glucose derived from sucrose), partially hydrolyzed starch, com syrup solids, dihydroch~lcones., monellin, steviosides, glycyrrhizin, and sugar alcohols such as sorbitol, ~ l, maltitol, hydrogenated starch hydrolysatesand I~ Lul~;s thereof may be utilized in ~m~-nntC. up to about 60%, and preferably from about 20% to about 50%, by weight of the suspension. Water-soluble artificial ~w~eL~.. ers such as soluble saccharin salts, i.e., sodium or c~lcium s~crh~rin salts, cyclamate salts, the so-linm, ~mm~ ninm or c~lcillm salt of 3,4-dihydro-6-methyl-1,2,3-oY~thi~7ine~-one-2,2-dioxide, the pot~C~cillm salt of 3,4-dihydro-6-methyl-1,2,3-CA 02218~39 1997-10-17 WO 96/37230 PCI~/USS~'0~301 ~Y .Ih; .,;..e 1-one-2,2-dioxide (~cesll1f~m~-K), the free acid form of saccharin, and the like may be utilized in ~LIllUllllki from about 0.00l% to about 5%, by weight of the SllCp~.nc~ion;
(f) flavoring agents such as those flavors well known to the skilled artisan, such as natural and artificial flavors and mints, such as peppermint, menthol, citrus flavors such as orange and lemon, artificial vanilla, cinn~m-n, various fruit flavors, both individual and mixed and the like may be utilized in ~lUUllt~ from about 0.5% to about 5%, by weight of the suspension;
(g) coloring agents such as pigm-onti which may be incorporated in ~wullls up to about 6%, by weight of the suspension. A p~er~llcd pigment, !;1; --;.--.l dioxide, may be incol~olated in alllUIIll~ Up to about 2%, and preferably less than about l %, by weight of the suspension. The coloring agents may also include natural food colors and dyes suitable for food, dIug and cosme~ic applications. These colorants are known as F.D.& C. dyes and lakes. The materials acceptable for the rul~gOillg 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 suspension;
(h) decolorizing agents such as sodium metabisulfite, ascorbic acid and the like may be incorporated into the suspension to prevent color çh~nges due to aging.
In general, decolorizing agents may be used in ~UllOUIl~ up to about 0.25%, and preferably from about 0.05% to about 0.2%, by weight of the suspension; and (i) solubilizers such as alcohol, propylene glycol, polyethylene glycol, and the like may be used to solubilize the flavoring agents. In general, solubilizing agents may be used in ~llOUIl~ up to about 10%, and preferably from about 2% to about 5%, by weight of the suspension.

The pharmaceutical suspensions of the present invention may be pl.~ d as follows:
(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 ~iic-rtorcion if the thiçlrener is not water soluble or a solution if the thiel~ner is water soluble;(B) admix the sweetening agent with water to form a solution;

WO 96/37230 PCT~/US~G~5~01 (C) admix the lh~pe~tic wound healing c~l.os;~ n with the thir~en~r-water ~ c to form a .I.. ;r.. thir~n~-t~le~ Lic wound healing cc~mrosib~n;
(D) combine the ~ ,t~"c- solllb~n with the th~ n~-th~la~c~lLic S wound healing composition and mix until ~--.;r~ , and (E) admix the optional adjunct m~t~r~ such as colc)ring agents, flavoring agents, decolorants, solubilizers, ~lLir~Ja, ll.g agents, buffers and ~ ition~l water with the ~lule of step (D) to form the suspension.

The ingestible therapeutic wound healing compositions of this invention may also be in chewable form. To achieve acceptable stability and quality as well as good taste and mouth feel in a chewable forml~l~bon several cnncicl~bons are important. These considerations include the amount of active substance per tablet, the flavoring agent employed, the degree of complcssibility of the tablet and the organoleptic properties of the composition.

Chewable therapeutic candy is prepared by procedures similar to those used to make soft confectionery. In a typical procedure, a boiled sugar-com syrup blend is formed to which is added a frappe If~lule. The boiled sugar-corn syrup blend may be prepared from sugar and corn syrup blended in parts by weight ratio of about 90:10 to about 10:90. The sugar-corn syrup blend is heated to temp~lalu,cis above about 120~C. to remove water and to form a molten mass. The frappe is generally prepared 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 ~ c.a~ to form an aerated sponge like rnass. The frappeis then added to the molten candy mass and mixed until homogeneous at temperatures between about 65~C. and about 120~C.

The ingestible therapeutic wound healing composition of the instant invention can then be added to the homogeneous Il~Lule as the temperature is lowered to about 65~C.-95~C. whereupon additional ingredients can then be added such as WO 96/37t30 PCT/US~ 301 nav~ g agents and coloring agents. The fi~rm~ hon is filrther cooled and formed into pieces of desired ~lim~n~ion~

A general discussion of the lozenge and chewable tablet forms of S confection~ry may be found in H.A. Lie~,.~- and L. T ~rhm~n, Ph~rm~ceuticalDosa~e Forrns: Tablets Volume 1~ Marcel Dekker, Inc., New York, N.Y. at pages 289 to 466, which tli~closllre is inco~ alcd herein by reference.

In accordance with this invention, th~r~pelltic~lly effective ~ UUIII~ of the therapeutic wound healing composit on~ of the present invention rnay be ~(lmixe~l into the hard and soft confectionery products. These ~ou~ are readily ~lct~-...;..ecl by those skilled in the art without the need for undue CA~JC~ t~ti~n In a plcrcllcd embo lim~nt, the ingestible therapeutic wound healing composition will comprise the therapeutic wound healing composition in an arnount from about 0.1% to about 10%and an ingestible vehicle, that is a ph~rm~ceutically acceptable carrier, in a quantity sufficient to bring the total amount of composition to 100%, by weight the ingestible therapeutic wound healing composition. In a more prcr~.lcd embo lim~nt~ the ingestible composition will comprise the therapeutic wound healing composition in an arnount from about 0.1% to about 5%, and in a most prcr~ d embolim~nt, the ingestible composition will comprise the therapeutic wound healing composition in an amount from about 0.1% to about 2%, and an ingestible vehicle in a quantity sufficient to bring the total amount of composition to 100%, by weight the ingestible therapeutic wound healing composition.

The present invention extends to m.-tho-ls of m~king the ingestible therapeutic wound healing compositions. In such methods, an ingestible therapeutic wound healing composition is pl~,~ducd by ~-lmixing a therapeutically effective amount of the therapeutic wound healing composition with a ph~rm~ce~ltically-acceptablecarrier. The ~pdldlus useful in accordance with the present invention comprises mixing and heating apparatus well known in the conrcclionery arts, and thc~cr~lci the selection of the specific d~pdldlUS will be dp~a~ to the artisan. The final ingestible WO 96137230 PCI~ 96J'~,J~Ol therapeutic wound healing compositions are readily prepared using mlo.tho l.~ general1y known in the co"~ilioneTy arts.

The therapeutic wound healing compositions may also be incorporated into chewing gums. In this form of the invention, the chewing gum composition cont~inc a gum base, a buL~cing agent, dhe illve.lliv~ therapeutic wound healingcomposition, and various additives.

The gum base employed will vary gready depending upon various factors such as the type of base desired, the coneict~ ncy of gum desired and the other components used in the composition to make the final chewing gum product. The gum base may be any water-insoluble gum base known in the art, and includes those gum bases utilized for chewing gums and bubble gums. Illustrative examples of suitable polymers in gum bases include both natural and synthetic elastomers and rubbers. For example, those polymers which are suitable as gum bases include, without limit~ho~, substances of vegetable origin such as chicle, crown gum, nispero, ros~,dinh~ jelntong, perillo, niger gutta, tunu, balata, gutta-percha, lechi-capsi, sorva, gutta kay, ~Lules thereof and the like. Synthetic Çl~ctnm~r~c such as bllt~rliene-styrene copolymers, polyisobutylene, isobutylene-isoprene copolymers, polyethylene, ll~ ulcs thereof and the like are particularly useful.

The gum base may include a non-toxic vinyl polymer, such as polyvinyl acetate and its partial hydrolysate, polyvinyl alcohol, and mi~ les thereof. When nhli7ecl~ the molecular weight of the vinyl polymer may range from about 2,000 up to and including about 94,000.

The amount of gum base employed will vary greatly depending upon various factors such as the type of base used, the consistency of the gum desired and the other components used in the composition to make the final chewing gum product.
In general, the gum base will be present in ~ b from about 5% to about 94%, by weight of the final chewing gum cc,ll,~osilion, and preferably in ~ ulll~ from about 15% to about 45%, and more preferably in amounts from about 15% to about 35~/O, WO 96t37230 PCTtUS9''~5301 and most preferably in ;~ from about 20% to about 30%, by weight of the final chewing gum colll~o~ilion.

The gum base composition may contain cul~ ;on~ tom~r solvents to aid in son~,.. i,.g the el~tom~r base cu ~ullent. Such els~tomer solvents may cu~ .ise terpinene resins such as polymers of Alpha-pinene or l~-pinene, methyl,glycerol or pentaery~ritol esters of rosins or m~ifiecl rosins and gums, such ashydrogenated, ~l;"-~,;,ed or polym~n7.~1 rosins or llliALulGs thereof. Examples of tomt~r solvents suitable for use herein include the pentaerythritol ester of partially hydrogenated wood or gum rosin, the pentaerythritol ester of wood or gum rosin, the glycerol ester of wood rosin, the glycerol ester of partially LllGli~;Gd wood or gum rosin, the glycerol ester of pol~,llG-i~Gd wood or gum rosin, the glycerol ester of tall oil rosin, the glycerol ester of wood or gum rosin and the partially hydrogenated wood or gum rosin and the partially hydrogenated methyl ester of wood or rosin, llliALulGs thereof, and the like. The elastomer 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 plastici_ers or softeners such as lanolin, palmitic acid, oleic acid, stearic acid, sodium stearate, potassium stearate, glyceryl tri~cet~te~ glyceryl lecithin, glyceryl monostearate, propylene glycol monostearate, acetylated monoglyceride, glycerine, IlliALulGs thereof, and the like may also be incorporated into the gum base to obtain a variety of desirable ICALU1GS and con~i~t~ncy pl-~c.Lies.
Waxes, for example, natural and synthetic waxes, hydrogenated vegetable oils, petroleum waxes such as polyurethane waxes, polyethylene waxes, paraffin waxes, microcrystalline waxes, fatty waxes, sorbitan monoste~rate, tallow, propylene glycol, llf.~Lu.Gs thereof, and the like may also be incorporated into the gum base to obtain a variety of desirable textures and c~>nii~t~ncy properties. These traditional additional materials are generally employed in ~l~ullL~ Up to about 30%, by weight of the gum base, and preferably in ~ UllL~ from about 3% to about 20%, by weight of the gumbase.

