CA2129732A1

CA2129732A1 - Cytoprotective compositions containing pyruvate and antioxidants

Info

Publication number: CA2129732A1
Application number: CA002129732A
Authority: CA
Inventors: Alain Martin
Original assignee: Individual
Current assignee: Warner Lambert Co LLC
Priority date: 1992-02-25
Filing date: 1993-01-13
Publication date: 1993-09-02
Also published as: ZA931297B; AU4275793A; WO1993016690A1; EP0627917A1; MX9301014A; JPH07505145A; AU3315397A

Abstract

2129732 9316690 PCTABS00025 The present invention pertains to cytoprotective compositions for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties. In one embodiment, the cytoprotective composition comprises (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof, and (b) an antioxidant. In a second embodiment, the cytoprotective composition comprises pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of mammalian cells. The cytoprotective compositions may be incorporated into a wide variety of pharmaceutically acceptable carriers to prepare pharmaceutical products. This invention also relates to methods for preparing and using the cytoprotective compositions and the pharmaceutical products in which the cytoprotective compositions may be used.

Description

W093/16690 PCT/US93~00260 ~-f ' ;
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CYTOPROTECTIVE COMPOSITIONS CONTAININ~ ~:
PYRU~ATE AND ANTIOXIDANTS :~
~";~

~.~'BAC~:ROllND Ol? T~ TIO~ `
~ " ' This application is a continuation-in-part o~
copendinga~plication serial no. 663,50a, filed l March l99l~

Fiela of the I~v~tio~

This invention pertains to cytoprotect.ive compositions for preventing and reducing injury to mammalian rells from a medicament having cytotoxic properties, and increasing the resuscitation and proliferation rates of the cells. In a first embodiment, the cytoprotective composition comprises (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof, and (b) an antioxidant. In a ~econd embodiment, the cytoprotective composition comprises pyru~ate 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 sa~urated and unsaturated fatty acids wherein WO9~16690 ~9~ ~3~ 2 - PCT/~S93/00260 the fatty acids are those fatty acids required for the repair of cellular m~mbranes and resu~citation of mammalian cells. The cytoprotective composi~ions may be incorporated into a wide variety of pharmaceutically acceptable carriers to prepare pharmaceutical products.
This invention also r~lates ~ me~hods for preparing and using the cytoprotec~ive compositions and the pharmaceutical products in which the cytoprotective compositions may be used.

D~cription of th~ B~ckg~ou~

1~ ~~ Cancer is a group of n~oplastic diseases af~ec~ing di~erent organs and systems in ~he body. A
common feature in all cancers is cellular mutation and abnormal and uncontr~lled cell growth usually at a rate greater than that of normal body c~ . Neither th etiology of cancer nor the manner in which can~e~ causes death is completely understood.

Significant advances have been made in tha chemoth~rapeutic treatment of cancer. Most anticancer agents act at specific phases of the cell cycle and are therefore only active against cells in the process of divisio~. Although differences in the duration o~ th~
cell cycle occur between different types of cells, all cells show a similar pattern during the division proc~ss which may be characterized as follows: (1) a presynthetic phase; (2) a DNA synthesis phase; (3) a post~ynthetic phase following termination of DNA synthesis; and (4) a mitosis phase, wherein the cell containing a double complement of DNA divides into two daughter cells. Most anti-neoplastic agents act specifically on processes such as the DNA ~ynthesis phase, the transcription phase, or the mitosis phase and are therefore considered cell-cycle specific agents.

: .

WO93t16690 ~ 32~ ,? ~ `; PCT/US93/00260 A problem with the chemotherapeutic treatment of cancer is that normal cells which proliferate rapidly~
such as those in bone marrow, hair follicles, and the gastrointestinal tract, are often damaged or killed by the anti neoplastic agents. This cytotoxicity problem :~
occurs because the metabolism of cancer cells i~ similar to that of normal cells and anticancer agents lack ~:
specificity ~or cancer cells. Because most of the metabolic differences between normal and neopla~tic cells are quantitative, antican~er drugs are usually e~ployed at or near the toxic range in order to obtain satisfactory therapeutic effects.

When cells are injured or killed as a result of ~-~a cytotoxic ~gent, a cytoprotective step is desirable to protect the cells from the cytotoxic agent, resuscitate the injured cells, and h~lp produce new cell. to replace the dead cells. Injur~d cells require low levels of oxygen in the initial stages of recovery to suppr~s oxidative damage and higher leve:Ls of oxygen in the later :~
stages of recovery to stimulate cellular viability and proliferation. . ;

Stressed and inj~ured mammalian cells are often exposed to activated oxygen species such as supexoxide ~2 ), hydrogen peroxide (H2O2), hydroxyl radical (OH-), and singlet oxygen (1023. In vivo, these reactive oxygen intermediates are generated by c2115 in response to aerobic metabolism, catabolism of drugs and other xenobiotics, ultraviolet and x-ray radiation, and the r~spiratory burst of phagocytic cells ~such as white blood cells) to kill invading bacteria. Hydrog~n peroxide, for examplel is produced during respiration of most livi~g organisms especially by ~tressed and injured cells. ~:

These active oxygen species can injure and kill cells. An important example of such damage is lipid peroxidation which involves the oxidative degradation of ;~

WOg3/16690 PCT/US93/00260 3 ' unsaturate~ lipids. Lipid peroxidation is highly detrimental to membrane structure and function and can cause numerous cytopathological effects. Cells defend against lipid peroxidation by producing radi al scavengers such as superoxide dismutase, catala~e, and peroxidase. Injured cells have a decreased ~bility to produce radical scavengers. Exce~s hydrogen peroxide can react with DNA to cause backbone br~akage, produce mutations, and alter and liberate bases~ Hydrogen peroxide can also react with pyrimidines to open the 5, 6-double bond, which reaction inhibits the ability of pyrimidines to hydrogen bond to compl~mentary base-~, Hallaender et al~ (1971). Such oxidative biochemical in~ury can result in the loss of .cellular membrane ~ ntegrity, reduced enzyme activity, changes in transport kinetics, chanyes in membrane lipid conten-t/ and leakage of potassium ions, amino acids, and other cellular material.

Antioxidants have been ~hown to inhibit da~age a~sociated with active oxygen specie~. ~or example, pyruvate and other alpha-ketoacids have b~en reported to react rapidly and stoichiometrically with hydrogen peroxide to protect cells from cytolytic effects, LC9~~LL~b~C~Y_$_~l., J- Exp. Med., 165, pp. 500-514 (1987).

United States patents no~ 3,920,835, 3~98~,556, and 3,988,470, all issued to an Scott et al., disclose m~thods for treating acne, dandruf~, and palmar keratosis, r~pectively, which consist of applying to the a~fected area a topical composition comprising from about 1% to about ~O% of a lower aliphatic c~mpound containing from two to six carbon atoms selected from the group consisting of alpha-hydroxyacids, alpha-ketoacids and esters thereof, and 3 hydroxybutryic acid in a pharmaceutical~y acceptable carrier. The aliphatic compounds include pyruvic acid and lactic acid.

W~93/16690 PCT/U~93/0~2~0 ~ 5 ~ ~ 7 ~7'~:' United States patents nos~ 4,105,783 and 4,197,316, both issued to Yu et al., disclose a method and composition, respectively, for treating dry skin which consists of applying to the affected area a topical :-composition comprising from about 1% to about 20~ of a compound ~elected from the group consisting of amid~s and ammonium salts of alpha-hydroxyacids, beta-hydroxyacids, and alpha-ketoacids in a pharmaceutically acc~ptable carrisr. The compounds include the amides and ammonium lo ~alts of pyruvic acid and lactic acid~

United States patent no. 4,234,599, issued to -~
Van Scott et al., di-~closes a method for tr~ating actinic and nonactinic skin keratvses which consists of applying 15 ~-~ to the affected area a topical composition comprising an effective amount of a compound sele~ted from th~ group consisting of alpha-hydroxyacids, beta-hydroxyacids, and alpha-ket~acids in a pharmaceutically acceptable carrier~
The acidic compound~ include pyruvic acid and lactic acid.

United states patent no. 4,294,852, issued to Wildnauer et al~, discloses a composition for treating skin which comprises the alpha-hydroxyacids, beta hydroxyacids, and alpha-ketoacids disclosed above by Van S ott et al. in combination with C3-C~ aliphatic ~;
alcohols. ;~
~.
United States patent no. 4,663,166, issued to Veech, disclose~ an electrolyte solution which comprises a mixture oP L-lactate and pyruvate in a ratio from 20:1 to 1:1, respectively, or a mixture of D-beta~
hydroxybutyrate and acetoacetate, in a ratio from 6:1 to 0.5:1, respectively. ~.
;~
Sodi~m pyruvate has been reported to reduce the number of erosions, ulcers, and hemorrhages on the gastric mucosa in guinea pigs and rats caused by acetylsalicylic acid. The analgesic and antipyretic WO93/16~90 PCT/US93/00260 ~ ; ~ 6 -propertie's of acetylsalicylic acid were not impaired by sodium pyruYatp~ Puschmann, Arzneimittelforschunq, 33, pp. 410-415 and 415-416 (19B3).

Pyruvate has been reported to exert a positive inotropic effect in stunned myQcardum, which i5 a prolonged ventricular dysfunction followin~ brief periods of coronary artery occlusions which doe~ not produce irreversible damage, Mentzçr et al., ~nn _~y~g~, 20g, pp. 629-633 (1989). ~::

Pyruvate has been reported to produce a relative stabilization of left ventricular pressure and work parameter and to reduce the size of infarctions.
~-~Pyruvate improves resumption of spontaneous beating of ~he heart and restoration of normal rates and pressure development, Bunqer et al., J ~ol. Cell. Cardiol., 18, pp. 423-438 (1986), ochizuki_et_al., J. Physiol.
~3riSl, 76,pp. 805-812 ~1980)~ Re~itz et al., Cardiovasc. Res., 1~, pp. 652-658 tl981~, -Giannelli at al~, Ann. Thorac._Sur~., 2l, pp~ 386-396 (1976).

Sodium pyruvate has been reported to act as an antagonist to syanide intoxification (presumably through the formation of a cyanohydrin) and to protect again~t 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 et al., Toxicol Appl. Pharmacol., 5Q, pp. 437-442 (1979~, Sabri et al., Brain Res., 483, pp. 1-ll (19B9).

A chemotherapeutic cure of advanced L1210 leukemia has been reported using sodium pyruvate to restore abnormally deformed red blood cells to normal.
The deformed red blood cells prevented adequate drug delivery to tumor cells, Cohen, Cancer Chemother.
Pharmacol., 5, pp. 175-179 (~981).

W093/l6690 PCT/~S93/~0260 ~ ~ 2373 Primary cultures of heterotopic tracheal transplant exposed in vivo to 7, 12 dimethyl-benz(a)anthracene were reported to be succes~fully maintained in enrichment medium suppl~mented with sodium pyruvate along with cultuxes of interleukin-2 stimulated peripheral blood lymphocytes, and plasmacytomas and hybridomas, pig embryos, and human bla~tocysts, Shacter, J. Immunol. ~ethod~, 9~, pp. 259-270 ~1987), ~archok et al., Cancer Res., 37, pp. 1811-1821 (1977), Davis, J. Reprod. Fertil. Su~pl., 33, pp. 115-124 (1~85), Okamoto et al,, No To_Shinkei, 38, pp. 593-59B ~1986), Cohen et al., J. In Vitro Fert. Embryo Transfer, 2, pp. 59-64 (19 15 ~
United States patents nos. 4,158,057, 4,351,835, 4,415,576, and 4j645,764, all iss~ed to .Stanko~ di~close methods for pre~enting the accumulation of fat in the liver of a mammal due to the ingestion of alcohol, for controlling weight in a mammal, for inhibiting body fat while in~reasing protein concentration in a ma~mal, and for controlling the deposition of body fat in a living being, respectively.
The methods comprise administering to the mammal a therapeu.ic mixture of pyruvate and dihydroxyacetone, and optionally riboflavin. United States patent no. 4~548,937, issuPd to Stanko, discloses a method for controlling the weight gain of a mammal which compris~s administering to the mammal a therapautically effective amount of pyruvate, and optionally riboflavin. United States patent no. 4,812,479, issued to Stanko, discloses a method for controlling the weight gain of a mammal which comprises administering to the mammal a therapeutically effective amount of dihydroxyacetone, and optionally riboflavin and pyruvate.