W096137230 PCT/US35/O~;~nl The gum base may include e~ ivci i........ ,l!~ of mineral adjuv~ such as c~lcillm carbonate, m~neeinm c~ul,olla~ min~ ~1.. ;.. " hydroxide, ~1,.,.. ;
silicate, talc, tric~lcillm phosFh~te~ ~iie~lr,jllm phosphate and the like as well as .~u.~s thereof. These mineral adjuv~ul~ may serve as fillers and textural agents.
These fillers or adjuvall~ may be used in the gum base in various ~uu,~. Preferably the amount of filler when used will be present in an amount up to about 60%, by weight of the chew-ving gum base.

The chewing gum base may ~ ition~lly include the c.,nv~ l ;on~l additives of coloring agents, ~ntioxi-i~nt~, prcse.valives and the like. For example, .,;"", dioxide and other dyes suitable for food, drug and cosm~h'c applic~ n~
knowvn as F.D. & C. dyes, may be ntlli7e~1 An ~ntic xi-l~nt such as butylated hydroxytoluene (BH~), butylated hydroxyanisole (BHA), propyl gallate, and llf,~lul~,s thereof, may also be inc11l~1e~1 Other con~,...l;--n~l chewing gum additives known to one having ordinary skill in the chewing gum art may also be used in the chevwing gum base.

The gum composition may include effective alllUul~ of collv~l;c)n~l additives selected from the group con~;~ting of sweetenin~ agents (sweeteners), plasticizers, softeners, ~-mlll~ifiers, waxes, fillers, bulking agents, mineral adjuva~
flavoring agents (flavors, flavorings), coloring agents (colorants, colorings), antioxidants, ~ci~llll~nti, thic~eners, I~ ul~,S thereof and the like. Some of these additives may serve more than one purpose. For example, in sugarless gum compositions, the sweetener, e.g., sorbitol or other sugar alcohol or ml~lul~s thereof, may also function as a bulking agent. ~imil~rly, in sugar co~ --;--g gum compositions, the sugar sweetener can also function as a bulking agent.

The plasticizers, softeners, mineral adjuv~ts, colorants, waxes and antioxidants discussed above as being suitable for use in the gum base may also be used in the gum composition. Examples of other collv~nlional additives which maybe used include t-mnl~ifiers, such as lecithin and glyceryl monostearate, thickeners, used alone or in combination with other softeners, such as methyl celllllose7 ~lEin~te~

WO 96/37230 PCT/US96J'~59Cl carrageenan, x~nth~n gum, gelatin, carob, tr~g~r~nth, locust bean, and calb~-y methyl c~llnlose, ~ci~ nt.c such as malic acid, adipic acid, citric acid, tartaric acid, fumaric acid, and ~ es thereof, and fillers, such as those ~ cll~se~l above under the C~ltg~3ly of mineral adjuvants. The fillers when used may be utilized in an arnount up S to about 60%, by weight of the gum composition.

Bulking agents (carriers, e~ten~l~qrs) suitable for use in chewing gums include ~wed~ g agents selected from the group con~i~ting of m--nos~r,rh~rides, disaccharides, poly-saccharides, sugar alcohols, and llfi~ s thereof; polydextrose;
maltodextrins; minerals, such as r~lcillm carbonate, talc, ~ llll tliox~ ir~ cillm phosphate, and the like. Bulking agents may be used in ~IOU~l~ up to about 90%, by weight of the final gurn composition, with ~ UIlll~ from about 40% to about 70%, by weight of the gurn composition being ~l~r~ d, with from about 50% to about 65%, by weight, being more plercllcd and from about 55% to about 60%, by weight of the chewing gum composition, being most ~r~;r~ ,d.

The ~weel~-;--g agent used may be selected from a wide range of materials including water-soluble ~wec;~ ers, water-soluble artificial sweeteners, water-soluble sweeteners derived from n~tllr~lly occurring water-soluble ~w~;d~.lers, dipeptide based sweeteners, and protein based sweeteners, inchl<ling llli~ s thereof. Without being limited to particular :,wec;L~.lers, lepres.,.l~live categories and examples include:
(a) water-soluble ~wec~ agents such as monos~cch~rides, disaccharides and polysaccharides such as xylose, ribulose, glucose (dextrose), mannose, galactose, fructose (levulose), sucrose (sugar), m~ltose~ invert sugar (a I. i~ e of fructose and glucose derived from sucrose), partially hydrolyzed starch, corn syrup solids, dihydrochalcones, monellin, steviosides, glycyrrhizin, and sugar alcohols such as sorbitol, Illallllilol, maltitol, hydrogenated starch hydrolysates and Irli~ s thereof;
(b) water-soluble artificial sweeteners such as soluble saccharin salts, i.e., sodium or calcium saccharin salts, cycl~le salts, the sodium, ~.1111~011;11111 or calcium saltof3,4-dihydro-6-methyl-1 ,2,3-ox~thi~ine-4-one-2,2~1ioxi-1e, thepot~ m CA 02218~39 1997-10-17 WO 96/37230 PCT/US96/0~,01 salt of 3~4-dihydro-6-methy~ 2~3-ox~th~ ne4-one-2~2-dioxide (~cçsnlf~m~q-K), thefree acid form of saccharin, and the like;
(c) dipeptide based ~wcGh,.lers, such as L-aspartic acid derived ~wcc~ rc, such as L-aspartyl-L-phenyl~l~nine methyl ester (A:~pal~l~e) and m~tr.ris~lc S described in United States Patent No. 3,492,131, L-Alpha-aspartyl-N-(2,2,4,4-tcL~ cLllyl-3-thietanyl)-D-alanin-amide hydrate (Alitame), methyl esters of L-aspartyl-L-phenylglycerineandL-aspartyl-L-2,5-dihydrophenyl-glycine, L-aspartyl-2,5-dihydro-L-phenylalanine; L-aspartyl-L-(l-cycl~hexen)-~l~nine7 and the like;
(d) water-soluble ~wcGLcllers derived from naturally occllrring water-l 0 soluble sweeteners, such as chlorinated derivatives of ordinary sugar (sucrose), known, for example, under the product dee~ tion of Snrr~lQse~ and (e) protein based sweetrners such as th~llm~-~ccous danielli (Th I and 1[).

In general, an effective amount of ~wccl~,ner is utilized to provide the level of bulk and/or sweetness desired, and this amount will vary with the sweetener selecte-l This amount of sweetener will norm~lly be present in ~ll~UIIt~ from about 0.0025% to about 90%, by weight of the gum composition, depending upon the sweetener used. The exact range of allloullL~ for each type of ~wecLc~er is well 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 independent from the flavor level achieved from flavor oils.

Plcrcllcd sugar based-sweeteners are sugar (sucrose), com syrup and llli~Lulcs thereof. Preferred sugarless sweeteners are the sugar alcohols, artificial sweeteners, dipeptide based ~wccLc~ers and mixtures thereof. Preferably, sugar alcohols are used in the sllg~rlecc compositions because these sweeteners can be used in amounts which are sufflcient to provide bulk as well as the desired level of sweetnecc Plcrcllcd sugar alcohols are selected from the group concisting of sorbitol, xylitol, rnaltitol, m~nnitrll, andl~ ulcs thereof. More preferably, sorbitol or a lLI~Lulc of sorbitol and ~ n~ )l is lltili7e-1 The gamma form of sorbitol is plcrcllcd. An CA 02218~39 1997-10-17 artificial sweetener or dipeptide based ~weG~GIler is preferably added to the gum composihon~ which contain sugar alcohols.

The coloring agents useful in the gum composi1~on~ are used in ~lluunl~
S crrG~i~ive to produce the desired color. These coloring agents include pigm~nt~ which may be incûrpûrated in ~luull~ up to about 6% by weight of the gum composition.
A ~lGr~llGd pigment, ~ ioxi~le, may be inco,~o.ah~d in a,l,uu,,l~ up to about 2%, and preferably less than about 1% by weight of the composition. The colorants may also include natural food colors and dyes suitable for food, drug and cosmetic applications. These colorants are known as F.D.& C. dyes and lakes. The materials acceptable for the foregoing uses are preferably water-soluble. Illustrative nonlimiting examples include the indigoid dye known as F.D.& C. Blue No.2, which is the o lillm salt of 5,5-indigohntli~lllfonic acid. Similarly, the dye known as F.D.& C.
Green No. 1 comprises a triphenylmethane dye and is the monosodium salt of 4-[4-(N-ethyl-p-sulfoniu",l~"~;ylamino'diphenylmethylene]-[l-(N-ethyl-N-p-sulro~ benzyl)-delta-2,5-cyclohexadieneimine]. A full recitation of all F.D.& C. colorants and their corresponding chemical structures rnay be found in the Kirk-Othmer Encyclopedia of Chemical Technolo~y, 3rd F~ihon, in volume 5 at pages 857-884, which text is incorporated herein by reference.
Suitable oils and fats usable in gum compositions include partially hydrogenated vegetable or animal fats, such as coconut oil, palm kernel oil, beef tallow, lard, and the like. These ingredients when used are generally present in~llOU~I~ up to about 7%, by weight, and preferably up to about 3.5%, by weight of the gum composition.

In accordance with this invention, therapeutically effective a~l~oL~ of the therapeutic wound healing compositions of the present invention may be admixed into a chewing gum. These ~llOull~ are readily determined by those skilled in the art without the need for undue GA~efi.. ~ l;on In a prer~"~;d emboflim~-nt, the final chewing gum composition will comprise the therapeutic wound healing composition in an amount from about 0.1% to about 10% and a chewing gum composition in a WO 96137230 PCT/USr.~ 01 quantity sufficient to bring the total amount of cc,lll~G~ilion to 100%, by weight of the chewing gum composition. In a more ~lGrtllGd embotlimpnt~ the final chewing gum composition will cQmrrice the the~f.c..l;c wound healing composition in an amount from about 0.1% to about 5%, and in a most ~lGrGlltd embo-lim~nt, the final chewing S gum composition will c~-mrriee the th~relltic wound healing composition in an amount from about 0.1% to about 2%, and a chewing gum composition in a quantity sufficient to bring the total amount of composition to 100%, by weight of the chewing gum composition.

The present invention extends to methols of m~king the therapeutic chewing gum compositions. The therapeutic wound healing compositions may be incorporated into an otherwise c~llve~ on~l chewing gum composition using standard techniques and eqllirmt~nt known to those skilled in the art. The apparatus useful in accordance with the present invention comrri~ees mixing and heating a~)~JaldlUS well known in the chewing gum m~nnf~ctu~ing arts, and therefore the se1ection of the specific a~palaLus will be apparent to the artisan.

For example, a gum base is heated to a ~Glll~L,.d~UlC sufficiently high enough to soften the base without adversely effecting the physical and chemic~l make up of the base. The o~ llulll temperatures utilized rnay vary depending upon thecomposition of the gum base used, but such tG~ cralu,cs are readily cletermined by those skilled in the art without undue cA~ ;orl.

The gum base is conven~ion~lly melted at Iclll~l_.alulcs that range from about 60~C. to about 120~C~. for a period of time sllfficien~ to render the base molten.
For example, the gum base may be heated under these con~itic-nc for a period of about thirty Ill;llllles just prior to being A~imixe~A inc,~ ly with the r~---A;-~ g ingredients of the base such as the plasticizer, fillers, the bulking agent and/or sweeteners, the softener and coloring agents to plAetici7e the blend as well as to modulate the hardness, viscoelasticity and formability of the base. The chewing gum base is then blended with the therapeutic wound healing composition of the present invention which may have been previously blended with other tr~li~icmAl ingredients. Mixing is continued =

WO 96t37230 PCTtUS35'0~01 until a irl, , ~LI~le of gum composition is obtained. TLc~ca~l the gum composition l.f.,.~ may be formed into desirable chewing gum shapes.

In a specific embo lim~-nt the invention is directed to a ther~p~ tic 5ph~rm~celltic~l composition for ~L~ e.,LI-g and redllcing injury to ,~ n cells,and increasing the r~enecit~tion rate of injured ~ ..l"l~ n cells, which CQmrrieçs (A) a therapeutically ~ ;Live amount of a therapeutic wound healing c~ o~ilion of Embodiment One (I) selecteA from the group cQn.eieting of 10(I.A) (a) pyruvate selected from the group con~ ng of pyruvic acid, ph~ ce~ltically acceptable salts of pyruvic acid, and I~ Lul~s thereof;
(b) an antioxidant; and (c) a Illi~lu-e of sdLulatcd and unsdlu~dl~d fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resll~çit~tiQn of ,~".. "",~ n cells;

(I.B) (a) pyruvate selected from the group con~i~ting of pyruvic acid, ph~rm~cel_tically acceptable salts of pyruvic acid, and l- i~Lu-~s thereof;
(b) lactate selectecl from the group cQn~i~ting of lactic acid, 20ph~rm~celltically acceptable salts of lactic acid, and l~ ,s thereof; and (c) a llfi~ of sdluldlcd and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular Ilwl~ es and resuscitation of ~ - -,. . "- - .~ n cells;

25(I.C) (a) an ~ntioxi-l~nt; and (b) a Illi~Lule of saturated and lmc~ t~d fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of l~"..,l,.l~ n cells;

30(I.D) (a) lactate selecte~l from the group co~ ting of lactic acid, ph~rm~celltic~lly acceptable salts of lactic acid, and I~Lules thereof;
(b) an ~ntioxi~l~nt and -(c) a llfi~Lulc of 5~ and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular lllcl~ es and reenscit~hon of ,,.~.,,,,.~li~n cells, and (B) a pharm~centically acceptable carrier.
s The ph~rm~celltir~lly acceptable carrier may be s~lectecl from the group c~n~ ng of ph~rm~c.elltical appliances, topical vehiclesl and ingestible vehicle.

In another specific embo lim~.nt, the invention is directed to a method for preparing a therapeutic ph~rm~ce~ltlc~l composition for ~lGvcllLillg and re~ ing injury to ~ n cells, and increasing the r~e ~ecit~t~on rate of injured ..li..l",.~ n cells, which comprises the steps of:
(A) providing a therapeutically effective amount of a therapeutic wound healing composition of Embodiment One (I.A-D) sel~cte~l from the group coneictlng of:
(I.A) (a) pyruvate selecte~1 from the group coneieting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and ~ Lul~S thereof;
(b) an antioxidant; and (c) a llli~LlllG of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular lllGllll~l~les and resuscitation of ",i."""~ n cells;

(I.B) (a) pyruvate selected from the group consisting of pyruvic acid, pharm~ce -tically acceptable salts of pyruvic acid, and llli~LulGs thereof;
(b) lactate selected from the group con~ietinE of lactic acid, ph~ ce~lhc~lly acceptable salts of lactic acid, and llliX.~UlGS thereof; and (c) a llli~Ul~ of s~ fA and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular lllGlllI)l~les and resuscitation of ~ n cells;

WO 96/37230 PCT/US9GJ~5901 (I.C) (a) an ~nh~ nt; and (b) a ~Lu-~ of s~tllr~tç~l and lln.~ ~l fatty acids where*n the fatty acids are those fatty acids le.~ dd for the repa* of cellular llW Lai~es and resll~-it~hon of m ~ n cells;

(I.D) (a) lactate scle~t A from the group con~;~ting of lactic acid, ph~rm~cellhc~lly acceptable salts of lactic acid, and ~Lul~,S thereof;
(b) an antioxidant; and (c) a ~ Lulc of SaLulalcd and unsaturated fatty acids where*n the fatty acids are those fatty acids le~ dd for the repair of cellular membranes and resuscitation of m~ n cells; and (B) providing a ph~rm~ceutically acceptable carrier; and (C) admixing the therapeutic wound healing composition from step (A) and the ph~rm~ce~lhcally acceptable carrier from step (13) to form a thc~à~ LiC pharmaceutical 1 5 composition.

Throughout this application, various publications have been referenced.
The ~iicclos~lres in these publications are incorporated herein by reference in order to more fully describe the state of the art.
The present invention is fulther illustrated by the following examples which are not intended to limit the crrccLivc scope of the claims. All parts andpercentages in the examples and throughout the specific~h~n and claims are by weight of the final composition unless otherwise specifi~
E. E~amples Of The Therapeutic Wound ~ 1 n~ Compositions Of Embodiment One (I.A-D) Study 1 ~ This study clen~n~trates a comparison of the viability of U937 monocytic cells after exposure of the cells to various antioxidants and combin~hon~

WO 96/37230 PCI~/US!15'~ 01 of antioxidants. This shudy also ~monetrate a c~ ;eon of the levels of hydrogen peroxide produced by U937 monocytic cells and ..._-...n~ n epic~ k~r~hnocytesafter C,~yO~ul~, of the cells to various ~nhoxi-l~nte and combinations of antioxidants.
The results of this study are illn.ct~t~l in Figures 1 4 and examples 1-26 below.
s ~ Tnm~ n epidermal keratinocytes and monocytes were employed to eY~mine the ability of various ~nh~xi-1~ntc to reduce levels of hydrogen peroxide in these cells. Hydrogen peroxide was measured after the cells were exposed to ulh-aviolet light in the wavelength range from 290 to 320 nm (UV-B) or to the infl~.,.. n~lo,y compound 12-o-tetr~(lec~noyl-phorbol-l3-acetate (TPA). ~o li~lm pyruvate was tested at various conc~ntr~t - ne to ~let~mine the effect of conc~ntr~tione of this ~ntioxitl~nt on the hydrogen peroxide pro(1ncti-)n by epidermal cells and monocytes. Magnesium pyruvate, calcium pyruvate, zinc pyluvdt~, and cul~illations of sodium pyruvate with ascorbic acid, lactic acid, and Vitamin E were then tested to cl~t~Tmine the effect of these salts and combin~tione of antioxidants on the hydrogen peroxide production by epidermal cells and monocytes.

M~mm~ n epidermal ker~tinocytes were isolated by t~ ;on of epithelial sheets and grown in modified basal MCDB 153 m~lillm supplem~nt~l withepidermal growth factor, bovine piLuiL~Iy extract, and hydrocortisone. Cells were ed in a humidified incubator with 5% carbon dioxide at 37~C. Keratinocytes were seeded in 60 mm culture dishes at a cell density of 3 x 105 cells per dish and the cultures were exposed to 1 M.E.D. dose of ultraviolet-B light (100 mJ/cm2) or treated with 100 ng/ml of TPA.
U937 monocytic cells are a cultured cell line grown in RPMI media with 10% fetal calf serum. Cells were ...~ .;..ed in a 60 mrn culture dish at 5% carbon dioxide at 37~C. at a seeding density not e~cee~ling 1 x 106 cells per dish.

Sodium pyruvate, lactic acid, ascorbic acid, and Vitamin E were dissolved in distilled water, with sufflcient s~ ct~nt. The concentrations of the sodium pyruvate solutions prepared were 1 mM, 10 mM, 50 mM, 100 mM, and -W O 96137230 PCTtUS96tO5901 200 mM. The c~nc~nh-ations of the lactic acid solutions ~ ul were 1.0%, 0.1%, and 0.05%. The conc~ntr~ti~nc of the ascorbic acid solllhonc ~ d were 1.0%, 0.1%, 0.05%, and 0.025%. The concentr~tionc of the Vitamin E solutions ~
were 1 U, 10 U, 50 U, and 100 U. The test solutions were adjusted to a pH value of 7.4 with 1.0N sodium hydroxide solution and then sterile filtered. The a~l~liateccncenhr~tion of test solution or co~ ;llaLion of test solllhonc was added to the cells ;~",,,~ ..ly prior to exposure of the cells to ult-raviolet light-B or TPA [lOOng/ml].
Stock solutions were prepared so that the vehicle did not con.chhlte more than 1% of the total volume of the culture media.
Intr~cçlh-l~r hydrogen peroxide prolllctiQn by .,.~ n epidermal ker~tinQcytes and U937 monocytes was measured using dichlorofluoresceill ~ cet~te (DCFH-DA, Molecular Probes, Eugene, Ore.). DCFH-DA is a non-polar non-fluorescent compound that readily diffuses into cells where it is hydrolyzed to the polar non-fluorescent derivative DCFH which then becomes trapped within the cells. In the presence of intracellular hydrogen peroxide, DCFH is oxidized to the highly fluorescent compound DCF. Hence, cellular fluorescence intensity is directly proportional to the level of intr~cçll~ r hydrogen peroxide produced. Cellular fluor~.sc~-nce intensity can be llluniLolGd by fluo.illl~Lly and by flow cytometry.
~mm~ n epidermal keratinocytes and U937 cultured monocytes (1 x 106 per dish) were incubated at 37~C. with 5 uM of DCFH-DA. Production of hydrogen peroxide was measured using a Coulter Profile analytical flow c~ cLel.
Linear and log intensity of green fluorescence data was collected. For each analysis, a quantity of 10,000 to 20,000 events was accnmlll~te(l Optical ~lignm~-.nt for the in~ was performed daily. Coefficients of variation for rulw~ angle light scatter and integrated green fluorescence were generally less than two. Each analysis was repeated three times and the qll~ntit~tion of fluorescence was expressed in terms of f~ ,-lloles (fmol, 10-15 moles) of DCF oxidized per cell, which is a direct measure of the inh~c~llular hydrogen peroxide produced. .Alt~rn~tively~ in the saturated and uns~ ed fatty acid examples in examples 27-52, fluorimetry was used to assess the DCF oxidation per cell.