Rats fed a calcium-oxalate lithogenic diet including sodium pyruvate were reported to develop fewer urinary calculi (stones) than control rats not given W093~16690 ~ 8 - PCT/US93/00260 sodium pyruvateJ O~awa et al., Hinyokika Ki~o, 32, pp. 1341-1347 (1986). ~-United States patent no. 4,521,375, is~ued to .
Houlsby, disclose~ a msthod for sterilizing surfaces ;~
which come into contact with living tis~ue. The method comprises sterilizing the surface with aq~eous hydrogen p~roxide and then neutralizing the surface with pyruvic acid.
United State~ patent no. 4,~16,9~2, issued to Tauda et al., discloses a method for decomposing hydrogen peroxide by reacting the hydrogen peroxide with a phenol ~-or aniline derivative in the presence of perox~ase.
15 ~
United States patent no. 4,696,~17, i~sued to Lindstrom et al.~ discloses an eye irrigation solution which compris~s Eagle's Minimum Essential Mediu~ with Earle~s saltsO chondroitin sulfate, a buffer solution, 2-mercaptoethanol, and a pyruvate~ The irrigation solutionmay optionally contain ascorbic acid and alpha-tocopherol. United State~ patent no. ~,725,586, issued to Lind~trom et al., discloses an irrigation ~olution which comprises a balanced salt solution, chondroitin sulfate, a buffer solution, 20mercaptoethanol, sodium bicarbonate or dextrose, 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 treatin~ fing~rnails and toenails which consists essentially of soybean oil or sunflower oil in combination with Vitamin E.

United States patent no. 4,847,069, issued to Bissetk et al., discloses a photoprotective composition comprising (a) a sorbohydroxamic acid, (b~ an anti-inflammatory agent selected from ~teroidal anti-inflammatsry agents and a natural anti-inflammatory W093/l6690 2 1 2 ~ i7-~ PCT/~S93/00260 agent, and (c) a topical carrier. Fatty acidis may be present as an emollient. United State~ p2tent no. 4,847,071, issued to Bissett et al., discloses a photoprotective composition comprising la) a ~ocopherol or tocopherol ester radical scavenger, (b) an anti-infla~matory agent selected from steroidal anti~
inflammatory agents and a natural anti-inflamm2~0ry agent, and (c) a topical carrier. Unit~d States patent no. 4,3~7,072, issued to Bissett e~_alO, dis¢loses a topical composition comprising not more than 25%
tocopherol sorbate in a topical carrier.

United States patent no. ~ 9 533,~37, issued to Yamane et al., discloses a culture medium which comprises ~a carbon source, a nucleic acid source precursor, amino acids, vitamins, minerals, a lipophilic nutri~nt, and serum albumin, and cyclodextrins. ~he lipophilic substances include unsaturated fatty acids and lipophilic vitamins such as Vitamin A, D, and E~ Ascorbic acid may also be present.

United Kingdom . patent application no. 2,196,348A, to Kovar et al., discloses a synthetic culture medium which comprises inorganic salts, monosaccharides, amino acids, vitamins, buffering agents, and optionally sodium pyruvate adding magnesium hydroxide or magnesium oxide to t~e emulsion. The oil phase may include chicken fat.

United States patent no. 4,284,630, is~ued to Yu et al~, discloses a method for stabilizing a water in-~il emulsion which comprises adding magnesium hydroxide or magnesium oxide to the emulsion. The oil phase may include chicken ~at.
Preparation HT~ has been reported to increa~2 the rate o~ wound healing in artificially created rectal ulcers. The active ingredients in Preparation-HTM are skin respiratory factor and shark liver oil, WO93116690 ~ lO - PCT/US93/00260 Subramanyam et al., Diqestive Diseases and Sciences, 2~, pp. ~29-~32 (1984).

The addition of sodium pyruvate to bacterial and yeast systems has been reported to inhibit hydrogen peroxide productiorl, enhance growth, and protect the systems against the toxicity of reactive oxygen int~rmediates ~ The unsaturated f atty ac:ids and saturated f atty acids contained within chicken f ~t enhanced 10 membrane repair and reduced cytotoxieity. The antioxidants glutathione and thioglycollate redured the injury induced by oxygen radical species, lMartin, Ph. ~.
thesis, ( 19 87 -8 9 ) .

15 ~~ United States patent no. 4,615,697, issued to Robinson, discloses a con~rolled release treatment composition comprising a ~reating agent and a bioadhesive agent comprising a water swellab:le but wat~r~insoluble, fibrous cross-linked carboxy-functional polymer.

2~
European patent application no. 0410696A1, to Kellaway~ et al., di~closes a mucoadhesive delivery syst~m comprising a treating agent and a polyacrylic acid cross-linked with from about 1% to about 20% by weight of a polyhydroxy compound such as a sugar, cyclitol, or lower polyhydric alcohol.

While the above ther~peut~c compositions are r~ported to inhibit the production of reactive oxygen intermediates, none of the above compositions are entirely satisfactory cytoprotective compositions. None of the compositions has the ability to simultaneously decrease cellular levels of hydrogen peroxide production, increase cellular resistance to cytotox.ic agents, increase rates of cellular proliferation, and increase cellular viability to protect and resuscitate ma~malian cells. The present invention provides such improved therapeutic cytoprotective compositions without the wo g3/l66go - 11 2 1~ 2 i ~ ~ PC~/US93~00260 disadvantages characteristic of previously known compositions.

~N~RY OF T~E I~V~TIO~
': ' The present invention pertains to cytoprotecti~e composition~ for preventing and reducing injury to mammalian c~lls from a medicament having cytotoxic properties. In a first ~mbodiment, the cytoprotective composition comprises (a) pyruvate elect~d from the group consi~ting of pyruvic acid, pharmaceutically acceptable salts of pyxu~ic acidr and '-~mixtures thereo~, and (b) an antioxidant. Xn a ~econd embodiment, the cytoprot~ctive composition comprises (a) pyruvate select~d from the group consisting of pyruvic acid, pharmac~utically acceptable salts of pyruvic acid, and mixtures thereof, (b) an antioxidant, and (c~ a mixture o~ saturated and unsaturated fatty ~cids wherein the fatty acids are those fatty acids required for th~
repair of cellular msmbranes and resuscitation of ma~malian cells.

The cytoprotective compositions of the present invention may be admini~tered to cells concuErently with a cytotoxic agent. The cytoprotective compositions may also be administered to cells prior to administration of a cytctoxic anticancer agent to sel~ctively protect non~
cancerous cells in the presence of cancerous cells.
-The cytoprotective compositions may be incorporated into a wide variety of pharmac~utically `~;~
acceptable carriers to prepare pharmaceutical produc~s. `~:
This invention also rela~es to methods for preparing and using the cytoprot~ctive compositions and the pharmaceutical products in which the cytoprotective :~
compositions may be used.

WO93/16690 - 12 - PCr/US93/002~0 BRII;:l? DE5CRIPl'ION OF THE FIG~

Figure 1, top portion, is a graph showing the viability of U937 mono ytic leukemia tumor cells ater 2 4 hours, as determinPd by tritiat~d thymidine i~corporation assay, following treatment oiE the cells with different dosage levels of Doxorubicin. Figure 1, bottom portion, is a graph showing th~ vlability o~ Ug37 monocytic l~ukemia tumor cells after 24 hours, as determined by exc-lusion of the vital dye trypan blue ;~
assa~r, following treatment of the ceils with different dosage levels of Doxorubicin. . `
15 ~
Figure 2 is a graph showing the viability of U937 monocytic leukemia tumor cell~ after 1 hour, as ~ ~:
determined by exclusion of the vital dye trypan blue assay, following treatment of the c:ells with different 2 0 dosage levels o~E Doxorubicin .

Figure 3 i~ a graph showing the viability of U937 monoc:ytic: leukemia tumor cells after 24 hours~ as determined by tritiated thymidine incorporation assay, following ~reatment of the cells with the cytoprotec:tive components of the present invention, alone and in combinations, at different dosage levels.

Figure 4 , top portion, is a graph showing the ~.:
30 viability of U937 monocytic leukemia tllmor cells in a wash-out study, as determined by tritiated thymidine inc:orporation assay, after 24 hour pretreat~ent of th~
cells wi~h 5 mM sodium pyruvate followed by administration of dif f erent dosage levels of Doxorubicin .
Figure 4, bottom portion, is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidin~ inc:orporation assay, after 24 hour pretreatment of the cell~ with 5 mM

WO93/16690 PCT/~S~3/00260 - 13 ~ 2 9 ~ ~ . J

sodium pyruvate followed by administration of different dosage levels of Doxorubicin.

Figure ~l top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wa~h-out ~tudy, as dete~mined by tritiated thymidine incorporation a~say, after 24 hour pr~treatm~nt of the cells with 0~5% fatty acids followed by admini~tration of different dosage levels of Doxorubici~ Figure 5, bo tom portion, i5 a graph showing the viability of peripheral blood monocytes in a wash-out study, a~ determined by tritiated thymidine incorporation assay, a~ter 24 hour pretreatment of the cells with 0~5% fatty acids followed by administration of different dosage level~ of 15 ~-~ Doxorubicin~

Figure 6, top portion, is 3 graph showinq the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated th~midine incorporation assay, after 24 hour pretrea~m~nt of the cells with 10 U Vitamin E followed by administration of different dvsage levels of Doxorubicin. Figure 6, bottom portion, is a graph showing the viability of p~ripheral blood monocytes in a wash-out study, as d~termined by tritiated thymidine incorporation assay, after 24 hour pretrPatment of the cells with 10 U Vitamin E followed by administration of different dosa~e levels of Doxorubicin, Figure 7, top portion, is a graph showing the viability of Ug37 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretr~atment of the cells with 50 U Vitamin E followed by administration of diferent dosage levels of Doxorubicin. Figure 7, bottom portion, is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E followed by administration of different dosage levels of Doxorubicin~

W093/16690 ~ 3~ ~3 - 14 - PCT/US~3J~0260 Fi~ure 8, top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated ~hymidine incorporation assay, aft2r 24 hour pretreatment of the cells with 5 mM sodium pyruvate and ~.5% fatty acid~
followed by administration of different dosage levels of Doxorubicin. Figure ~ bottom portion, is a graph showing the viability of peripheral blood mono~yteg in a wash-out study, as determined by tritlated thymidine incorporation assay, after 24 hour pretreatment of the c~lls with 5 mM sodium pyruvate and 0.5~ fatty aci~-~
followed by admini~tration of different dosage levels of Doxorubicin.
15 ~~ :
Figure 9, top portion, is a graph showing the viability vf U~37 monocytic leukemia tumor cells in a wash-out ~tudy, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatme~t of the cells with 5 mM sodium pyruvate and 10 U Vitamin E
followed by administration of different dosage levels of Doxorubicin. Figure 9, bottom portion, is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated th~midine inc~rporation assay, after 24 hour pretr~atment of the cells with 5 mM sodium pyruvate and 10 U Vitamin E
followed by administration of different dosage levels of Doxorubicin.

Figure lO, top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 50 U Vitamin E
followed by administration of different dosage levels o~
Doxorubicin. Figure lO, bottom portion, is a graph ~howing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the WO93/1669~ - 15 ~ PCT/~S93/00260 cells with 5 mM sodium pyruvate and 50 U Vitamin E
followed by administration of different dosage level~ of Doxorubicin.

Figure 11, top portiont is a graph showing the viability o* U937 monocytic leukemia tumor cells in a wash-out s~udy, as determined by tritiated thymidine `:
incorporation assayO aft~r 24 hour pretreatment o~ the cells wi~h lO U Vitamin E and 0.5% fatty acids follow~d by admini~tration of different dosage levels of :~
Doxorubicin. Figure 11, bottom portion, is -a graph showing the ~iability of peripheral blood monocyt~s in a wash-out study, as determined by tritiated thymidine inc~rporation assay, after 24 hour pretreat~ent of the 15 ~-~cells with lO U Vitamin ~ and 0.5% fatty acids followed .
by administration of different dosage levels of Doxorubicin. -~

Figure 12, top portiont is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24.hour pretreatment of the cells with 50 U Vitamin E and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin. Figure 12, bottom portion, is a graph showing the viability of peripheral blood monocyt~s in a wash out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E and 0~5% fatty acids followed by administration of different d~sage levels of Doxorubicin.

Figure 13, top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out s~udy, as determined ~y tritiated thymidine incorporation a~say, after 24 hour pretreatment of the cells with 5 ~M s~dium pyruvate, 10 U Vitamin E, and 0.5%
fatty acids rollowed by administration of different do~age levels of Doxorubicin. Figure 13, bottom portion, WOg3/16690 ..~ PCT/US93/00 ~ ;? ~ 16 is a graph showing the viability of peripher~l blood monocytes in a wash-~ut study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with ~ ~ sodium pyruvate, 10 U Vitamin E, and 0.5~ f~tty acids followed by administration of di~ferent dosage levels of Doxorubicin.

Figure 14, top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 2~ hour pr~treatment of the cells with 5 mM sodium pyruvate, 5U U Vitamin E, and 0.5%
fatty acids followed by administration of different do~age levels of Doxorubicin. Figure 14, bottom portion, 15 ~ïs a graph showing the viabillty of peripheral blood monocytes in a wash-out study, as determined by tritiated t~ymidin~ incorporation assay, after 24 hour pretreatment of the cells with 5 mM ~odium pyruvate, 50 U Vitamin E, and 0 . 5% fatty acids followedi by administration oP
20 different dosage levels of Doxorubicin.

Figure 15, top portic:~n, is a graph showing the viability of Ug37 monocytic leukemia tumor cells in a co-culture study, a5 determined by tritiated thymidine incorporation assay, after 24 hour pretreat~ent of the cells with 5 mM odium pyruvate followed by administration of different dosage l~vels of Doxorubicin.
Figure 15, bottom portion, is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorpoxation assay, after 24 hour pretreatment of the cells with 5 mM
sodium pyruvate followed by administration of diff~rent dosage levels of Doxorubicin.

Figure 16, top portion, is a graph showing the viability of U937 monocytic leukemia tumor rells in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of t~e cells with 0.5~ fatty acids followed by administration of WO 93~ 1 6690 PCI`/USg3/0~260 -- 17 ~-, ~ 2 ?~ J ''~' different dosage levels of Doxorubicin. Figure 16, bottom portion, is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, 5 after 24 hour pretreatment of the cells with 0 . 5% fatty acid~ followed by administration of different dosage levels of Doxorubicin.

Figure 17, top portion, is a graph showing the viability of U937 monocytic leuk~mia tumor cells in a co-culture study, as d~termined by tritiat@d thymidin~
incorporation assay, after ~4 hour pretr~atment of the cells with 10 U Vitamin E followed by admini~tration of different dosa~e levels of Doxorubicin. Fi~ure 17, 15 ~-~ bottom portion~ is a graph showing the viability o~
periph ral blood monocytes in a co-culture ~tudy, as determin~d by tritiated thymidine incorporatiDn assay ~
a~ter 24 hour pretreatment of the cells with 10 U
Vitamin ~ followed by administration of different do~age l~vel~ of Doxorubicin.

Figure 18, top portion, is a graph showing the viability of U9~7 monocytic leukemia tumor cells in a co~
culture study, as determined by tritiated thymidine 25 incorporation a~;say, after 24 hour pretreatment of the cell~ with 50 U Vitamin E followed by administration of ~iffer~nt dosage le~els of Doxorubicin. Fi~ure 1~, bottom portion, is a graph showing the viability of peripheral blood monocytes in a co~culture ~tudy, as determined by tritiated thymidine incorporation as~ay, after 24 hour pretreatment of the cells with 50 U
Vitamin E followed by administration of different dosa~e levels of Doxorubicin.

Figure 19, top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 0~5% fatty acids WO93/16690 ~ 8 - PCT/US93tO0260 followed by administration of different dosage levels of Doxorubicin~ Figure 19, bottom portion, is a graph showing the viability of perip~eral blood monocytes in a co-culture ~tudy, as determined by tritiated thymidine incorporation assay, after 24 hour pr~txeatment of the cells with 5 mM sodium pyruvate and 0.5% fat~y acids followed by administration of different dosage levels of Doxorubicin.

Fi~ure 20, top portion, is a graph showing the viability of U937 mon~cytic leukemia tumor cells in a co-culture study, as determined by tritiated thymidin~
incorporation assay, after 24 hour pretreatmant of the cells with 5 mM sodium pyruvate and lO U Vitamin E
15 ~ ollowed by administration of different do~age levels of Doxorubicin. Figure 20, bottom portion, is a graph showing the viability of peripheral blood monocytes in ~
co-culture ~tudy, as detPrmined by tritiated thymidine incorporation assay, after 24 hour pretr~atm~nt of the cells with 5 mM ssdium pyruvate and lO U Yitamin E
fsllowed by administration of different dosage levels o Doxorubicin.

Figur~ 21, top portion, is a graph showing the viability of U~37 monocytic leukemia tumor cells in a co-culture study, as determined by tritiated thymidine inccrporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 50 U Vitamin E
followed by administration of different dosage levels of Doxorubicin. Figure 21, bottom portion, is a graph showing the viability of peripheral blood monocyte~ in a co-cultuxe study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and S0 U Vit~min E
followed by administration of different dosage l~vels of Doxorubicin.

F'igure 22, top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a co-WO93/166~0 - 19 ~ 2 ~`37 ? ~ PCT/US93/00260 culture study, as determined by tritiated thymidine incorporation as~;ay, after 24 hour pretreatment of the cells with 10 U Vitamin E and O . 5% fatty acid~ followed by administration of different dosage levels of 5 Doxorubicin. ~igure 22, bottom portiorl, is a graph ~howing the viability of peripheral blood monocyt~s in a co-culture study, as de ermined by txitiated thymidine incorpor~tion assay, a~ter 24 hour pretreat~ent of the cells with 10 U Vitamin E and 0. 5% fatty acid~ followed by administration of differ~nt dosage lev~ls of Doxorubicin.

Figure 23, top portion~ is a graph showing the viability of U937 monocytic leukemia tumor cells in a co-~~ culture study, as determined by tritiated thymidineincorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E and O~% ~atty acids followed by administration of different dosage level~ of Doxorubicin. Figure 23, botto]m portio~ is a graph showing the viability of periphe.ral blood monocyte~ in a CD culture study, as determined by ~ritiated thymidine incorporation assay, a~ter 24 hour pretreatment of the cells with 50 U Vitamin E and 0.5% fatty acids followed by a~ministration of different do~age levels of Doxorubicin.

Figure 24, top portion, is a graph showing the viability of U937 monocytic leukemia ~umor cells in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretr~atment of the cells with S mM ~odium pyruvate, 10 U Vitamin E, and O.5%
fatty acids followed by administration of different dosage levels of Doxorubicin. Figure 24, bottom portion, is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determin~d by tritiated thymidine incorporation assay, after 24 hour `~`
pretreatment of the cells with 5 mM sodium pyruvate, lO U
Vitamin E, and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.

W0~3/1669~ PCT/US93tO0260 ~ 20 -~7~

Figure 25, top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a co cul~ure study, as determined by tritia1:ed thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate, 50 U Vitamin E, and 0.5~
fatty acids followed by administra~ion of di~ferent dosage levels of Doxorubicin. Figur~ 25, bottom portion, is a graph showing the Yiability of p~ripher~l blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 m~ sodium pyruvate, 50 U
Vitamin E, and 0.5~ fatty acids followed by adminis~.ration of different dosage levels of Doxorubicin.
15 ~-D~AIL~D D~BC~IP~ION OF T~ I~VB~TIO~

Applicant has discovered cytoprotective compositions for protecting mammalian cells from a medicament having cytotoxic properties by preventing and reducing injury to the cells. Cells treated with the cytoprotective compositions o~ the present invention show decreased levels of hydrogen peroxide production, increased resistance to cytotoxic agents, increased rates of proliferation, and increased viability. The cytoprotective compositions may be administered to cells concurrently with a cytotoxic agent or the cytoprotective compositions ~ay be administered to cells prior t~
administration of a cytotoxic anticancer agent to selectively protect non-cancerous cells in the presence of canc~rous cells. Because cancerous cells have a rapid metabolism, cancerous cells will rapidly consume the protective cytoprotective composition and will not be protected by the cytoprotective compositions when the chemotherapeutic medicament is subsequently administered.

W O 93/1fi690 PCT/US93/00260 ~ / h 3 ~J ~ J
The term "injured cell" as used herein means a cell which has (a) injured membranes so that transport through the membranes is diminished resulting in an increase in ~oxins and nsrlmal cellular wastes inside the cell and a decrease in nutrients and other component~
necessary 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 produc antioxidants and enzy~ne~, and (c~ damaged D~A, ~UNA, and ribosomes which mus~ be repaired or replac~d before norlnal cellular functi4ns can be resuumed. The tex~n ~'resuscitation~' of injured maDu~alian cells a~ u~ed herein means the reversal of cytotoxicity~ the stabilization of the cellular membrane, an increase in ~ he proliferation rate of the cell, and/or the normalization of cellular func~ions such as the ~ecretion of gro~rth factors, hor~nones, and the likeO The ter~n "cytotoxicity'l as used herein means a condition cau~d by a cytotoxic agent that injures the cell. Injured cells do not proliferate because injured cells e~pend all energy on cellular repair. Aiding cellular repair promotes cellular proliferation.

Epidermal keratinocytic cells and monocytic cells have multiple oxygen generating mechanisms and the degree to which each type of mechanism functions diff~rs in each type of cell. In monocyte~, for example, the r~spiratory bursting process is more pronounced than in epidermal keratinocytes. Hence, the compo~ents in the cytoprotective compositions of the pr~se~t inv~ntion may vary depending upon the types of cells involved in the condition being treated.

In a first embodiment, the therapeutic cytoprotective composition for treating mammalian cell~, preferably epidermal keratinocytes, comprises (a~
pyruvate, ~b) an antioxidant, and (c~ a mixture of saturated and unsaturated fatty acids.

WO93/16690 ~c?~ 22 PCT/US93/00260 While not wishing to be bound by theory, applicant believes that pyruvate (or pyruvic acid) can be transported inside a cell where it can act as an antioxidant to neutralize oxygen radicals in the cell.
Pyruvate can also be used inside the cell in th~ citric acid cycle to provide enerqy to increase cellular viability, and as a precursor in the syntheci~ of important biomolecules to promote cellular proliferation.
In addition, pyruvate can be used in the multi~unction oxidase system to reverse cytotoxicity. Antioxidant~ D
especially lipid-soluble antioxidants, can be absorbed into the cell membrane to neutralize oxygen radicals and thereby protect the membrane. The combination of pyruvate inside the cell and an antioxidant in the ~cellular m~mbrane functions in a sy~ergistic manner to reduce hydrogen peroxide production in the cell to l~vels lower than can be achieved by use of either type of component alon~.

The saturated and unsaturated ~atty acids in the present invention are those :Eatty acids required for the repair of cellular membranes and resuscitation vf mammalian cells. Hence, the fatty acids in the cytoprotective composition, which may be i~ the form of mono-, di-, and/or triglycerides or free fatty acids, are readily available for the repair of injured cells and the production of new cells to replace dead cells. Cells injured by oxygen radicals need to produce unsaturated fatty acids to repair cellular membranes~ However, the production of un~aturated fatty acids by cells requires oxygen. Thus, the injured cell needs high levels of oxygen to produce unsaturated f atty acids and at the same time needs to reduce the level of oxygen within the cell to reduce oxidative injury. By pro~iding the cell with the unsaturated fatty acids needed for repair, the need of the cell to produce unsaturated fatty acids is reduced and the need for high oxygen levels is also reduced. The presence of mixtures of saturated and unsaturated fatty acids in the cytoprotective composition significantly W093/l6690 - ~3 ~ P~T/U~93/00260 enhanc~s the ability of pyruvate and the antioxidant to inhibit reactive oxygen production. By stabilizing the cellular membrane, unsaturated fatty acid~ also improve membrane function and enhance pyruvate transport into the cell. By improving the viability of the cells, u~saturat d fatty acids also improve the repair o~
cellular membranes rate of the cellsO Hence~ the three components in the cytoprotective composition function tog~th~r in a syn~rgi~tic manner to prevent and reduce injury to mammalian cells, increa~2 the resu~citation rate of injured cells, and increase the production of new cells.

In a second embodiment, the therapeutic ~-~cytoprotective composition for treating mammalian c~lls, preferably epidermal keratinocytes, comprises (a) pyruvate, (b) lactate, and (c) a mixture of saturated and wlsaturated fatty ac:ids. In this embodiment, lactate i~
employed instead of an antioxidant. Antioxidants react 20 with, and neutralize, oxygen radicals after the radic:als are already f ormed . Lactate, on the other hand, is a component in the cellular feedback m~chanism and inhibits the re~piratory bursting process to suppress the production of active oxygen species. The combination of pyruvat to neutralize active oxygen species and lactate to suppress the respiratory bursting process functions in a synergistic manner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved by use of either type of component alone. The presence of mixtures of saturatad and unsaturated fatty acids in the cytoprotective composition significantly enhances the ability of pyruvate and lactate to inhibit reactive oxygen production. Hence, the ~hree components in the cytoprotective composition in this embodiment func~ion together in a synergistic manner to prevent and reduce injury to mammalian cells, and increase the proliferation and resuscitation rate of the cells.