CA 02218~39 1997-10-17 The viability of the U937 monocytic cells after exposure of the cells to various ~ntioxirl~nt~ for 24 hours was measured. The viability of the cells was ~letf ...;..e~l by exposing the cells to the dye propidium iodide. Permeable cell ~w~ es which absorbed the dye were not considered viable. The viability of the S cells was represented as the percentage of cells that ~YC~ propidium iodide.
Figure 1 depicts in bar graph format the viability of U937 monocytic cells afterexposure of the cells to no ~ntiQxill~nt (Example 1, control), to sodium pymvate(Example 2), to ascorbic acid (Example 3), to lactic acid (Example 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 combin~ti-n~ of antioxidants.
Specifically, the viability of U937 monocytic cells was measured after exposure to no antioxidant (Example 6, control), to ascorbic acid and lactic acid (Example 7), to ascorbic acid and Vitamin E (Example 8), to sodium pyruvate and ascorbic acid (Example 9), to sodium pyruvate and lactic acid (Example 10), to sodium pyruvate and Vitamin E (Example 11), to lactic acid and Vitamin E (Example 12), and to sodiumpyruvate, ascorbic acid, and lactic acid (Example 13).

Figure 1 shows that ascorbic acid is cytotoxic to monocytes at concentrations as low as 0.25%. Figure 2 shows that the cytotoxicity of ascorbic acid was reversed by the ?~d-1itlQn of 10 mM of sodium pyruvate. Figures 1 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 cytotoxicity of ascorbic acid.

Sodium pyruvate was then tested at various concentrations to dettormine the effect of concentrations of this antioxidant on the hydrogen peroxide production by epidermal cells and monocytes. M~rnm~ n epidermal keratinocytes and monocytes were exposed to (a) I M.E.D. dose of ultraviolet light-B and ~b) 100 ng/ml of 12-O-tetr~dec~noylphorbol-13-acetate (TPA) in the presence of sodium pyruvate at the following concentrations: 200 mM, 100 mM, 50 mM, 10 mM, 1 mM.

WO 96t37230 PCT/US96/05901 The ~ illlUIll cQnc~ntr~tion of sodium pyruvate to reduce the hydrogen peroxide production by epidermal cells and monocytes was found to be 10 mM.
Concentrations of sodium pyruvate of 50 rnM and above were cytotoxic to both epiderma1 keratinocytes and monocytes.
~nesillm pyruvate, c~lcillm pyruvate, zinc ~yluv~e, ascorbic acid, lactic acid, and Vitamin E, and combinations of sodium pyruvate with ascorbic acid, lactic acid, and Vitamin E were then tested to ~etermine the effect of these salts and combin~hQn~ of ~ntioxi~l~nt~ on the hydrogen peroxide production by epidermal cells and monocytes. The following test solutions were prepared.

(a) sodium pyruvate [10 rnMl;
(b) zinc salt [10 mM];
(c) magnesium salt [10 mM];
(d) calcium salt [10 mM];
(e) sodium pyruvate [10 mM] and ascorbic acid [0.025%];
(f) sodium pyruvate [10 mMl and lactic acid [0.05%];
(g) sodium pyruvate [10 rnMl, 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%];
(.1) Vitamin E [1 U, 10 U, 50 U, and 100 U]; and (k) vehicle solvent controls.

There was no significant di~,.ci.,ce among the zinc, magnesium, and calcium salts of pyruvic acid on the hydrogen peroxide production by epidermal cells and monocytes. The zinc and c~k illm salts of pyruvic acid in~lllce~l di~ h~n ofkçr~hnQcytes. For convenience, the sodium salt was used in subsequent tests.

WO 96/37230 PCT/U~ .5~1 The ~ UIll conc~.nh~hon of lactic acid to reduce the hydl~
peroxide production by epiderrn~l cells and monocytes was found to be 0.05%. Theoplilll~ conc~?ntration of ascorbic acid was found to be 0.025%. The higher conc~ntr~hon~ of both of these co~vul-ds were found to be cytotoxic to both types S of cells. The opLi~u ll concPnh~hon 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 after c.~l,o~u c of the cells to no ~nhoxi~nt (Example 14, control), to sodium pyruvate (Exarnple 15), to ascorbic acid (Example 16), to lactic acid (Example 17), and to Vitamin E (Example 18). Sodium~yluv~LG and Vitarnin E ~ignific~ntly reduced the hydrogen peroxide production by monocytes.

Figure 4 depicts in bar graph format the levels of hydrogen peroxide produced by U937 monocytic cells after exposure of the cells to various combin~hon~
of ~nhoxirl~nt~ Specific~lly~ the levels of hydrogen peroxide produced by U937 monocytic cells were measured after exposure to no ~nhoxi~nt (Example 19, control), to 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 ~yluvalc 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 c~"~illation of lactic acid (0.05%) and Vit~min E
(50 U) ~ignific~ntly reduced the hydrogen peroxide pro~lnchon by monocytes.

The morphological alterations in epidermal keratinocytes were observed in conhrol culhures and in cultures exposed to ulhraviolet-B. Cells in the layer closest to the demlis are basal keratinocytes. These cells proliferate and migrate into the spinous and granular layers of the epidermis where the cells begin to dirrc~cllLi~Lc. The dirrclclltiation pattem results in cells ~-nllcle~hng and fomling comified envelopes at the uppermost portion of the epirlermi~ the stahum corneum. The dirrc~ hon of keratinocytes is conhrolled by the levels of c~lci--m, m~gne~ m, and other clc..,~-t~ in the ~ A;I~ Cells in culhure systems ~ oLillg dirr~ hcn appear as an epidermal WO 96/37230 PCT/US9Gi(~53~il sheet forming ~ cl~ or tight junctions with each other. K~ cytes that become nonadherent or float in the media were con~ ~ed responding to a cytotoxicevent.

S The following morphological alterations in the ~ r~.qli~n epidermal keratinocytes were observed for the following control cultures:

10 mM Sor~ m Pyruvate: Tight junctions of cells were formed and the proliferation rate of the cells was higher than the rate of the control cells.
0.025% Ascorbic Acid: Cells were floating in a cytotoxic response to ascorbic acid.

0.025% Ascorbic acid and 10 mM Sodium Pyruvate: Few tight junctions of cells were observed and cells appeared similar to the cells in the sodium pyruvate culture.
0.05% Lactic Acid: Cells appeared ~l~m~ l1y altered as an epidermal sheet and asflat granular cells.

0.05% Lactic Acid and 10 mM Sodium p~rruvate: Cells formed an epiderrnal sheet but appeared smaller than the cell in the lactic acid culture.

50 U Vitamin E: Cells appeared the same as the cells in the control culture.

50 U Vitarnin E and 10 mM Sodium Pyruvate: Cells increased in IlUlll~ and changed in appearance resembling the cells in the sodium pyruvate culture.

The following morphological alterations in the .~.,.. ~~li~n epidermal keratinocytes were observed for the corresponding cultures exposed to ultraviolet light-B, 100 mJoules, for 24 hours:
10 mM Sodium P~rruvate: Cells proliferated more rapidly than the cells in the control culture.

CA 02218~39 1997-10-17 WO 96137230 PCI~/US96/05901 0.025% Ascorbic Acid: Cells were non~lh~rent and floating in a cytotoxic response to ascorbic acid greater than the cytotoxic response of the cv~ ollding cells without ultraviolet-B light exposure.

0.05% Lactic Acid: Cells formed an epidermal sheet and were more granular than cells in the control culture without ultraviolet-B light exposure.

50 U Vitamin E: Cell growth was inhibited but cells appeared similar to cells in the control culture without ultraviolet-B light exposure.
50 U Vit~min E and 10 mM Sodium P~uvate: Cells appeared similar to cells in the control culture and proliferated to a greater extent than cells in the control cultures without ultraviolet-B light exposure.

Morphological alterations in the U937 monocytic cell line were also observed for control cultures and cultures exposed to ultraviolet light-B, 100 mJoules, for 24 hours. The following compounds and combination of compounds, at the concentr~tions set out below, significantly inhibited the levels of hydrogen peroxide produced by U937 monocytic cells Sodium pyruvate at 10 mM and 50 mM;
Vitamin E at 50 U and 100 U, and Lactic acid at 0.05% and Vitamin E at 50 U.

~.Y~ - Of The Therapeutic Wound Healing Compositions Of Embodiment One a.A-D) Study 2 This study demonstrates a comparison of the levels of hydrogen peroxide produced by U937 monocytic cells and epiderrnal keratinocytes after exposure of the cells to various combinations of antioxi~l~nte with and without a mi~Lule of saturated W O 96137230 PCTrUSg~ Ol and uns~ al~d fatty acids. The results of this study are illustrated in Figures 5-7 and eY~mples 27-52 below.

~mm~ n epidermal keratinocytes and U937 monocytic cells and the test solutions of sodium pyruvate, lactic acid, ascorbic acid, and Vitamin E were ed as describe above for Examples 1-26. Intr~cell~ r hydrogen peroxide production by the l.,;3.,....~1i~n epidermal keratinocytes and U937 monocytes was also measured as described above.

A mi~Lule of fatty acids derived from chiçlrPn fat was prepared for ~Miti/~n to the cultured cells by mixing 0.1% of the chicken fat with the culture media.
At the le~ e-atule of the culture media, 37~C., the chicken fat was miscible. This r,hir.~.n fat .~ L~; was added to cultures of cells pIior to exposure of the cells to ultraviolet-B light or TPA tre~tm~nt As set out in examples 1-26, ~ ",~ n epiderrnal keratinocytes and monocytes were exposed to (a) 1 M.E.D. dose of ultraviolet light-B and (b) 100 ng/ml of 12-O-tetradecanoylphorbol-13-acetate in the presence of various antioxidants and combinations of antioxidants with and without a ~ lule of saturated and unsatul~lt;d fatty acids [0.1%, 0.5%, and 1.0% rhiç~en faq.

Figure S depicts in bar graph format the levels of hydrogen peroxide produced by U937 monocytic cells after exposure of the cells to various combin~tion~
of antioxidants with and without a ~ of saturated and unsaturated fatty acids.
Specifically, the levels of hydrogen peroxide produced by U937 monocytic cells were measured after exposure to lactic acid and Vitamin E without fatty acids (Example 27) and with fatty acids (Example 28), to ascorbic acid and lactic acid without fatty acids (Example 29) and with fatty acids (Example 30), and to ascorbic acid and Vitamin E
without fatty acids (Example 31) and with fatty acids (Example 32). The ability of the ~~ .ations of lactic acid and Vitarnin E, ascorbic acid and lactic acid, and ascorbic ~ acid and Vitamin E to reduce the hydrogen peroxide pr~lnr,tion by monocytes was increased in the presence of fatty acids. The most effective combination to reduce the WO 96137230 PCT/US9GJ~ Ol hydrogen peroxide production of monocytes was lactic acid (0.05%) and Vitamin E
(50 E) in the presence of a ~lule of s~ ~ and lmc,.l...,~ l fatty acids (0.5%).

Figure 6 depicts in bar graph format the levels of hydrogen peroxide S produced by epidermal keratinocytes after exposure of the cells to various ~nhoX~ nt~
with and without a l~luut; of s;~ ecl and ~m~ ed fatty acids. SFeci~c~lly, the Ievels of hydrogen peroxide produced by epidermal keratinocytes were meds ued after exposure to no ~nho~ nt without fatty acids (Example 33, control) and with fattyacids (FY~nnple~ 34), to sodium pyruvate without fatty acids (Example 35) and with fatty acids (Example 36), to ascorbic acid without fatty acids (Example 37) and with fatty acids (Example 38), to lactic acid without fatty acids (Example 39) and with fatty acids (Example 40), and to Vitamin E without fatty acids (Example 41) and with fatty acids (Example 42). The ability of sodium ~ uv~Le and Vitamin E to reduce the hydrogen peroxide production by epid~rm~l k~-,r~hnocytes was increased in the presence of fatty acids. The most effective combin~fion~ to reduce the hydrogen peroxide production of epidermal keratinocytes were sodium pyruvate in combination with aLule saturated and nn~c~ ed fatty acids and Vitamin E in combination with a Lule of saturated and unsaturated fatty acids.

Figure 7 depicts in bar graph format the levels of hydrogen peroxide produced by epidermal keratinocytes after exposure ofthe cells to various combin~hon~
of antioxidants with and without a ~ Lul~i of s~ ed and unsaturated fatty acids.Specifically, the levels of hydrogen peroxide produced by epidermal keratinocytes were measured after exposure to no antioxidant without fatty acids (Example 43, control) and with fatty acids (Example 44), fo sodium pyruvate and ascorbic acid without fatty acids (Example 45) and with fatty acids (Example 46), to sodium pyruvate and lactic acid without fatty acids (Example 47) and with fatty acids (Example 48), to sodium pyruvate and Vitamin ~ without fatty acids (Example 49) and with fatty acids (Example 50), and to ascorbic acid and Vitamin E without fatty acids (Example 51) and with fatty acids (Example 52). The ability of all combin~ of ~ntioxi-l~nti to reduce the hydrogen peroxide production by epid~rm~l keratinocytes was increased in the presence of fat~ acids. In order of potency, the most t;rreclive combinations to reduce the hydrogen peroxide pro~ c~ion of epidermal k~ nocytes were sodium pyruvate and Vitamin E, sodium pyruvate and lactic acid, and Vitamin E, each in combination with a nli~lul~ of salulal~d and unsàlulalcd fatty acids (0.5%).

S Because of the cytotoxicity of cells towards ascorbic acid described above, the ascorbic acid CC"~ alions without sodium pyruvate were not con~i~lt?red ~igni~c~ntly di~ele.1t from the control test solution.

Summary Analysis Of The Data From Studies 1 and 2 Human epiderrnal keratinocytes were isolated by trypsini7~on of epithelial sheets and grown in modified base MCDB 153 m~linm supplc~ l with epidermal growth factor and bovine ~iluil~y extract. Cells were seeded in culture dishes at a density of 3 x 105/dish. Prior to exposure to W B light (lOOmJ/cm2) or .t with l OOng/ml TPA, the cultures were treated with the ~rop,iate concentration of wound healing components. Intr~cel~ r production of hydrogen peroxide was measured using DCFH-DA, a nonpolar compound that readiiy diffuses into cells, hydrolyzad to a nonpolar derivative. In the presence of intracellular hydrogen peroxide, DCFH is oxidized to a highly fluorescent compound DCF. Thus, cellu1ar fluorescence intensity is directly proportional to levels of hydrogen peroxide produced and can be monitored by flow cytometry. Hydrogen peroxide is cytotoxic,therefore lower levels of hydrogen peroxide production is desirable for cellularviability.
In all cases, the three component wound healing composition surpassed the predicted outcomes, clearly demonstrating u,~lGdicted synergy.

WO 96137230 PCT/US9G105~01 Results 1 - Control 250 250 0 2 - Fatty Acids 250 230 -20 (0.5%) 3 - Sodium Pyruvate 250 490 +240 (1 OmM) 4 - Vitamin E 250 400 +150 (50 units) 5 - Pyruvate & 250 430 +180 Fatty Acids 6 - Vitamin E & 250 200 -50 Fatty Aci~is 7 - Pyruvate & 250 290 +40 Vi~n~n E

8 - Pyruvate & 250 120 -130 Vitamin E & Fatty Acids Column I shows the dirrelel" L~ t groups.
Column 2 shows the production of H202 in control cells (fmoVcell).
Column 3 shows the production of H202 after trç~ ont with wound healing components.
Column 4 shows the .lirre.ellce in production of H202 from control after the trç~ nt.

All comparisons were ~esse~l against the controls, which produced 250 H202 fmoVcell. The positive numbers represent H202 production in excess of the control and the negative l~ represent H202 production below the control. These results are set out in Figure 8.

CA 02218~i39 1997~10~17 WO ~6137230 PCT/US961'~5~01 ~r I ' ~'- of Single I~ ' Effects Fatty Acids (-20) & Vitamin E (+150) & Pyruvate (+240) +370 Is The Predicted Three Component Effect -130 Is The Wound healing composition Actual Effect 500 Is The Difference Between Predicted Effect minus Actual effect (Synergy) Combination of Paired and Single Ingredients 10Pyruvate & Fatty Acids (+ 180) & vitamin E (+ 150) +330 Is The Predicted Three Component Effect -130 Is The Wound healing composition 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 composition Actual Effect 20320 Is The Difference between Predicted Effect minus Actual Effect (Synergy) Pyruvate & Vitamin E (+40) & Fatty Acids (-20) 25+20 Is The Predicted Three Component Effect -130 Is The Wound healing composition Actual Effect 150 Is The Difference between Predicted Effect minus Actual Effect (Sy: ~Y) In all cases, the three component wound healing composition surpassed the predicted outcomes clearly demonstrating unpredicted synergy.

Examples Of The Therapeutic Wound Healing Compositions of F.~ho~iiment One ~I.A-D) Study 3 s This study ~leTIl- netr~t~s a co..~ on of the wound healing abilities of the therapeutic wound healing compositions of the present invention versus cullve.-L;on~l wound healing compositions. The results of this study are i~ ts~1 in examples A-D.
The wound healing compositions of Examples A-D were prepared having the cornpositions set out in Table A.
FY~rnrl~ !
Ingredient A B C D
Prep_HTM
sodiurn pyrubate -- 2% -- --vitamin E 1%
chicken fat -- 2%

shark liver oil 3% 3% 3%
petrolatum in 64% 66.5% 68%
mineral oil all~ou~lts22.53% 25.03% 26.8%
paraffin totaling 5% 5% 5%
t~rnl~l.cifit-r 100% 0.2% 0.2% 0.2%
These components are present in Pl~dldlion HTM

Wound healing composition A was coll..lwlcially available ~,~dlion HTM. Wound healing composition B was a petrolatum base fonm~ on conl;t;.,;.,g live yeast cell derivative, shark oil, and a llli~LLu-e of sodium pyruvate, CA 02218~39 1997-10-17 WO 96t37230 PCI~/US96/05901 vitamin E, and chick~n fat. Wound healing composition C was a petrolatum base forrnnl~tion co..l~i..;..g live yeast cell derivative and shark oil. Wound healing composition D was a petrolatum base form~ h- n only.

S Wound healing studies were carried out using hairless mice (SKR-1, Charles River) 6-8 weeks in age. One group of mice were ul~llGaled as a control group and were referred to as Example E. In each group there were 6 mice for ev~ hc-n at either day 3 or day 7 for a total number of 60 ~nim~l~ in the study. The mice were anestheti7~1 with ether and a midline 3 cm full thickness longit~l~lin~l incision was made with a number 10 scalpel blade. Incisions were closed using steel clips at 1 cm intervals.
Forml-l~hons A-D set out above were applied in a randomized blinded study to thewounds on day 0 at 2 hours following wounding and reapplied at 24 hour intervals during the 7 days of the study. The wounds were ~x~mined daily and scored on a basis of 0-S for closure on each day of the study, with a score of 5 reprçs~nting the wound best 1 5 healed.

The ~nim~l~ were sacrificed on day 3 and day 7 using cervical dislocation. The dorsal skin including the incision was dissected without the subcutaneous tissue. The skin was placed in neutral buffered formalin and subsequently sectioned and stained with hematoxylin and eosin. The wounds were examined microscopically and ~ ,scntativetissue sections were photographed.

On each day of the experiment, the score and rank order of the formlll~hons for closure of wounds and speed of healing were as follows:
B (5) >> D (4) >> C (2) >/= E, Control (2) > A (1) Photographs of the wounded mice on day 4 are set out in Figures 9A-9D and 10.

Figures 9A-9D and 10 show that Form~ h~n B, which was a petrolatum base f~rmlll~hion cc..l~;~.i..g live yeast cell derivative, shark oil, and a ~ Lule of sodium pyluval~, vitamin E, and chicken fat, was a significantly better wound healing agent WO 96/37230 PCT/US95.'~5~01 than the other form~ tlon.c These results are ~up~ Gd by the subjective grading of the wound closures and the speed of healing on each day (1-7) of the GA~ . as well as on the objective histological toY~min~tinn of tissue secti~ne to measure the extent of inflz.. i~ y cell infiltr~te within the wound and the extent of epitheli~li7~tion at the wound edges. The final result was that less scar tissue was present at day 7 on the mice treated with Fonnlll~ti- n B.

F~-rmnl~tiQn D, which was a white petrolatum fiormlll~hon only, was judged to be significantly more effective to pl~>mole healing than either Fo~nlll~fion C, which was a petrolatum base formlll~tlon ~II~ illg shark liver oil and live yeast cell derivative, or Fnnn~tin A, which was ~h.,.l;..n HTM. The superior ability of Formlll~tlon D over Formlll~tion C to i~ uve healing may result from a delay in the healing process caused when the live yeast cell dt.-vaLivG is depleted and the cells shift to an ~lt~ tive nutrient source. The presence of the n.lAlulG of sodium pyruvate, vitarnin E, and chicken fat in FolTnlll~h~ n B a~al.. -lly offsets the depletion of the live yeast cell derivative.

Formulation C, which was a petrolatum base formnl~tiQn ~ ~ lg live yeast cell derivative and shark oil, was judged comparable to the control (u~ alGd wound) in speed of wound closure and extent of healing. Formlll~tion A, which was P~ Lion HTM, appeared to be the least GrrG~iLivG healing formulation by both subjective grading of wound healing and by objective ~ rnin~tion of tissue sections.
The superior ability of Form~ ti(-n D and Formlll~ti-)n C over Forrmll~tion A tolUV~ healing may be due to their ability to act as an occlusive wound dressing that prevents transepidermal water loss and thus p~ t;s healing and wound closure. The poQr ability of Formulation A to improve healing may be due to the potential CytQtoXiCity of phenylmercuric nitrate present in Preparation HTM as a preservative.