Wo93/16690 ~`~13~` ~ 24 - P~T~US93/00260 In a third embodiment, the therapeutic cytoprotective composition for treating mammalian cells, preferably epidermal keratinocytes, comprises (a) ~n antioxidant, and (b) a mixture of ~aturat~d and unsaturated fatty acids. The presence of mixtures oP
sa~ura~ed and unsaturated ~a~ty acid~ in the cytoprotective composition in this embodiment significantly enhances the ability of the antioxidant o inhibit reactive oxygen production. The co~bination of an antioxidant to neutralize active oxygen species and fatty acids to rebuild cellular membranes and reduce the need of the cell for oxygen functions in a ~ynergistic manner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved.by either type of 15 ~-~ component alone. ~ence~ the component~ in the cytoprotective composition in this embodiment function together in a ~ynergistic manner to prevent and reduce injury to mammalian cells, and increase the proliferation and resuscitation rate of the cells.
In a fourth embodiment, the therapeutic cytoprotective composition for treating mammalian cells, preferably monocytes, comprises (a) lactate~ (b) an antioxidant, and (c) a mixture of saturated a~d unsaturated fatty acids. In ~hîs embodiment, lactate is employed because the respiratory bursting process is more pronounced in monocytes than in epidermal kerati~ocytes.
The combination of lactate to suppre~s the respiratory bursting process and an antioxidant to neutralize actiYe oxygen species functions in a synergi~tic manner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved by either component alone.
The presence of mixtures o saturated and unsaturated fatty acids in the cytoprotective composition in this embodiment significantly enhances the ability of lactate and the antioxidant to inhibit r~active oxygen production. Hence, the three components in the cytoprotective composition in this embodiment function together in a synergistic manner to prevent and reduce WO 93/16690 - 2 5 ~ 7 Pcr/us93/00260 injury to mammalian cells, and increase the proliferation and resuscitation rate of the cells.

In a f if th embodiment, the therapeutic 5 cytoprotective composition for treating mammalian cells, preferably epidermal keratinoc:ytes, c:~smprises (a) pyruvate, and (b~ an antioxidan~ hen ~he therapeutic cytoprotective composition in thi~; embodim~nt is administ~red to cells before the cytotoxic agent is 10 aslministered, the c:ombination of pyruvat:e inside the cell and the antioxidant in the cellular membr~ne functions in a synergistic manner to redllce hydrogen peroxide produc:tion in the cell and ~hereby prevent injury to the cell. When injury to the cell is prevented and the cell 15 '~-f ~ aes not require resuscitation, the mixture of saturated and unsaturated f atty acids need not be employed in the c:ytoprotective composition. Hence, the two component~; in the cytoprotective compositis:~n in this embodiment ~Eunction together in a synergistic manner to pr~vent and 20 reduc~ injury to mammalian cells..

Accordingly, the combination o~ ingr~di~3nts set out in the above embodiments function~ together in an enhanc:ed manller to prevent and reduce injury to mammalian 25 cells and to increase the proliferation and resuscitation rate of mammalian cells. The therapeutic effect of the combinatic~rl of the components in eac:h of the a}: ove embodim~nts is markedly greater than that expected by the mere additic:~n of the individual therapeutic eomponents.
30 Hence, applicant' s therapeutic cytoprotec:tive compositions have the ability to decrease intracellular levels of hydrogen peroxide productiorl, increase cellular resistance to cytotc~xic agents, increa~e rates of cellular prolieration, and increase cellular viability.
The cells which may be treated with the c:ytoprotective compositions in the present invention are mammalian cells. Although applicant will de~cribe the present cytoprotective compositions as useful for , .
,;.

W093/16690 P~T/~S93/00260 ~ ~ 26 -b~ ?~
treati~g ~ammalian epidermal keratinocytes a~d mammalian monocytes, applicant contemplates that all mammalian cells which may be protected or resuscitated by applicant's cytoprotective compositions may be used in the present invention. Keratinocytes are represe~tativ. :~-of normal mammalian cells and are the f astest proliferating cells in the body. The correlation b~tween the reaction of keratinocytes to injury and ~herapy and that of mammalian cells in general is very high.
Monocytes are representative of specialized ~a~malian -~
cells such as the white blood cells in the i~mune system : ~.
and the organ cells in liver, kidney, heart, and brain.
The mammalian cells may be treate~ i~ viv~ and in vitro.

15 ~-~ Epidermal keratinocytes are the specializ~d epithelial cells of the epidermis which synthesizQ
keratin, a scleroprotein which is th~ principal constituent of epidermis~ hair, nails, horny tissue, and the organic matrix of the enamel of teeth. Mammalian --epidermal keratinocytes constitute about g5% of the epidermal cells and together with melanocytes form the binary system of the ep'.dermis. In its various ~uccessive stages, epidermal keratinocytes are also known as basal cells, prickle cells, and granular cells~
Monocytes are mononuclear phagocytic leukocytes which undergo respiratory bursting and are involved in reactive oxygen mediated damage within the epider~
Le~k~cytes are white blood cells or corpuscles which may be classified 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 and monocytes. Phagocyte cells are cells which ingest mi.croorganisms or other cells and foreign ~.
particles. Monocytes are also know~ as large mononuclear leukocytes, and hyaline or transitional leukocytes. :.

WO 93/16690PCr/lJS93/1)0260 f-~7 f I 1 ~ 7~1 J b ,~

Pyruvic acid (2-oxopropanoic acid, alpha-ketopropionic acid, CH3COCOOH) or pyruvate (at physiological pH) isa fundamental int~3rmediate in 5 protein and carbohydrate metabolism and in the citric:
ac:id cycle. The citric:~ acid cycle (tricarboxylic acid cycle, Xreb's cycle) is the major reaction ~equenc~ which executes the reduction of oxygen to gerlerate adenosine triphosphate (ATP) by oxidizing organic compounds in 10 respiring tissues to provide electrc)ns to the transport system. Acetyl coenzyme A ( "active acetyl'~ oxidized in this proce~s and is thereaft:er utilized in a variety of biolc:~gical process~s and is a precu~sor in the biosynth~sis of many fatty acids and sterol~. The two 15 ~-~ major sources of acetyl coenzyme A are deriv~d ~rom the metabolism of glucose and fatty acids. Glycolysis consists of a series of transformations wherein each gluco e ~molecule is transformed in the c~llular cyt:opla~m into two molecules of pyruvic acid. Pyruvic acid may 20 then enter the mitochondria where it is oxidiz~d by co~nzyme A in the pres~nce of lenzymes and cofactors to acetyl c:oerlzyme A. Acetyl coenzyme A can then ent~r th~
citric ac:id cycle.

2~ In muscle, pyruvic acid (derived from glycogerl) i~ reduced to lactic acid during exertion. Lactic acid is reoxidized and partially retransfor~ned to glycogen during rest~. Pyruvate can also act as an antioxidant to neutralize oxygen radicals in the cell and can be used in 30 the multifunction oxidase system to reverse cytotoxicityO

The pyruvate in the present invention may be selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof. In general, the pharmaceutically acceptable salts of pyruvic acid may be alkali salts and alkaline earth sal~s~ Preferably, the pyruvate is selected ~rom the group consisting of pyruvic acidr sodium pyruvate, potassium pyruvate, magnesium pyruvate, s. ~ ~ 28 - ~CI/US9310~260 calcium pyruvate, z inc pyruvate, manganese pyruvate, and mixtures thereof . More pref erably, the pyruvate is selected from the group of ~alts consisting of sodium pyruvate, potassium pyruvate, magnesium pyruYate~ c:alcium 5 pyruvate, z inc: pyruvate, manganese pyruvate, and ~aixture~;
thereof. P~o~t preferably, the pyruvate is ~;odium pyruvate.

The amount of pyruvate pres~nt in the 10 cytoprotec:tiv~ c:ompositiorls of the pr~sent invention is a th~rapeutically effective amount. A therap~utically effective amount of pyru~ate is that amount of pyruvate necessary to increase the proliferation and resuscitation rate of mammalian cells. The exact amount of pyruvate is 15 ~-~a matter of pref erence sub j ect to such f ac:tors as lthe type of condition being treatecl as well as the other ingr~dients in the composition. When t}le c:ytoE)rotectisr~
compo~ition contains two components, pyruvate is preferably pr~sent in the cytopro~ective c:omposition in 20 an amount from about 10% to about 75%~ pre~erably from about 20% to about 60%, and more pr~ferably from about 25% to about 55%, by weight of the cytoprotective compositior~. When the cytoprotectiv~ composition contains three componerlts, pyruva~e is pref erably present 25 in the cytoprotective composition in an amount from about 1096 to about 50%, preferably from about 20% to about 45%, and more preferably from about 25% to about 40~6, by weight of the cytoprotective c:omposition.

L Lactic acid ((S)-2 hydroxypropanoic acid, (~) alpha-hydroxypropionic acid, CH3CHOHCOOH) or lactate occuxs in small quantities in the blood and muscle fluid of mammals. Lactic acid concentration i~crea~es in mu~cle and blood after vigorous activity. Lactate is a component in th~ cellular feedback ~echanism and inhibits the natural respiratory bursting process of cell~ thereby suppressing the production of oxygen radicals.

WO93/166~0 - 29 - ,-";~ PCT/US93/00260 ,' S :J ~ ,1 The lactate in the present invention may be selected from the group consisting of lactic acid, pharmaceutically acceptable salts of lactic acid, and mixtures thereof. In ~eneral, the pharmaceutically acceptable salt~ of lactic acid may be alkali salts and alkaline earth salts. Preferably, the lactate i~
selected from the group consisting of lactic acid, sodi~m lactate, potas~ium lactate, magnesium lactate, calcium lactate, zinc lactate, mangane~ lactate, and mixtures thereof. ~ore preferably, the lactate is selected from the group consisting of lactic acid, sodi~ lactate, potassium lactate, magnesium lactate, calcium lactate, zinc lactate, manganese lactate, and mixtures thereof.
Most preferably, the lactate is lactic acid.
15 ~-~
Th~ amount of lactate present in thecytoprotectiYe compositions of the present invention is a therapeutically effective amount. A therapeutically effective amount of lactate is that amount of lactate necessary to increase the proliferation and resuscitation rate of ma~malian cells. For a composition, a therapeutically effective amount of lactate is that amount ne~essary to suppress the respira~ory bursting process of white blood cells to protect and resuscitate ~5 the mammalian cells. In general, a therapeutically effective amount of lactate in a compo~ition i~ from about 5 to about lo times the amount of lacta~e n4rmally found in serum. The exact amount of lactate is a matter of preference subject to such factors as the type of condition being treated as well as the other ingredients in the composition. In a pr~ferred embodiment, lactate is present in the cytoprotective 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 o~ the cytoprotective composition.

Antioxidants are substances which inhibit oxidation or suppress reactions promoted by oxygen or peroxides. Antioxidants, especially lipid-soluble WO93/16~90 ~ PCT/US93/00260 ~ ' ~ 30 -antioxidants t ca~ be absorbed into the cellular membrane to neutralize oxygen radicals and thereby protect the membrane. The antioxidan~s useful in the present invention may be selPcted from the group consisting o Yitamin A (retinol), Vitamin A2 (3, 4-didehydroretinol), all forms of carotene such as ~lph~ carotene, betaD
carotene (beta, beta carotene), gamma-carotenef delta carotene, Vi~amin C (ascorbic acid, L ascorbi~ acid), all forms of tocopherol such as Vitamin E (alpha-tocoph~rol,

3,4-dihydro-2,5,7,8-tetramethyl-2-~4,~,12~trimethyltri decyl)~2H-1-b~nzopyran 6-ol), beta~tocopherol, gamma-tocophexol, and del ta tocopherol, and mixtures thereof.
Preferably, the antioxidant is sele~ted from the group of lipid soluble antioxidants consisting of Yitamîn A, beta~
15 ~-~carotene, Vitamin E, and mixtures thereof. More preferably, the antioxidant is Vitamin E.

The amount of antioxidant present in the cytoprotective compositions of the present invention is a therapeutically effective amount. A therapeutically effective amount of antioxidarlt is that amount of antioxidant nece~sary to increase th~ proliferation and resuscitation rate of mammalian cells. The exact amount ~f antioxidant is a matter of preference subject to such factors as the type of condition being treated as well as the other ingredients in the composition. When the cytoprot~cti~e composition contains two components, the antioxidant is preferably present in the cytoprote::tive composition in an amount from about 10% to about 7596, 30 preferably from about 20% to about 60%, and more preferably from about 25% to about 55%, by weight of the cytoprotective composition. When the cytoprotective composition c:ontains three components, the antioxidant i~
preferably is present in the cytoprotective composition 35 in an amount from about 1~% to about 50%, preferably ~rom about 2 0% to about 4 5g6, and more pref erably f rom about 25g6 to about 40g~, by weight of the cytoprotective compositic)n .