These results show that the wound healing compositions of the present invention which comprise a ~liALul~; of sodium py~lv~k;, vitamin E, and ~iGlrt?n fat increase the proliferation and resllecit~tion rate of ",i1."".~ n cells. The wound healing compositions m~ te low levels of oxygen in the initial stages of healing to CA 02218~39 1997-10-17 ~u~ress oxidative damage and higher levels of oxygen in the later stages of healing to ~lo~ e collagen formation.

II. Immunostimulating-Wound Healing Compositions S A. Embodiment Two (IA-D I X) Applicant has discovered therapeutic immunostim~ hng-wound healing compo~ihon~ (I.A-D + X) which comprise an immllnoshmnl~hng agent ~X) and the wound healing compositions of Embodiment One (I.A-D). Preferably, the wound healing composition comprises (a) pyruvate, (b) an antioxidant, and (c) a llfi~ ; of satul~led and unsaturated fatty acids. TmmllnoshmlllAhng agents can stimlllAte the immune system in a patient to kill an infecting organism but do not ~ ~ the wound healing process. Wound healing compositions can increase the resuscitation rate of injured I~A~ A1;A11 cells and the proliferation rate of new l"A."~I,AliAn cells to replace dead cells but do not kill infecting org~ni~m~ Applicants have found that the combination of an imml-noshmlllAhng agent and a wound healing composition results in an immlln~stimlllAhng-wound healing composition which acts synergistically toenhance wound repair in both the upper and lower portions of the skin. The therapeutic immlmos~imlll~hng-wound healing compositions are superior in stim--lAhng both the extent of re-epitheli~li7Ahi~ n as well as the extent of tissue integrity and leukocyte infiltration in the dermis.

The combination of the imm.-noshiml-lAhng agent and the wound healing compositions of the present invention provides a phArmAcellhcal composition useful for healing infected wounds and having an enhanced ability to prevent and reduce injury to ~IAll~ liAn cells and further increase the resuscitation rate of injured m~mmAliAn cells. The tissue damage associated with many infectious ~ e~es is believed to be caused by the production of cellular produced active oxygen species. Combination of the immllnoshmlll~hng agent and the wound healing compositions may suppress suchreactive oxygen-linked tissue injury.

Tmmnncshmlll~hng agents are c~ ~~oullds which shmnl~t~ the immune system to send macrophages to the infected site. Tmm lnoshml-l~hng agents are generally cc,~ ents of bacteri~ or yeast which react with roving macrophages to induce the immune system to respond to the foreign body. The immllnoshmlll~ting agents in the immlmoshimlll~ting-wound healing compositions of the present invention may be selected from a wide range of therapeutic agents and I~ Lul~s of therapeutic agents. Nonlimihn~ illustrative c~tegories of such imm~noshm~ hn~ agents includeinterleukin 1 agonists, interleukin 2 ~gonictC, ill~elr~ agonists, RNA synthesisinhibitors, T cell stimlll~hng agents, and adjuv~ll~. Adjuva~ such as Freund's complete adjuvant, are substances added to antigens to intensify the immune response to the antigen when the ~ lui~ is ~-lTnini.ct~.red to an animal. Nonlimihng illustrative specific examples of such immlln~ stimnl~ting agents may be selected from the group con.cichng of b~ ar~ illTM and Freund's complete adjuvant. Preferably, the immllnostimnl~ting agent is betafectinTM.
BetafectinTM (PGG-glucan, Alpha-Bert Technology, Inc. Worcester, MA) is a glucose polyrner that stimnl~tes and enhances specific hnm~ r~l and cellular responses to challenge by infectious or~nicmc- BetafectinTM (poly(1-6)-3-D-glucopyranosyl-(1-3)-B-D-glucopyranosyl) belongs to a class of compounds kno~-vngenerically as J3glucans and is a highly purified, soluble, active molecule derived from a nonrecombinant yeast strain of Saccharomyces cerevisiae. BetafectinTM lacks invivo pyrogenic and infl~ t~.~y effects rçclllhng from cytosine induction, but retains potent immlmo~timlll~tl ry properties. BetafectinTM has a high afffinity for B-glucan receptors of human monocytes and neutrophils and binds competitively to the receptor in a dose-dependent manner at concentrations cignific~ntly below those required for other natural glucan preparations derived from bakers' yeast. BetafectinTM
significantly increases human neutrophils and macrophage in vitro microbicidal activity against Staphylococcus aureus without directly stim~ ting synthesis of the cytokines, interleukin-1, or tumor necrosis factor.
13-Glucans are complex polysacch~ricles composed entirely of glucose subunits. When polymerized, glucose can form a wide array of macr~-m--lecnl~r WO 96137230 PCI'/US~'0!;~01 structures with dramatically dirr~ biologic functions. For eY~mple, when glucose;jubu,.il~ are bound together between the 1 and"the 4 poeiti~ne~ ct~ lose is formed, but a 1 to 3 connection results in the fiorm~hon of 13-glllc~n.e ~'Tlllr,ose polymers are not always straight chain molecules. In the case of ~-glucan, short branches off the main S backbone are also found. As extracted from the yeast, J3-glucans are not very soluble in water. In an aqueous ~ v~ they will exist in a ~lule of cQnfi~r~h~n.
ranging from random coils to aggr~3al~s of triple helices. BetafectinTM is not an activator but rather a primer of the immune response. When a cell is primed, the cell is pushed into a state whereby it is ready to respond when an activator is present but the primer itself does not induce any obvious change in the cell's behavior.

The amount of immllnoshm~ hng agent used in the present invention may vary depending upon the therapeutic dosage recc?mm~.n(l~d or p~ d for the par~icular immllnc)shm~ hing agent. In general, the amount of immllnoshimlll~hing agent present is the ordinary dosage required to obtain the desired result. Such dosages are known to the skilled pr~ctihoner in the m~ l arts and are not a part of the present invention. In a ~,er~.lcd embo lim~nt, the immlm~stimlll~hng agent in the immllnoshmnl~ting-wound healing composition is present in an amount from about 0.001% to about 10%, preferably from about 0.01% to about 1%, and more preferably from about 0.1% to about 0.5%, by weight.

b. Methods For Making The ImmunostimuLating-Wound Healing Compositions Of Embodiment Two (I.A-D + X) The present invention extends to m~thor1.c for making the therapeutic immllnoshmlll~hng-wound healing compositions (I.A-D + X). In general, a therapeutic immunoshmlll~hng-wound healing composition is made by forrning an ad~ Lu~; of the wound healing components of Embodiment One (I.A-D) and an jmmllnoshml~ hng agent. In a first aspect of Embodiment Two (I.A + X), an immllnoshmlll~hng-woundhealing therapeutic composition is made by forming an ~.l.,,;x~ of an immllnostimlll~hing agent and a wound healing composition I.A, comprising (a) a WO 96/37230 PCI~/US96J~5~01 pyruvate, (b) an ~ntioxi-l~nt, and (c) a ~lulc of c~ and lmc~tnr~te(l fatty acids.
In a second aspect of Embo-limt-nt Two (I.B + X), an immlmoshmlll~ting-wound healing the-d~culic composition is made by fo~ming an ~ ;x~ c of an immlmoshmlll~ting agent and a wound healing composition I.B, comprising (a) a ~y~uvdlc;, (b) a lactate, and (c) a ll~Lul~ of s~h~r~tçd and nnc~hlr~ted fatty acids. In a third aspect of Embodiment Two (I.C + X), an immllnos-hmlll~hng-wound healing therapeutic composition is made by forming an adLui~ulc of an immlmoshmlll~hng agent and a wound healing composition I.C, comrricing (a) an ~nhnxicl~nt, and (b) a Lulc of saturated and unsaturated fatty acids. In a fourth aspect of Embodiment Two (I.D + X), an immllnostimnl~hng-wound healing therapeutic composition is _ade by forming an ~ ;xllllc of an immllnoshmlll~hng agent and a wound healing composition I.D, comprising (a) a lactate, (b) an antioxidant, and (c) a llli~tU c of salul~lcd and uns~lu.~ted fatty acids.

In a ~-Crt;llcd embo-limtont, the invention is directed to a method for preparing a therapeutic immlm~!shmlll~hng-wound healing composition (I.A + X) which comprises the steps of admixing the following ingredients:
(A) a therapeutically effective amount of an immllnoshmlll~hng agent; and (B) a wound healing composition which comprises:
(a) pyruvate selected from the group conciching of py~uvic acid, pharmaceutically acceptable salts of pyruvic acid, and ~ tUUCS thereof;
(b) an antil-xi~l~nt; and (c) a ~ urc of satul~led and lm~hlr~terl fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of ., li1,, ,, . .~li~n cells.

c. Methods For Employing The Immunostimulating-Wound He~l;ng Compositions Of Embo~l;~cnt Two a.A-D
The present invention extends to m~tho-lc for employing the therapeutic imm--noshim--l~hng-wound healing compositions (I.A-D + X). In general, a therapeutic CA 022l8539 l997- lO- l7 cc..l.~o~iLion is employed by contacting the therapeutic composition with a wound. In a ~ ,r~ d embo~limpnt~ the invention is ~ .ltd to a m~thc--l for healing an infected wound in a ."i.."...~l with an immllnoshmlll~hng-wound healing composition (I.A + X) which comprises the steps of:
S (A) providing a therapeutic immllnoshmlll~hng-wound healing composition which comprises:
(1) a wound healing composition which comprises:
(a) pyruvate s~lectecl from the group cnn~i~ting of pyruvic acid, ph~rm~cellhcally acceptable salts of pyruvic acid, and l~ UUGS thereof;
(b) an antioxidant; and (c) a ~ Lu-e of saturated and unsaLu-~ted fatty acids wherein the fatty acids are those fatty acids re~lui~cid for the repair of cellular ~ es and (2) a therapeutically effective amount of an immllnoshimlll~hng agent;
andresuscitation of ...i., .... ,~li~n cells; and (B) cont~cting the immllnoshmlll~hng-wound healing composition with the infe.;Led wound.

d. ~ugr~ led Imm~ ~sti~ulating-Wound Healing Compcs;'i~ns Of Embodiment Two (I.A-D I X I M) In another aspect of Embodiment Two, the immllnoshmll1~hng_wound healing compositions (I.A-D + X) of the present invention may be further combined with m~lic~mt-.nt~ useful for treating wounds (M) to form ?,ngmt-,ntç~l imm--noshmlll~hng_wound healing compositions (I.A-D + X + M). In this embo-lim~nt, the combination of the immllnoshmlll~hng-wound healing composition of the present invention and the m~ic~m.o.nt useful for hreating wounds provides an ~llgm~nte~l immunostimlll~hng-wound healing composition having an enhanced ability to increase the proliferation and resuscitation rate of ",i.""l,~ n cells. For example, ~ the therapeutic compositions of the present invention may be used in combination with medics.. ~ useful for hreating wounds such as other immunoshmlll~hng agents (BetafectinTM), antiviral agents, ~ntiker~tclytic agents, anti-infl~.. ~tc,.y agents, antifungal agents, hretinoin, sunscreen agents, ~1~rm~tc~logical agents, topical WO 96137230 PCI~/US96/05901 ~nhhi.et~mine agents, ~nfl1b2~cteri~1 agents, bio~Ah~cive agents, ~ ilaLuly bursting inhibitors (lactic acid, ~ nocine)~ inhibitors of prosf~g1~nAin synthesis (ibu~lur~,.l, aspirin, indomethacin, l,.eclof~,.,omic acid, retinoic acid, p~Aim~te O, ...~c1u.~
oxyben_one), steroidal anti-infl~..l...~to.y agents (corticosteroids inr.1nAing synthetic analogs), ~ntlmiçrobial agents (neosporin ~;.. 1.. ~.. 1, silvadine), antiseptic agents, anesthetic agents (~ e hydrorhl- riAe, 1i~loc~ine b~n7oc~ine)~ cell nnfnent media~
burn relief m~Air~fionc, sun burn m~Aic~fionc~ acne preparations, insect bite and sting medications, wound ~1e~n.ct-rs, wound drescingsJ scar re~1cing agents (vitamin E), and the like, and ll~x.LulcS thereof, to further enhance the proliferation and res11crit~tlon rate of ,... ~.. .~li~n cells. Preferably, the mt-Aic~m~nt useful for treating wounds is sçlecte from the group consisting of imm11nostim111~t1ng agents, antiviral agents, ~nfltkrr~t~lytic agents, anti-infl~ y agents, antifungal agents, tretinoin, sunscreen agents,Açrm~tc)logical agents, topical ~nhhict~mine agents, ~nflih?~ctt-ri~1 agents, bio~Ahrcive agents, lc~hàt~ly bursting inhibitors, inhibitors of prost~g1~nAin synthesis, antimicrobial agents, cell nutrient media, scar reA~1cing agents, and ll~LulcS thereof.
More preferably, the m~Aic~m~.nt useful for treating wounds is selected from the group con.cicting of immnn~stim111~tinE~ agents, antiviral agents, antikeratolytic agents, anti-infli..... ~t~.,y agents, antifungal agents, acne treating agents, sunscreen agents, Aerm~tc)logical agents, ~nflihict~mine agents, ~nhib~cterial agents, bioadhesive agents, and l~ es thereof.

In a plcrcl~cd embodiment, the invention is directed to an ?~11gm~.nt~1 immnnostim1l1~ting-wound healing composition a.A + X + M) which comprises:
(A) a therapeutic immnnostim111~hng-wound healing composition which comprises:
(1) a therapeutically crrc-;Live amount of an immnnostim111~ting agent; and (2) a wound healing composition which comprises:
(a) pyruvate s~ ct~ from the group con.cicting of pyruvic acid, pharm~celltically acceptable salts of pyruvic acid, and ",i~lu,es thereof;
(b) an antioxidant; and WO 96/37230 PCT/US961'~5~01 (c) a llfi~lul~ of ~hlr~ and lln~hlr~te~l fatty acids wherein the fatty acids are those fatty acids re~luil~d for the repair of cellular . ~ es and resuscitation of l.. i.. ~li~n cells, and (B) a m~-lic~n-~nt useful for hreating wounds.
The present invention extends to m~fh~1~ for making the a~
immlmoshmlll~hng-wound healing compositions. In general, the ~ . "~ A
compositions are made by ~lmixing the t~ a~.ulic imm~mostimlll~hng_wound healingcomposition with the medicament useful for hreating wounds to pr~ e the ~n~,lllr..l~cl immllnoshmlll~ting-wound healing composition.

The present invention also extends to methods for employing the allgmPnt~l immunostimlll~hing-wound healing compositions. In general, an ~llgm~ont~
immunostimnl~ting-wound healing composition is employed by cont~cting the composition with a wound. In a ~ r~llcd embo-lim~ont, the invention is directed to a method for healing an infected wound in a ~ 1 with an 7~ngmPnt~(l immllnoshmlll~hng-wound healing composition (I.A + X + M) which comprises the steps of:
(A) providing a therapeutic ~nglll...llecl immllnoshmlll~hng_wound healing composition which comprises:
(1) a therapeutically effective amount of an immnnoshmlll~hng agent;
(2) a wound healing composition which comprises:
(a) pyruvate selectecl from the group con~i~hng of pymvic acid, ph~rm~cellhcally acceptable salts of pyruvic acid, and llfi~lul~;s thereof;
(b) an antioxidant; and (c) a Illi~Lu~e of saluuated and unsaturated fatty acids wherein the fatty acids are those fatty acids re.luil~;d for the repair of cellular membranes and resuscitation of ~",~.""~~ n cells; and (3) providing a medicament useful for hreating wounds; and (B) cont~chng the ~g.. ~ <l immnnoshmlll~hng-wound healing composition with the infected wound.

WO 96/37230 PCI/US~ 5301 The ypes of wounds which may be healed using the immunostimulating-wound healing compositions and the augmented immllncshimlll~hng-wound healing compositions of the present invention are thosewhich result from an infected injury which causes epidermal damage. The topical Sthc.a~ ic compositions may be used orally in the form of a mouth wash or spray to protect and accelerate the healing of injured oral tissue.

Methods for healing a wound comprise topically ~-lmini.et~ring the compositions of the present invention directly to a wound site to increase the healing rate of the wound. The composition is .,.,.;.. ~ ~l in contact with the wound for a period of time sufficient to increase the proliferation and resuscitation rate of the cells.

e. Formulations Of 15The Immunostimulating-Wound ~e~!;n~ CO roQ;~ions Of Embodiment Two (I.A-D ~ X) and (I.A-D I X I M) Once prepared, the inventive therapeutic immunoshmlll~fing-wound healing compositions and ~ngmt-.nte(l immllnc~stimlll~ting-wound healing compositions 20may be stored for future use or may be formnl~tçd in effective ~llllUUlll::i with ph~rm~celltic~lly acceptable ca~Tiers such as ph~rm~cellti~l appliances and topical vehicles (oral and non-oral) to prepare a wide variety of ph~rm~celltical compositions.
The ph~rm~ce~hcally acceptable carriers which may be employed and the methods used to prepare the ph~rm~ce~ltic~l compositions have been described above in 25cnnnectic-n with the formlll~tione of the wound healing compositions of Embodiment One (I.A-D).

In a ~r~r.,ll~d embo~limt~nt~ the invention is directed to an immnnQstimlll~ting-wound healing ph~rm~ceutical composition which compriees 30(A) a therapeutic immnnn~etimlll~tlnsg-wound healing composition (I.A + X) which comprises:
(1) a therapeutically effective amount of an immnnQstlmlll~ting agent; and WO 96137230 PCTtUS96J'()~Ol (2) a wound heal*ng composition which comprises:
(a) pyruvate selecte~l from the group con~ictin~ of pyruvic acid, ph~rm~ce~lhc~lly acceptable salts of pyruvic acid, and ,~lu,~,s thereof;
(b) an ~n~ioxir1~nt and S (c) a lr.i~lu,~ of s~ t~ and nnc~t~lr~t~l fatty acids wherein the fatty acids are those fatty acids lc~lui~cd for the repa* of cellular Illcl~lalles and resuscitation of .. ,.. ~ n cells; and (B) a ph~rm~celltically acceptable carrier selected from the group cQncichng of ph~rm~celltical appliances, bioadhesives, and occlusive vehicles.
In another p~cr~l~l embo-lim~nt, the invention is directed to a method for preparing a ph~rm~celltic~l composition for increasing the proliferation andresuscitation rate of ~ n cells, which comrrices the steps of:
(A) providing a therapeutically effective amount of an immllnostimlll~ting-wound healing composition (I.A + X) which comprises: ( 1 ) a n immllnostimlll~hng agent; and (2) a wound healing composition comprising:
(a) pyruvate selected from the group con.eichng of pyruvic acid, ph~rm~ceutically acceptable salts of pyruvic acid, and llfi~lu~cs thereof;
(b) an antioxidant; and (c) a mi~lulc of saluu~tcd and unsaturated fatty acids wherein the.fatty acids are those fatty acids required for the repair of cellular membranes and r~sllccit~tion of ~ ...""~ n cells;
(B) providing a ph~rm~ceutically acceptable carrier; and (C) admixing the immllnostimlll~ting-wound healing composition from step (A) and the ph~rm~ce~tically acceptable carrier from step (B) to form a pharmaceutical composition.

WO 96/37230 PCT/US96J'\0!;901 f. E~mples Of The Immu, ~ sting-Wound HeJing Compositions Of Embodiment Two a.A-D ~ X) Study 1 This study ~lernonehates a co~ ;e~.~ of the wound healing abilities of the therapeutic wound healing compositions of the present invention in infecte~ and noni"rt;~ ;d rnice versus CWIV~ ..I;on~l wound healing composihone The results of this shudy are illueh~te~l in examples A-I.
The wound healing shudies were carried out using hairless mice. Five form~ hons were examined in a r~n-lnmi7~1 double blind study. There were 6 mice in each shudy group for ev~h-~hon at either day three or day seven. A mi~llin~ 3 cm, full thickness longih~ n~l incision was made on the ~nestheh7~1 mice. The creamsl 5 were applied 2 hours following the wounding and reapplied at 24 hour inteTvals during the 7 day shudy. BetafectinTM, the immllnoshmlll~hn~ agent, was applied 20 ...;..~ s before the application of the creams. Wounds were examined daily and scored for closure on a scale of 0-5 with 5 in~lic~hng the most healed. The animals were sacrificed and the tissue samples were examined histologically. Both infected and noninfected wounds were used. Regardless of the presence of infection, the compositions with BetafectinTM with live yeast cell derivative (LYCD) and the wound healing composition were significantly better as wound healing agents then the other tested fcrmlll~c This wound healing ability was rl~mr~neh~t~l by subjective grading of healing through wound closure and objective histological e~min~hons of tissuesections. The order for wound healing efficacy following the BetafectinTM with live yeast cell derivative and wound healing composition was as follows: 2) Betafectin; (3) wound healing composition with live yeast cell derivative; 4) Neosporin and wound healing composition; and 5) u~lLl~;ated conhrol.