WO93/16690 31 - ~ 2 ~ 7 ~ PcT/us93/oo26o The mixture of saturated and unsaturated fatty acids in the present invention are those fatty acids required for the repair of mammalian cellular membranes and the production of new cells. Hence, the fatty acids 5 are readily incorporated into the cell and are immediately available for the repair of injur d cell~ and the proliferation of new cells. By providing the cell with the unsaturated fatty acids needed for repair t the need of the ::ell f or unsaturated f atty acid~ is reduced 10 and the need for hiyh oxygen levels is also reducPd.
Accordingly, the presence of the mixtures of saturat~d and unsaturated fatty acids in the cytoprotective compositions significantly enh~nces the ability of pyruvate, lactate, and the antioxidant to inhibit 15 ~-~ reactive oxyge~ production.

Fatty acids are carboxylic acid compounds found in animal and vegetable fat and oil. Fatty ~cids ar~
classified as lipids and are composed of chains of alkyl groups c~ntaining from 4 to 22 carbon atoms and 0-3 double bonds and characterized by a texminal carboxyl group, -COOH. Fatty acids may be saturated or unsaturated and may be solid, semisolid, or liquid. The most common saturated fatty acids are butyric acid (C4), lauric acid (Cl2)~ palmi~ic acid (C16), and stearic acid (C18). Unsaturated fatty acids are usually derived from veg~tables and consist of alkyl chains containing from 16 to 22 carbon atoms a~d 0-3 double bonds with the characteristic terminal carboxyl group. The most c~mmon unsaturated fatty acids are oleic acid, linoleic acid, and linolenic acid (all C18 acids).

In general, the mixture of saturated and unsaturated fatty acids required for the repair of 35 mammalian cellular membranes in the present invention may be derived from animal fats and waxes. Cells produce the chemical components and the energy required for cellular viability and store excess energy in the form of fat.
Fat is adipose tissue stored between organs of the body WO93/16690 . PCT/US93/00260 3 ~ - 32 to furnish a reserve supply ~f energy. The preferred animal fats and waxes have a fatty acid composition similar to that of human fat and the fat contained in human breast milk. The preferred animal fats ~nd waxes may be selected from the group consisting of human fat~
chicken fat, cow fat (defined herein as a bovine domestic animal regardless of sex or age), sheep ~at, horse fat, pig fat, and whale fat. The more preferred animal fats and waxes may be selected from the group consiæting o~
human fat and chicken fat. The most preferred ani~al fat is human fat. Mixtures of other fats and wax~s, such as vegetable waxe~, marine oils (~specially shark liver oil), and syntlletic waxes and oils, which have a fatty acid composition similar to that of a~imal fats and ~-~axes, and preferably to that of human fats and waxes, may also be amployed. The mixture of saturated and unsaturated fatty acids may also be derivad from .nimal and vegetable fats and waxes, and mixtures thereof.

In a preferred embodiment, the mixture of saturated and un6aturated fatty acids has a composition similar to that of human fat and comprises the following fatty acids: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, myristQleic acid, palmiti~ acid, palmitoleic acid, stearic, oleic acid~ linoleic acid, linolenic acid, arachidic acid, and gaddoleic acid. Preferably, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic, ol~ic acid, linoleic acid, linolenic acid, arachidic a~-id, and gaddoleic acid are present in the mixture in about the following percentages by weight, re~pectively (carbon chain number and number of unsaturations are shown parenthetically, respectively):
0.2%-0.4~ (C4), 0.1% (~6)~ 0.3%-0.8~ (C8), 2.2%-3.5%
( 10)~ ~-9% 5-5% (C12), 2.8%-~.5% (C14), 0.1~-0.6%
(Cl~ 13, 23.2%-24.6% (C16), 1.8%-3.0% (C16 1), 6.9%-9.9%
(Cl8), 36-0%-36-5% (C18 1), 20%-20.6% (~1~ 2)~ 7.5-7.8 (C18:3)~ 1-1%-4-9% ~C20), and 3.3%-6.4% (C20 1).

WO93/16690 PCT/U~93/00260 - 33 ~ 9 ! ~
S'.~ .L ;~ J

In another pre.ferred embodimen~, ~he mixture o~
saturated and unsaturated fatty acids is typical~y chicken fat comprising the following fatty acids: lauric acid, myristic acid, myristoleic acid, pentad~canoic acid, palmitic acid, palmitoleic acid, margaric acid, margaroleic acid, stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, and gaddoleic acid.
Preferably, lauric acid, myristic acid, myriætol~ic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid, margaroleic acid, stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, and gaddoleic acid are present in the mixture in about the following percentages by weight, respectively: a~
15 ~-~(C12), 0.8% (C14), 0.2% (C14 1), 0.1~ (~15)~ 25~3% (Cl6)~
7.2% (C16 13, 0.1% (C~7~, 0-1% (~17~ 6-~% (C18)~
(~18~ 20-6~ (~18:2)t 0-8~ (IClg; 3) t 0.2% (C2~), and 0.3% (C2~ 1), all percentages ~/~ 10%.

The above fatty acids and percentages thereof present in the fatty acid mixture are gi~en as an example. The exact type of fatty acid present in the fatty acid mixture and the exact amount of fatty acid employed in the fatty acid mixture may be varied in order to obtain the result desired in the final product and such variations are now within the capabilities of those skilled in the art without the need for undue experimentation.

The amount of fatty acids present in the cytoprotective compositions of the presient invention is a therapeutically effective amount. A therapeutically effective amount of fatty acids is that amount of fatty acids n~cessary to increase the repair of cellular membranes and resuscitation rate of mammalian cells. The exact amount of fatty acids employed is subject to such factors as the type and distribution of fatty acids employed in the mixture, the type of condition being treated, and the other ingredients in the composition.

wo 93/ 1 6690 ~ 3 ~ 34 - PCT/US93/00260 h~
When the cytoprotective composition contains two components, the fatty acids are preferably present in the cytoprotective compo~ition in an amount from about 10% to about 75~, preferably from about 20% to about 60%, and ~ore preferably from about 25% to about 55%, by weight of the cytoprotective composition. When the cytoprotective compositio~ contains three components, the fatty acid~ are preferably present in the cytoprotective composition in an amount from about 10% to about 50%, preferably from about 20% t~ about 45%, and more preferably from about 25~ to about 40%, by weight of the cytoprotective composition.

In accord with the pre-~ent invention, the 15 ~-~therapeutic cytoprotective compositions for preventing and reducing injury to mammalian cell~ from a medicament having cytotoxic properties may b~ selected from the group consisting of ~
(1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; :~
(b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids ~-required for the repair of cellular membranes and resuscitation of ma~malian cells; ~:
(2) (a) pyruvate selected from the gr~up consisting of pyruvic acid, pharmaceutically acceptable salts of pyruYic acid, and mixtures thereof;
(b) lactate selected from the group consisting of lactic acid, pharmaceutically acceptable salts of lactic acid, and mixtures thereof; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and re~uscitation of mammalian cells;
(3) (a) an antioxidant; and (b) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids ~ 35 ~ 2 ~ 3r!lf3ll required for the repair of cellular membranes and resus~itation of mammalian cells;

(4) (a) lactate selected from the group consisting of lactic acid, pharmaceutically acceptable salts of lactic acid, and mixturQs thereof;
(b) an antioxidant; and ~c) a mixture of saturated and un~aturat~d fatty acids wherein the fatty acids are those ~atty acids ~-required for the repair of cellular membrane~ and resuscitation of mammali~n cells; and

(5) (a~ pyruvate selected from the group consisting o~ pyruvic acid, pharmaceutically acceptable s~lts of pyru~ic acid, and mixtures thereof; and ~b) an antioxidant.
15 ~
In a preferred embodiment, the cytoprotective compositions for treating ma~malian cells, preferably ~pidermal keratinocytes, may be selected from tha group consi~ting of:
(1) (a) pyruvate ~elected from the group , consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
(b) an antisxidant; and (c) a mixture o~ saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of ellular membranes and resuscitation of mammalian cells;
(2) (a) pyru~ate s~lected from the group consistîng of pyruvic arid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
(b) lactate selected from the group c~nsisting of lactic acid, pharmaceutically acceptable salts of lactic acid, and mixtures thereof; and (c3 a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids r~quired for the repair of cellular membranes and re~cuscitation of mammalian cells; and (3~ (a) an antioxidant; and WO93/16690 - 36 - PCT/U~93/00260 (b) a mixture of saturated and unsaturated fatty acids wherein the fatty ac:ids are those fatty acids required for the repair of cellular membranes and resuscitation of mammalian cell~; and ~:
(5) (a) pyruvate selected from the group consisting of pyruvic acid, pha~maceutically acceptable salts of pyruvic acid, ~nd mixtures thereof; and (b) an antioxidant.

In ~ more preferred embodiment~ the cytoprotectiYe compositions for treating mammalian cells, ~-~
preferably epidermal keratinocytes, may be selected from ::
the group consisting of~
(1) (a) pyruvate selected from the group 15 ~ consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
(b~ an antioxidant, and `~`
~c~ a mixture of aturated and unsaturated :~
fatty acid~ wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of mammalian cells; and (~) (a) pyruvate selected from th~ group .
consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b~ an antioxidant~ :

In a most preferred embodiment, the cytoprotective compositions for treating mammalian cells, preferably epidermal keratinocytes, comprîse:
(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 resu~citation of mammalian cells. `:

WO 93/16690 PCr/US~3/00260 3 7 ,. ~

The present invention extends to methods for makiny the therapeutic cytoprotective compositions. In general, a cytoprotec:tive composition is made by ~orming an admixture of the components of the compositionO In a 5 f irst embodiment, a cytopro~e~tive compo:~;ition is mad~ by f orming an admixture of ( a ) a pyruvate, (b~ an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids~ In a second embodi~ent, a cytoprotective composition is made by forming an 10 admixture of (a) a pyruvate, (b) a lactate, and (c) a mixture of saturat~d and unsaturated f atty acids . In a third embodiment, a cytoprotective composition is made by forminq an admixture of (a) an antioxidant, and (b) a mixture of saturated and unsaturated f atty aciàs . In a 15 ~-~ fourth embodiment, a cytoprotective coDIposition is made by forming an admixture of ~a) a lactate, ~b~ an antioxidant, and (c) a mixture of ~;a~urat~d arad unsaturated fatty ac::ids. In a fifth em2~odiment, cytoprl~tectiv~ composition is made by forming an adrnixture of (a~ a pyruvate, and (b) an antioxidan~

For some applications, the admixture may be formed in a solvent such as water. If necessary, the pH
of the ~lvent is ad justed to a range from about 3 . 5 to about 8 . O, and preferably from abc~ut 4 . 5 to about 7 . 5, and more prePerably about 6 . O to about 7 . 4 . The admixture is the~ sterile f iltered . Other ingredients may also be incorporated into the c:ytoprotective composikion as dictated by the nature of the desired composition as well known by those having ordinary skill in the art. The ultimate cytoprotective compositions are readily prepared using methods generally known in the pharmaceutical arts.

once prepared, the inve~tive therapeutic cytoprotective compositions may be stored for future use or may be formulated in effective amounts with a cytotoxic agent to form cytoprotective pharmaceutical compositions. The combination of the cytoprotective WO 93/1669(~ PCI/US~3/0026U
~ 3 $ --compositions of the present invention and the medicament cytotoxic to cells provides a cytoprotec~ive ;
pharmaceutical composition having the ability to prevent and reduce injury to mammalian cells from the cytotoxic medicam~nt and increase the r2susci~ation rate of injured mammalian cells. The dose level of the cytotoxic medicament in the cytoprotective pharmaceutical composi ion may thereby be raised to higher than normal levels.
The cytotoxic agents which may be use~ in the cytoprotective pharmaceutical compositions of the pres~nt invention may be selected from a wide variety of medicaments. For example, medicaments taken on a long 15 ~ erm regimen tend to cause liver, kidney, tissue, and other toxicity problems. In addition, certain cytotoxic medicam~nts, such as potent chemc)~herapeutic medica~ents used t~ treat malignant tissues, are believed to stimulate rel~ase of significan~ amounts of reactive oxygen species by mammalian tissue~ which can cause oxidative injury. Combination of the cytoprotective compositions of the present in~ention with ~uch cytotoxic medicaments may inhibit induction of reactive oxygen production while simultaneously decreasing side effects of such medicaments. ~y decreasing the side effects of sueh medicaments, the dosage levels of the medicaments may be increased thereby increasing the therapeutic e f f ect of the medicaments. For example, the cytoprotective compositions may be used in topical cytoprotective pharmaceutical compositions in combination wi~h cytotoxic medicaments such as epithelial cell cohesiveness reducers such as tretinoin ~Retin A), dermatological abradants, and anti-inflammatories, to protect and enhance the resuscitatio~ rate of the injured mammalian cells. The cytoprotective compositions may also be u ed in ingestible cytoprotective pharmaceutical compo~itions in combination with medicaments that cause cytotoxic side effects such as anti-tumor, anti-viral, and antibacterial medicaments including the lipid WO93/l6690 _ 39 _ 2 . 2 ~ 7 ~.` i'J PCT/US93/00260 regulating agents gemfibrozil and lo~astatin, ~entrally acting anticholinesterases such as tacrine, chemotherap~utic medicaments such as the anthracycline antibiotic doxorubicin, gastric irritants such as acetylsalicylic acid and ibuprofen, to protect and enhance the resuscitation rate of the injured mammalian cells.