A detailed ~Illlllll;1ly of shudies is provided below that were performed to establish that formlll~hione including BetafectinTM, the immnnoshmlll~hng agent, alone and in combination with fiormlll~h~ne including Prep~r~hQn HTM or a Neosporin base with live yeast cell derivative (LYCD) and the wound healing composition ofcompounds, sodium pyruvate, Vitamin E (alpha-tocopherol) and fatty acids .onh~nce wound h~?~linE

There were 9 groups of mice, with N=6 per group, and the groups had the following ll~n~ l A) wounded, no l~ t...~ (control); B) B~l~r~.;lil,TM alone, 2 ~lg/mouse per day, added topically in Phosph~te Burr~l~,d Saline (PBS); C) Pl~alalion HTM with wound healing composition; D) P~ .~alion HTM with BetafectinTM; and E) Neosporin form~ ti~7n with wound healing composition. Groups F-I conci~te~l of groups of 6 mice each that were treated with a culture of 107 Staphylococcus 30 minutes after wounding and 2 hours prior to trç~tmpnt with anyformulation. Groups F-I con~tit]t~l the infection model to tlett~Tmine the ability of BetafectinTM as well as the Neosporin forrn~ tic-n to enhance wound healing in the presence of an infection.
Studies were carried out using hairless SKH-1 inbred mice, 6-8 weeks of age. Mice were placed in cages of 6 each. Mice were anesthetized with ether and a midline 3 cm full thickness longitll~lin~l inci~i~7n was made with a nu lll)c. 10 scalpel blade, producing a full thickness wound that did not penetrate the underlying fascia.
Incisions were closed using steel clips at 1 c~";.. ~ intervals. Forml~l~tions were applied 30 minl-t~?~ following wounding and reapplied at 24 hours intervals during the 7 day post-operative period. Wounds were examined daily and rank ordered for effectiveness of formulation for enhancement of visual based wound healing each day.

Animals were photographed at day 3 or 4 of the cA~ ental protocol for docllm~nt~tion of visible effects of the ll~;Z I - ~ ~~ - ~t.~ Animals were sacrificed using ether e~lth~n~ on day 3 and day 7. The dorsal skin including the incision was ected without the subcutaneous tissue. The skin was placed in neutral buffered forTn~lin and subsequently sectioned and stained with ht~ ylin and eosin.
Mi~ ,scopic ex~min~tion of the wounds was performed and represe"lalivt; tissue sectionç~l photographed.

WO 96137230 PCT/US9G1~5 The T~ roups were as follows:

A. Control-wounded, no L~ l.r,.. t B. Freshly prepared P~ ion HTM fonn~ h~n col.l;.;ll;llg live yeast cellS dc.;v~liv~; (LYCD) and the wound healing composition of sodium pyruvate, Vitamin E, and 1% fatty acids.
C. Neo~o~ formulation with the wound healing c~ o~ition.
D. Betafectin~ in a solution of Phosphate Burr~ d Saline, added topically to the wound.
E. Betafectin~ and Preparation HTM with the wound healing composition. The Betafectin~M was added topically in an aqueous solution 20 minutes prior to the application of the fiorrn~ hion with wound healing composition and live yeast cell d~liv~liv~.
F-I. These groups were treated as set out above, but with the ~d~ihon of 107 Staphylococcus aureus as a bacterial infection agent applied 2 hours prior to the formlll~hons to be tested.

Photographs of the wounded mice are set out in Figures 11-14.

Figures 1 IA-l lD are photographs of wounded mice after 4 days of treatment with: no composition (Fig. 1 lA); BetafectinTM with Preparation HlM with the wound healing composition (Fig. 1 lB); BetafectinTU (Fig. 1 lC); and live yeast cell derivative with the wound healing composition.

Figure 12 is a photograph of a wounded mouse after 4 days of llr~ -t with a petrolatum base formulation only (Example D).

Figures 13A-13D are photographs of the histological results of wounded mice after 3 days of ~ llt with: no composition (Fig.llA); BetafectinTM with Pl~al~Lion H~M with the wound healing composition (Fig.l lB); BetafectinTM (Fig.l lC);
live yeast cell derivative cont~ining the wound healing composition (Fig.l lD).

=

Wo 96/37230 PCT/US96/05901 Figure 14 is a photograph of the histological results of a wounded mouse after 3 days of ll~ nt with neosporin con~ .g the wound healing composition (D).
Results: Regardless of the presence of infection, the rank order for wound healing efficacy (wound healing and closure, as well as histological correlation of rate of wound healing) was:

(E) wound healing composition and Bcl~rGcLinTM > (D) BetafectinTM > (B) wound healing composition = (C) Neosporin >> U~lleatcd.
tol D~,ic&l Description of Wounded Tissue at Day 3 From Each of the Treatment Groups A) Control: Wounded-No Tre~ .l Lower portion of the wounded area, i.e., dermis has heavy infiltration with both Iymphocytes and monocytes/macrophages.
The le e~ ç1i~1i7~tion that occurs at the outer most layer of the skin, the epidermal layer, is not complete. The tissue section shows that the dermal tissue is weak, in that the tissue integrity was not ...~ it;lled when it was sectioned.

B) Preparation HTM form~ hon with wound healing composition: From the thicker outer layer of the epidermis, the re-epith~ 1i7~tion process has progressed further than was ~p~,.lL in the ul~LIc~tcd control wounded tissue. Although there is infiltration of the dermis with monocytes and Iymphocytes, it does not appear to be as diffuse or as heavy as with the control u~lt~c~lcd wound tissue.
C) Neosporin formn1~tic n with the wound healing composition: The results with this formulation show that the re-epith~ 1i7~tion was extensive and rapid. In contrast, the infiltration of the macrophages and Iymphocytes within the derrnis was less than observed in either the control ullt~caled wound tissue or the wound tissue treated with the Plcpd-~lion HTM CO~ l;llg the wound healing composition ~ combination of agents. It appears that the fionn111~hl n~ c~ ;.. ;ng the combination of WO 96137230 PCT/US96J'~!;gOl Vitamin E, sodium pyruvate, and fatty acids shm~ tes proliferation of the outer layer of the epicl~rmi~

D) BetafectinTM in phosphate burreiçd saline: The histology of the wounded tissue S i.~ol~t5d from mice hreated for 3 days with B~ r~ TM show that there is a heavy infil~h-)n of macrophages and lymphocytes and that there is ~y~.l~ greater integrity of the tissue than that observed with any of the other l1Ç~ The re-epith~ li7~h-m is not, however, co~ ble to that observed with either the Neosporin or the Preparation HTM formlll~hon~ that contain the wound healing composition.
E) BetafectinTM and Preparation HTM wound healing composition form~ hon The histological sechon~ isolated from tissue taken from mice hreated with this formlll~h~m was by far superior in terms of both the extent of re-epitheli~li77hon as well as the extent of tissue integrity and leukocyte infilh~hon in the lower portion of the skin, the dermis. It is apparent that this combination of ingredients is able to act synergistically to Pnh~n~e wound repair in both the upper and lower portions of the skin.

The combination of Bci~r~,c~ TM and Preparation HTM wound healing composition stim~ tecl both an increase in wound healing within the lower portion of the wound, within the dermal layer, as well as shmlll~te<l the re-epitheli~li7~hon of the outer layer of the wound. These two activities resulted in a rapidly healed wound at day 3 following wounding. The difference in the activities of the other ffirmlll~hr~nc is because complete and rapid healing of wounds requires stimlll~hc n of healing in the outer and lower areas of the skin. From histological ~Y~min~h~n of the wounds, it is apparent that both the BetafectinTM alone as well as the wound healing composition alone shmlll~t~d the dermal area for wound hç~ling In contrast, the Neosporin formulation shmlll~tecl the re-epitheli~li7~hon~ that is shmlll~tecl the outer components of the skin to enhance wound closure. The combination of BetafectinTM with the wound healing composition in a P~alalion HTM like formlll~hon resulted in a synergistic enhancement of wound healing that was .~ignific~ntly better than any of the agents alone.

-WO 96/37230 PCT/US9~ 01 The mice that were infected with S. aureus and not treated with any agents were sacrificed at day 3, since the infection was not treated with antibiotics and r~m~inç~l purulent. The histology of tissue i~ol~tecl from wounds that were treated with ffirm~ hon~ of BetafectinTM, Ple~ n HTM, and wound healing comro~ihQn or S neo~ and wound healing composition, did not appear to differ with any of the except for the presence of polymo~phonncle~r cells within the dermis, where in normal uninfected tissue, the pre~lo...i.~;~..l cell type was monocytes and lymphocytes.

In ~....n~y, these studies show that there are several cc,~ ollents necessary to EJl~JalG an effective fiorm~ hon to enhance wound he~ling- (1) enhance the activity of the epithelial cells within the outer layer of the skin so that re-epithçli~li7~ion occurs at a rapid and efficient manner; (2) stimlll~te the activity of the infl~.. ;.~,ly and immune cells that come into the lower part of the skin, the dermis, in the early portion of the wound healing process; (3) prepare a stable formlll~ti~)n that is c~ mp~ihle with the BetafectinTM and the wound healing composition combination with live yeast cell derivative.

Claims

I Claim:

1. A therapeutic immunostimulating-wound healing composition which comprises a therapeutically effective amount of an immunostimulating agent and awound 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 immunostimulating agent is selected from the group consisting of betafectinTM and Freund's complete adjuvant.

3. The composition according to claim 2, wherein the immunostimulating agent is BetafectinTM.

4. The composition according to claim 1, wherein the pyruvate is selected from the group consisting of pyruvic acid, lithium pyruvate, sodium pyruvate, postassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate, methyl pyruvate, .alpha.-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 .alpha.-carotene, .beta.-carotene, gamma-carotene, and delta-carotene, all forms of Vitamin C including D-ascorbic acid and L-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 immunostimulating agent is present in the therapeutic wound healing composition in an amount from about 0.001% 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. A method for healing an infected wound in a mammal with an immunostimulating-wound healing composition which comprises the steps of:
(A) providing a therapeutic immunostimulating-wound healing composition which comprises:
(1) a therapeutically effective amount of an immunostimulating agent; 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 immunostimulating-wound healing composition with the infected wound.

16. A method for preparing a therapeutic immunostimulating-wound healing composition which comprises the steps of admixing the following ingredients:

(A) a therapeutically effective amount of an immunostimulating agent; 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.

17. An augmented immunostimulating-wound healing composition which comprises:
(A) a therapeutic immunostimulating-wound healing composition which comprises:
(1) a therapeutically effective amount of an immunostimulating agent; 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.

18. The augmented immunostimulating-wound healing composition according to claim 17, wherein the medicament useful for treating wounds is selected from the group consisting of other immunostimulating agents, antiviral agents, antikeratolytic agents, anti-inflammatory agents, antifungal agents, acne treating agents, sunscreen agents, dermatological agents, antihistamine agents, antibacterial agents, bioadhesive agents, respiratory bursting inhibitors, inhibitors of prostaglandin 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.

19. A method for healing an infected wound in a mammal with an augmented immunostimulating-wound healing composition which comprises the steps of:
(A) providing a therapeutic augmented immunostimulating-wound healing composition which comprises (1) a therapeutically effective amount of an immunostimulating agent;
(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 immunostimulating-wound healing composition with the infected wound.

20. An immunostimulating-wound healing pharmaceutical composition which comprises:
(A) a therapeutic immunostimulating-wound healing composition which comprises:
(1) a therapeutically effective amount of an immunostimulating agent; 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 pharmaceutical appliances, bioadhesives, and occlusive vehicles.