Xn one preferred embodiment, the medicament having cytotoxic properties in the cytoprotactîve pharmaceutical compositions is selected from the group consisting of doxorubicin, gemfibrozil, lovastatin, and tacxine. In a mora prefexred embodiment~ the medicament having cytotoxic properties in the cytoprotective ~pharmaceutical compositions is selected from the group consisting of doxorubicin, gemfibrozil, and tacrine. In a most preferred embodiment, the medicament having cytotoxic properties is dox~rubicin. Doxorubicin :-~
(Adriamycin) is a cytotoxic alnthracyclins antibio~ic reported to produce regre sion in di~seminated n~opla~tic conditions such as in various leukemias, tumors, neuroblastomas, sarcomas, and. carcinom~s. G~mfibrozil (Lopid) is a lipid regulating agent which lowers el~vated serum lipids primarily by decreasing serum triglyceride with a variable reduction in total serum cholesterol.
Lo~astatin tMevacor) is a cholesterol lowering 2gent which inhibits the enzymatic biosynthesis of cholesterol.
Tacrine ~Cognex, 1,2,3,4-tetrahydro-9-acridinamine) is a centrally active anticholinestera~e useful a~ ~ cognition activator. Tacrine has undergone clinical trials for use in treating severe Alzheimer's disease (presenile dementia). :~

In another preferred embodiment, the medicament having cytotoxic properties in the cytoprotective pharmaceutical compositions is an anticancer agent.
Nonlimiting examples of anticancer agents include chemically reactive druys having .nonspecific action, anti-metabolites, antibiotics, plant products, hormones, WO93/16690 ~J~ ~3~) _ 40 _ P~T/US93/00260 and other miscellaneous chemother~peutic agents.
Chemically reactive drugs having nonspecific action include alkylating agents and N-alkyl-N-nitroso compounds. Examples of alkylating agents include nitrogen mustards, azridines (ethylenimin~s), sulfonic acid esters, and epoxides. Anti metabolites are compounds that interferP with the f ormation or utilization of a normal cellular metabolite and include amino acid antagonists, vitamin and coenzyme antagonists, and antagonists of metabolites involYed in nucleic acid synthesis such as glutamine antagonists, folic acid antagonists, pyrimidine antagonist~, and purine antagoni~t~. Antibiotics are compounds produced by microorganisms that have the ability to inhibit the ~~ growth of other organisms and include actinomycin~ and relat~d antibiotics, glutarimide antibiotics, ~arkomycin, fumagillin, streptonigrin, tenuazonic acid, actinogan, peptinogan, and anthracyclic antibiotics ~uch a~
doxorubicin. Plant product~s includ~ colchicine, podophyllotoxin, and vinca alkaloids. Hormones include those steroids used in breast and prostate cancer and cortic~steroids used in leuke~.ias and lymphoma~. Other mi~cellaneous chemotherapeutic agents i~clude urethan, hydroxyurea, and related compounds; thiosemicarbazones ~5 and related compounds; phthalanilide and related compounds; and triazenes and hydrazines. In a pr~ferred embodiment, the anticancer agent is an antibiotic. In a more preferred embodiment, the anticancer ag~nt is doxorubicin. In a most preferr~d embodiment, the anticancer agent is doxorubicin.

In a specific embodiment, the invention is directPd at a cytoprotective pharmaceutical composition for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties which comprises:
(A) a cytoprotective composition selected from the group consisting of:

WO93/16690 - 41 ~ ?,~ 7r`~ ~ Pcr/us93/oo (1) ~a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically accepta}:)le salt~; of pyruvic acid , arld mixtures thereof;
(b) an antioxidant; and ( c ) a mixture of ~;aturated and unsaturated fatty acids wherein the fatty acids are tho~;e fatty acids required for the :resuscitation of injured mammaliar c: ells; and ~2) (a) pyruvate selected from the group consisting of pyruvic ac:id, pharmaceutic:ally acceptable salt~ o~ pyruvic as:~id , and mixtures thereof; and (b) an antioxidant; and (B) a medicament ha~ring cytotoxic prc~pertie~.

15 ~ In another form of this embodi~rlt, the cytoprotective compositions of the pres~nt invention, may be cc~bined in an immediate release form with an anticanGer medicament having cytotoxic properties in a timed rel@ase f orm to provide a tila~d relea~e 20 cytoprotective pharmaceutical composition. In thi~
embodiment, the timed release composition relea~;~s the cytoprotective composition substantially immediately and releases the cytc~toxic ::hem~therapeutic medicam2nt after a suitable period of timeI for example from one to 24 hours after releasing the cytoprotective composition, to sel~ctively protect non-cancerous aells in the presence of cancerous cells against the cytotoxic chemotherapeutic medicament. Cancer cells, unlike normal cells or b~nign tumor cells, exhibit the properti~s of invasion and metastasis and are highly anaplastic.
Because cancerous cells have a rapid metabolism, cancerous cells will rapidly consume the protective cytoprotective composition and will not be protected by th~ cytoprotective compositions when the chemotherapeutic 3S medicament is subsequently released. Non-cancerous cells which do not have such a rapid metabolism will not rapidly consume the cytoprotective compositions and will be protected when the chemotherapeutic medicame~t is subsequently released.

WO 93/16690 PCr/US93/0~260 ~ ~, o ;~ 13 ~ -- 4 2 In a specif ic embodiment, the inventiorl is direct at a timed-relPase cytoprotective pharmaceutical composition for selectively protecting non-cancerous 5 mammalian aells in the presence of cancerous ~ammalian cells from an anticarlcer medicament having cytotoxic properties which comprises:
(A) a cytoprotective composition in immediate release ~o~ selected ~rom the group consisting of:
(l) (a) pyruv~te select~d 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 15 ~fat~y acids wherein the fatty acids are those fatty acids re~uired for the resuscitation o~ injured mammali~n cells; and (2) (a) pyruvate selected from the group consisting o~ pyruvic acid, pharmaceutically acceptable salts of pyruvic arid, and mixtures thareof; and (b) an antioxidant; and (B3 an anticancer medicament having cytotoxic properties in timed-release form;
wherein the cytoprotective composition is released substantially immediately and the anticancer medicament is released after a period of time sufficient such that thP cancerous rells have substantially metabolized the cytoprotective composition and th~ non-cancerous cells have not substantially metabolized the cytoprotective composition.

A suitable or sufficient period of time is that period of time wherein the cancerous cells have substantially metabolized the cytoprotective composition and the non-cancerous cells have not substantially metabolized the cytoprotective composition. The period of time should not be so long that the non-cancerous cells substantially metabolize the cytoprotective composition and are unprotected. The exact time i5 WO 93/16690 4 3 2 ~ 2 t ' ~ ~J ; PCr/US93/00260 su~ject to such factors as khe type and quantity of cytoprotective composition employed, the medicament having cytotoxic properties used, and the type of cancerous cells and nc:~n-cancerous cells being treated.
5 Thus, the period of time may be varied in order to ob1:ai the result desired and such vax iations are within the capabilitie~ of those skillPd in the art without the need f or undue experimentation .

The present invention exte~ds to mekhod~; for making the cytoprotective pharmaceutical co:mposition. In general, a cytoprotective pharmaceutic:al compo ition is made by f orming an admixture of the components of he s:omposition. The cytoprotective compositions may be 15 ~prepared using standard techniqu~s and equipm~nt known to those skilled in the art~ The apparatus useflll in accordanc:e with the present inY~ntion c~Dlpri~;es apparatus well known in the chemical ancà bioc:hemical art~, and therefore the selection of the specif i apparatu~ will be 20 apparent to the artisan.

In one emb~diment, a cytoprotective pharmaceutical composition is made by forming an admixture of the cytoprotective composition ansl the 25 medicamerlt having cytotoxic properties. In a second embodiment, a timed release cytoprotective pharmaceutical c:omposition i5 made by f orming an admixture of the cytoprotective composition in immediate release form and the anticancer medicament having cytotoxic properties in 30 timed-release form.

The presen~ invention extends to methods for - using the therapeutic cytoprotective compositions. In one embodiment, the cytoprotective composition~ of the present invention may be administered to c~lls concurrently with a cytotoxic medicament. In another embodiment, the cytoprotective compositions of the present invention may be administered to cells prior to the administration of a cytotoxic anticancer medicament WO 93/16690 PCr/lJS93fO0260 ?~ 3 to sele~tively protect non-canceroUs cells in the presenc:e of cancerous cells against the anticancer agent.

In a specif ic embodimen~ I the invention is 5 diracted at a method for pro~ecting ma~alian cells from a medic:ament having cytotoxic properties which c:ompri~es the steps of:
(A) providing a cytoprotective compo ition ~;elected ~rom the ~roup consi~ting of:
( 1) ~a) pyruvate selected from the group consi~ting of pyruvic acid, phar~aceutic::ally aceeptable salts of pyruvic acid , and mixtures th~reof;
(b) an antioxidant; and ( c ) a mixture of saturated and un~aturated 15 ~-~f atty acids wherein the fa~ty acids are tho~e fat~y acids res~uired for the resuscitation of in~ured mammalian cells; and (2) (a~ pyruvate selected from the group consisting of pyruvic acid, pha:rmaceutically acc~ptal:ile 2 0 salts of pyruYic acid , and mixturies thereof; and (b) an antioxidant; and (B) providin~ an anticarlcer medicament having cytotoxic properties; and (C) administering the c:ytoprotective compc~sitior 25 from st~p (A) and the medicament from st~p (B) concurrently to mammalian cells to protect the mammalian cells from the medicament having cyt~toxic properties.

In another specific embodiment, the invelltion 30 is direct at a method for selectiv~ly protee:ting non-cancerous mammalian cells in th~ presenc::e of cancerous mammalian cells from an anticancer medicament having cytotoxic: properties which cornprises the ~teps of:
(A) providing a cytoprc)tectiv~ comE: osition in an 35 immediate release form selected from the gr~up consisting of :
(1) ~a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable sallts of pyruvic acid, and mixtures thereo~;

WO93t166gO P~T/US93/00260 - 45 ~ ~ ~ 2 ~ ~ r7 ~ ~

(b) an antioxidant; and (c) a mixture of saturated and unsa~urated fatty acids wherein th~ fatty acids are those fatty acîd~
required for the resuscitation of injured mammalian cells; and (2) ~a) pyruvate select d from the group consisting of pyruvic acid, pharmaceutically acceptable sal1:s of pyruvic acid , and mixtures therec)f; and (b) an antioxidant, and o (B) providing an anticancer medic:ament having cytotoxic properties in timed-release form~ and (C) administering the cytoprotective compo~ition from step (A) and the medicament from step (B~
concurrently to ma~malian cell~ to selectively protect ~non-cancerous mammalian cells in the presence o~
cancerous mammalian cell~ from the anticancer medicament having cytotoxic properties;
wher~in the cytoprotective composition i8 r~l~ased substantially immediate.ly and the anticancer medicament is released after a period of time sufficient such that the cancerous cells have substantially metabolized the cytoprotective.composition and the non-cancerous cells have not substantially metabolized the cytoprotective composition.
In yet another specific embodiment, the in~ention is direct at a method for selectively protecting non-cancerous mammalian cells in the presenc~
of ca~cerous mammalian cells from an anticancer medicament having cytotoxic properties which compri~es the steps of:
(A) administering to mammalian cells a cytoprotective composition to prevent and reduce injury to the mammalian cells selected from the group consisting o~:
(1) (a) pyruvate selected from the gr~up consisting of pyruvic acid, pharmaceu~ically acceptable sal~s of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and WO93/16690 PCT/US93/~0260 ~ ~' (c~ a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of iniured ma~malian cells; and (2) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and ~b) an antioxidant; and ~B) waiting a period of time sufficient ~uch that the cancerous cells have subst~ntially metabolized th~
cytoprotective composition and the non cancerous cells have not substantially metabolized the cytoprotective composition; and (C) a~ministering the cytotoxic anticancer 15 ~ medicament to the mammalian cells to treat the canc~rous cells which are unprotected by the cytoprotective compo~ition and the non canc~rou~ cells which are protected b~ ~he rytoprotective composition to there~y increase the therapeutic eff~e t of the anticancer medicament.

Methods for administering the cytoprotectivQ
compositions of the pr~sent invention to mammalian cells will vary depending upon the particular condition being treated and the cytotoxic agent employed. In general, the cytoprotective compositions will be administered in the same manner as the cytotoxic agent. Of course, the t~pe of carrier will vary depending upon the mode of adminiRtration d~sired for the pharmaceutical composition as is conventional in the art.

The cytoprotective compositions of the present invention may be administered parenterally, in the form of sterile solutions or suspensions, such as intrav~nously, intramuscularly, or subcutaneously. The cytoprotective compositions may also be administered topically. Non-oral topical compositions employ non-oral topical vehicles, such as oils, petrolatum bases, emulsions, lotions, crsams, gel formulations, foams, WO93~lh690 PCTtUS93/00260 4 7 ~ ,~ I rt r~

ointments, sprays, salves, and films, which are intended to be applied to the skin or body cavity and are not intended to be taken by mouth. Oral topical ompositions employ oral vehicles, such as mouthwashes, rinses, oral spray~, suspensions, bioadhesives9 and dental gels, which are i~te~ded to be taken by mouth but are not intended to be in~ested. The cytoprotective composition~ may also be administered orally, in the form of pill~, tablets, capsules, troche~, and the like, as well as sublingually, rectally, or tr~nscutaneously with a suitable pharmaceutically acceptable carrier for that particular mode of administration as is conventional in th~ artO

It is especially advantageous to formulate the ~-~pharmaceutical compositions in dosage unit forms for ease of administration and uniformity of dosage. The term dosage unit ~orms as used herein refers to physically di~crete units suitable for use as a unitary dosage, e~ch unit containing a predetermined quantity of active ingredient calculated to produce the desired therap~utic effect in association with the pharmaceutical carrier.

For parental therapeutic administration; the cytoprotectlve ccmpositions of the present invention may be incorporated into a sterile solution or ~uspension.
These preparations should contain at least about 0.1% of the inventive composition, by weight, but this amount may be varied to between about 0.1% and about 50% of the inventive composition, by weight of the par~ntal composition. The exact amount of the inventive composition present in such compositions is such that a suitable do~age level will be obtained. Preferred compo itions and preparations according to the pre~snt invention are prepared so that a paranteral dosage unit contains ~rom between about 0.5 milligrams to about lOO milligrams of the inventive composition.
"-Suitable carriers inclu~e propylene glycol-alcohol-water, isotonic water, sterile water for ~ 3;?~ - 48 -injection (USP), emulphorTM-alcohol-water, cremophor-ELTM
or other suitable carriers known to those skilled in the art. The sterile solutions or suspensions may also include the following adjuvants: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerine t propylene glycol, or other synthetir. solvent; antibacterial agents, such a~
~enzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium metabisulfite; chelating agent~, such as ethylenediaminetetraacetic acid (~DTA~; buffer~, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity~ such as sodium chloride or dextroseO The parental preparations may be enclosed in ampules~ disposable syringes, or multiple dose vials made 15 ~-~'of glass or plastic.

In another form of the invention, the therapeutic cytoprotective composition is incorporated into a nonoral topical vehicle which may be in the fonm of oils, petrolatum bases, emu:Lsions, lotions~ creams, gels ~ormulations, foams, ointments, sprays, salves, and films, and the like. Non-oral topical vehicles include water and pharmaceutically acceptable wat~r-miscible organic solvents such as ethyl alcohol, isopropyl alcohol, propylene glycol, glycerin, and the like, and mixtures of these solvents. Typical non-toxic non~oral topical vehicles known in the pharmaceutical arts may be used. The non oral topical cytoprotective compositions may also contain cvnventional additives employed in those products. Conventional additives include humectants, emollients, lubricants, stabilizers, dyes~ and perfumes, proYiding the additives do not interfere with the therapeutic properties of the cytoprotective composition.

3 5 Xn another f orm of the invention, the c:ytoprotective composition is incorporated into an oral topical vehicle which may be in the form of a mouthwash, rinse , oral spray , suspension , dental gel , bioadhesive , and the like. Typical non-toxic oral vehicles known in WO 93/16690 PCl/US93/00260 ~ 2 ~ 7 . `
the pharmaceutical arts may be used in ~he pre~;ent . invention. The preferred oral vehi les are water, ethanol, and water ethanol mixtures. Th~ water-ethanol mixt~lres are generally employed in a weight ratio from 5about 1:1 to about 20:1, preferably frs:~m about 3:1 to about 2O: 1, and mos'c preferably from about 3: 1 to about 10 :1 , respect:ively . The pH value of the oral vehicle is generally from about 4 to about 7, and pre~erably from about 5 to about 6 . 5~, An oral topical vehic:l~ having a lOpH value below about 4 is generally irritating to the oral cavity and an oral vehicle having a pH vallle greater than about 7 generally results in an unplea~;ant mouth feel~ The oral topical cytoprotective comp~sitio~ ~ay al~o contain conventional additives normally employed in 15 ~-~those 1?roducts. Conventional additivas ins:lude a ~luorine providing compound, a sweetening agent ~ a flavoring agent, a colcring agent, a humectant, ~ buff~r) and an emulsif ier, providing th~ additives do not in~erf ere wilth the therapeut:ic properties oP the 20 cyltoprotective composition.

In accordarlce with this invention "
therapeutically effective amounts of the cytoprotective compositions of the present in~ention may be admixed with 25 a topical vehicle to form a topic:al cytoprotective composition. These amounts are readily determined by those skilled in the art without the n~ed îor undue experimerltation., In a preferred embodiment, the topical cytoprotective compositions will comprise the cytoprotective composition in an amount from about 0.~%
to about 10% and a topical vehicle in a quantity sufficient to brin~ the total amount of composition to 100%, by weight of the topical cytoprotective composition. In a more preferred embodiment, the topical cytoprotective compositions will comprise the cyt~protective composition in an amount f~om about O.l~
to about 10%, and in a most preferred embodiment, the ~opical cytoprotecti~e compositions will comprise the cytoprotectiv~ composition in an amount from about 0~1%

W~:) 93/16690 ~ P~/US93/00260 to about 8%, and a topical vehicle in a quantity sufficien~ to bring the total amount :7f composition to 100%, by weight of the topical cytoprotective compositiQn.
The present invention extends to method~ ~Eor preparing the topical cytoprotectiv~ compositions. In such a method, the topical s~ytoprotectiv~ composition iE;
pr~pared by admixing a therapeutically effsctive amount 10 of the cytoprotective comp~sition of the present invention, the cytotoxic agenk, and a topical vehicle.
The f inal compositions are readily prepared using standard methods and apparatus generally known by those skilled in the pharmaceutical arts. The apparatus useful 15 ''~in accordance with the present invention comprises mixing apparatus well known in the pharmacelltical arts, and ther~fs:~re the s;election of the specif ic apparatus will be apparent to the artisan.

In a specific embodiment, the invention is directed at a cytoprotective pharmaceutical composition which comprises a pharmaceutically acceptable carrier and a theraE~eutically effe~::tive amount of a cytoprotective compositicsn for preventing and reduc:ing injury to mammalian c:ells from a medicament having cytotoxic prs7perties, wherein the cytoprotec:tive composition is selected from the group consisting of~
(1) (a~ pyruvate selected from the group consisting o~ pyruvic acid, pharmaceutically accept~ble salts of pyruvic acid, and mixtures thereo~;
(b) an antioxidant; and ~c) a mixture of saturat~d an~ unsaturated fatty acids wherein the fatty acids are those fatty acids required ~or the resuscitation of injured mammalian cells; and (2) (a) pyruvate selected ~rom the group consisting of pyruvic acid, pharmaceutically acceptable ~alts of pyruvic acid, and mixtures thereof; and (b) an antioxidant.

W093/16690 P~r/US93/00260 2 ` 2 ~ ~ `

In ~nother specific embodiment, the invention is directed at a method for preparing a cytoprotective pharmaceutical composition for protecting mammalian c~lls from a medicament having cy~otoxic properti~s which comprises the steps of:
(A) providing a therapeutically effective amount of a cytoprotective compo~ition which compri~e~
(l~ (a~ pyruvate ~lected 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 unsaturat~d fatty acids wherein the fatty acids are.those fatty acids ~-~eguired for the resuscitation of injured ma~malian cells; and (2) (a) pyruvate s~lected from the group consisting of pyruvic acid, pha~naceutically acc~ptable salts of pyruvic acid, and mixtures th~reof; and (b~ an antioxidant; and (B) providing a pharmaceutically acceptable carrier;
and (C) admixing the cytoprotective composition ~rom step (A) and the pharmaceutically acceptable carrier from step (B) to form a pharmaceutical composition.

Throughout this application, various publications have been referenced. The disclosures in the e publications axe incvrporated her~in by reference in order to more fully describe the state of the art.

The present invention is further illustrated ~y the following exampl~s which are not intended to limit the effective scope of the claims. All parts and percentages in the examples and throughout the specification and claims are by weight of the final composition unless otherwise specified.

WO93/16690 PCT~US93/00260 b~'~ ? ~t~ 52 -E~MPL~

These example~ demonstrate the cytoprokective abilities of the therapeutic cytoprotective compo~itions of the present invention.

~etho~

Isolation of Peripheral Blood Monocytes Peripheral blood was obtained from a normal healthy volunteer by venipuncture using an EDTA-containing Vacutainer (Becton Dickinson Mountain View, Ca.). A total of ~0 ml of peripheral blood wa~ mix~d in ~-~a ratio of 1:1 with Dulbecco's Minimal Essential ~edium (DM~M, Grand Island Biologicals, GIBCO, Grand I~land, N.Y.). The mixture wa divided into 2 ml por~ion~ a~d each portion was layered onto ~ ml o~ Ficoll~H~pagu~
gradient mixture (Pharmacy, Inc.y Piscataway, N.J.) and centrifuged in a B~ckman T~J6 refrig~rated centrifuge for 30 minutes at 1500 rpm and 4 C. After the cells were washed twice with phosphate buffered saline, the cells ;:
were resuspended in Hank's Balanced Salt Solution without ~a++/Mg++ (GIBCO). ::
Culture of U9~7 and Peripheral Blood Monocytes Periph~ral blood monocytes and U937 monocytic leukemia tumor cells w~re placed in sterile cultur~
flasks and maintained in culture using Dulbecco's Minimal Essential Medium, with 10% fetal calf serum, supple~ented with 2mM glutamine and Pen/Strep. The cytotoxi~ity of the cytotoxic a~ent on the cells was analyxed by propidium iodide exclusion technique~ and flow cytometric quantitation. Viability of the cells was ~uantified as the number of cell~ that excluded the ~ital dye trypan blue.

WO~3/166~0 2 1 2 ~ CT/U~93/00260 Preparation of Chemicals Sodium pyruvate was dissolved in distilled water and the solution was adjusted to pH 7.4 with lN sodium hydroxide c~lution. Solutions were sterile filtered. Stock solutions were prepared so khat the vehicle would not be more than 1% of the total volume o tha culture media.

A mixture of fa.ty a~ids derived from chicken fat was prepared by mixing 0.1% chicken fat with mineral oil to form an emulsifi~-d solution. Tween 80 was added to separate cultures of cells at similar conc~ntrations :~
and to examine possible vehicle e~fects~
Alpha-tocopherol phosphate tSigma Chemiaal Company, St~ Louis, MO) was added directly to the culture me~ium.
~, 3H-Thymidine Radiosoto~ic Incorporation Measurement of Cytotoxity ~"
Cells were pla~ed into 96 well dishes at a concentration of 1O6 cells/well. Tritiated thymidine ;~
(1 uCi/well) was added and the cells were incubat~d for ~ hours at which time the cells were harvested using a Cambridge cell harvestor. The samples were then placed ~::
in scinti.llation vials containing scintillation fluid and -~
counted~ These studies yielded a measure of the ability of the cell~ to proliferate, which is a measure of viability.

The results from the tritiated thymidine incorporation assay, a measure of DNA synthesis and cellular proliferation, correlated directly with the results from the dye exclusion viability assay. Because the tritiated thymidine incorporation assay is a more quantitatiYe assay, the tritiated thymidine incorporation assay was used for the remainder of the studies.

WO93~16690 PCT/US93/00260 ~ - 54 -A dose response curve for Doxorubicin (Adriamycin) alone was constructed. Doxorubicin is an anthracycline antibiotic used as a f irst line agent in a number of neoplastic condi~ions and is a well characterized cytotoxic agent. Dose~ and times examined ranged from 0.1, 0.5, 1, 5, 10, 25, and 50 ug/ml of Doxorubicin for 20-60 minutes and 24 hour~. The range of optimal concentrations for cytotoxity of ~oxorubicin was established for u937 monocytic tumor c~ to be 0.5, 1 and 5 ug at 24 hours and ~0 ug at 1 2 hours, s~e Figures 1 and 2.

The cytoprotective agents (sodium pyruvate, ~s ~-~Vitamin E, and fatty acids) alone, and in co~bination, were examined for their ability to decrease the cytotoxity of Doxorubicin to U937 monocytic leukemia . :
cells and normal peripheral blood mo~ocytes. Optimal concentrations of the single ingredients of sodium pyruvate, Vitamin E, and fatty acids were examined. The optimal concentrations of the agents that were able to protect cells against Doxorubicin induced cytotoxity were as follows: 10-50 U Vitamin E, 0.5~ fatty acids, and 5 mM of sodium pyruvate, see Figure 3 Window of susceptibility studies were conducted to dekermine the optimal treatment time of the cells with the cytoprotective agents prior to treatment of the cells with the cytotoxic agent. The normal cells and U937 leukemic tumor cells were pretreated separately in ~Iw~sh out" studi2s with the single agents alone, and in combination, at the optimal concentration described above for ~arious time periods, washed with fresh medium to remove the agents, and treated with the cytotoxic agent.
The co-culture of normal and U937 leukemic tumor calls was treated essentially in the same manner except that the sells were not treated separately, but co~cultured.
The optimal pretreatment time of the cells with the cytoprotective agents was found to be 24 hours prior to WO ~)3/16690 5 5 ~ ~ ~J ~ PClr/US93~00260 treakment of the cells with Doxorubicin. The cells were then placed in culture medium without the protective agents. The length of time that the cytopr3tection 1 sted was 24 hours following Doxorubicin treatment. At this time, peripheral cell viability is a limiting factor because these cells are normal cells and do not remain in culture for extended periods of timeO ~-~

Normal and U937 tumor cells were co-cultured and the cytotoxity of Doxorubicin on the cells wa~
determined by viability assays which examined the differential ability of the cytoprotective compositions alone, and in combinations, to protect the no~mal cells from the cytotoxity of the chemotherapeutic ag~nt 15 ~
The cells were isolated and ex~mined for morphologic~l evidence of cytotoxicity or prevention of ~-~ytDtoxicity. These studies determined the cytoprotective effect of the single agent~ and th~
combination of agents ~n the normal and tumor c~ DNA
synthesis s~udies using 3H-th~midine (1 uCi/well) were ~-carried out 4 hours prior . t~ termination of the experiment to determine the effect of the formulations on the proliPeration of the cells as a measure of the pr~v~ntion o~ CytotoxiGity and the extent of Doxorubicin induced cytotoxicity. Propidium iodide exclusion analysi~ was carried out for direct qu~ntitation of the cytotoxicity and the pre~ention of cytotoxicity. Each set of studies was performed in triplicate ~o th~t statistical analysis of the significant differ~nces between the treatment groups could be conducted.

The effect of the cytoprotective agents on the co-cu~ture of tumor and normal cells was very differe~t from the e~fect of these agents on the individual cell types alone. An interaction between the normal cells and the tumor cells must cause the viability of the tumor cells ~o be significantly diminished. The cytoprotective combination of 5 mM sodium pyruvate, 0.5% fatty acids, W0 93/16~90 ?,~ ,,9' ~ 56 - Pcr/us93/oo26o and lo U vitamin E provided signif icant protection to the normal peripheral monocytes and did not protect the tumor cells from the ~ffects of the cytotoxic: agent.

Wash-out studies were conducted to det~rmine viability of the peripheral blood monocytes co c:ultured with U937 monocyt;ic leukemia cells after 24 hour pretre~tment s~f lthe cells with the cytoprotective agent~
f ollowed }:>y admini tration of Doxorubic:in . With no ~:
lo Doxorubicin treatment, the viability of the control normal peripheral cells was enhanced from 5596 to 68% with , the use of 5 ~aM sodium pyruvate and 0.5% fatty acids, ~ee Fis~ure 30 With no ~oxorubicin treatlaent, the viability of the control U937 cells was enhanced from 439c to 62%
15 ~-~with the use of the combination of the cytoprotective compositic>n, 5 mM sodium pyruvate, ~0 U vitamirl E, and 0 . 5% fatty acids, s~e Fiqure 3 .

Pretreatment with a combin~tion of 10 U
2 0 Vitamin E and 5 mM sodium pyruvate prevented cytotoxity to normal peripheral blood monocytes with a conc~ntration of 0 ., 5 ug/ml Doxorubicin ~ 53~ to 68% viable), see Figure 9. Pr~treatment with a combination of 5 ~M sodium pyru~rate, 10 U Vitamin E, and 0. S~ fatty acids preverlt~d 25 cytotoxity to peripheral blood monvcytes with a concentration of 1 ug/ml Doxorubicin ~47% to 69% viable), s~e Figure 13. Pretreatment with the single agent 50 U
Vitamin E preven~ed cytotoxity to U937 tumor cells ... -.
induced by 1 ug/ml Doxorubicin (42% to 62~ viable), see Figure 7.

The viability of cultured peripheral monocytes without Doxorubicin was 66% and increased to 75% with the cytopro~Qctive combination of 5 mM sodium pyruvate, 10 U
35 Vitamin E, and 0. 5% fatty acids, see Figure 13. The viability ~f cultured peripheral monocytes treated with 0.5 ug/ml Doxorubicin was 47% and increased to 63.5% when pretreated with the cytoprotective combination of 5 mM
sodium pyruvate, 10 U Vitamin E, and 0.5% fatty acids,

6~0 P~T/US93/00260 ~. ~ 2 ~ 3~
see Figure 13. The viability of cultured peripheral monvcytes treated with 1 ug/ml Doxorubicin was 42~ and increa~ed to 66% when pretreated with the cytoprotective combination of 5 mM sodium pyruvate, 10 U Vitamin E, and 0.5~ fatty acids, see Figure 13.

The viability of cultured U937 tumor cell~
without Doxorubicin was 67% and did not increase when treated with any of the agents, see Figur~ 13. The viability of cultured U937 turnor cell~; with 0. 5 ug/ml Doxorubicin treatment was 47% and the highest increa~;e in ~iability occurred with pretreatment o~ SO U Vitamin E
and Q.5~ fatty acids, ~;ee Figure 12.. The ~riability of cul~ured U937 tumor cells with 1 ug~ml Doxor~bic:in 15 ~-~treatlaent was 45% and the highest incxease in viability oc:curred wil:h pretreatment of 10 U Vitamin E and û. 5%
f atty acids, see Figure 12 .

Optimal concentrations of the cytoprotectiYe agents to prevent Doxorubicin-induced cytotoxity were found to be 5 mM sodium pyruva~e, 10050 U Vitamin E, and 0.5% fatty acids. In wash-out studie~ the cytoprotective combination of sodium pyruvate, Vitamln E, and fatty a~ids and the combination of SmM sodium pyru~a~e and 10 U Vitamin E protected the no~mal peripheral blood monocytes from Doxorubicin-induced cytotoxityt see Figure 13. Vitamin E alone and fatty acids alone prevented the cytotoxîty of Doxorubicin in U937 cells, see Figure 11. When norm~l peripheral blood monocytes were co-cultured with U937 monocytic leukamia tumor cells, the cytoprotective combination of 5 mM
sodium pyruvate, 0.5% fatty acidsJ and 10 U Vitamin E
provided significant protection to the normal peripheral monocytes from Doxorubicin-induced cytotoxity and did not protect the t~mor cells from the effects of the cytotoxic agent, see Figure 24.

These results shvw that the combination of agents 5 mM sodium pyruvate, 0.5% fatty acids, and 10 U

c~ ?j9 - 5 8 - PCI'/US93/00260 and 50U Vitamin E are useful as selective cytoprotective agents for use with compounds th~t are toxic to normal cells as well as tumor cells.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope o~ the imrention and all ~uch modif ication~ are intended to be included within the sc:ope of the following claims.

, -- ~

Claims

I claim:

1. A cytoprotective composition for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties which comprises:
(a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b) an antioxidant.

2. The cytoprotective composition according to claim 1, wherein the pyruvate is selected from the group consisting of pyruvic acid, sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate, and mixtures thereof.

3. The cytoprotective composition according to claim 2, wherein the pyruvate is sodium pyruvate.

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

5. The cytoprotective composition according to claim 4, wherein the antioxidant is alpha-tocopherol.

6. The cytoprotective composition according to claim 1, wherein pyruvate is present in the cytoprotective composition in an amount from about 10% to about 75%, by weight.

7. The cytoprotective composition according to claim 1, wherein the antioxidant is present in the cytoprotective composition in an amount from about 10% to about 75%, by weight.

8. A cytoprotective composition for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties 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.

9. The cytoprotective composition according to claim 8, wherein the mammalian cells comprise epidermal keratinocytes.

10. The cytoprotective composition according to claim 8, wherein the pyruvate is selected from the group consisting of pyruvic acid, sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate, and mixtures thereof.

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

12. The cytoprotective composition according to claim 8, wherein the mixture of saturated and unsaturated fatty acids comprises animal and vegetable fats and waxes.

13. The cytoprotective composition according to claim 12, wherein the mixture of saturated and unsaturated fatty acids comprises human fat, chicken fat, cow fat, sheep fat, horse fat, pig fat, and whale fat.

14. The cytoprotective composition according to claim 13, wherein the mixture of saturated and unsaturated fatty acids comprises lauric acid, myristic acid, myristoleic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid, margaroleic acid, stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, and gaddoleic acid.

15. The cytoprotective composition according to claim 8, wherein pyruvate is present in the cytoprotective composition in an amount from about 10% to about 50%, by weight.

16. The cytoprotective composition according to claim 8, wherein the antioxidant is present in the cytoprotective composition in an amount from about 10% to about 50%, by weight.

17. The cytoprotective composition according to claim 8, wherein the mixture of saturated and unsaturated fatty acids is present in the cytoprotective composition in an amount from about 10% to about 50%, by weight.

18. A cytoprotective pharmaceutical composition for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties which comprises:
(A) a cytoprotective composition selected from the group consisting of:
(1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured mammalian cells; and (2) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b) an antioxidant; and (B) a medicament having cytotoxic properties.

19. A timed-release cytoprotective pharmaceutical composition for selectively protecting non-cancerous mammalian cells in the presence of cancerous mammalian cells from an anticancer medicament having cytotoxic properties which comprises:
(A) a cytoprotective composition in immediate release form selected from the group consisting of:
(1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured mammalian cells; and (2) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b) an antioxidant; and (B) an anticancer medicament having cytotoxic properties in timed-release form;
wherein the cytoprotective composition is released substantially immediately and the anticancer medicament is released after a period of time sufficient such that the cancerous cells have substantially metabolized the cytoprotective composition and the non-cancerous ells have not substantially metabolized the cytoprotective composition.

20. A method for preparing a cytoprotective composition for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties which comprises the steps of admixing the following ingredients:
(a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid , and mixtures thereof; and (b) an antioxidant.

21. A method for preparing a cytoprotective composition for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties which comprises the steps of admixing the following ingredients:
(a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of mammalian cells.

22. A method for preparing a cytoprotective pharmaceutical composition for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties which comprises the steps of:
(A) providing a cytoprotective composition selected from the group consisting of:
(1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured mammalian cells; and (2) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b) an antioxidant; and (B) providing an anticancer medicament having cytotoxic properties; and (C) admixing the cytoprotective composition from step (A) with the medicament from step (B) to prepare the cytoprotective pharmaceutical composition.

23. A method for preparing a timed-release cytoprotective pharmaceutical composition for selectively protecting non-cancerous mammalian cells in the presence of cancerous mammalian cells from an anticancer medicament having cytotoxic properties which comprises the steps of:
(A) providing a cytoprotective composition in an immediate release form selected from the group consisting of.
(1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured mammalian cells; and (2) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b) an antioxidant; and (B) providing an anticancer medicament having cytotoxic properties in timed-release form; and (C) admixing the cytoprotective composition from step (A) with the medicament from step (B) to prepare the timed-release cytoprotective pharmaceutical composition;

wherein the cytoprotective composition is released substantially immediately and the anticancer medicament is released after a period of time sufficient such that the cancerous cells have substantially metabolized the cytoprotective composition and the non-cancerous cells have not substantially metabolized the cytoprotective composition.

24. A method for protecting mammalian cell from a medicament having cytotoxic properties which comprises the steps of:
(A) providing a cytoprotective composition selected from the group consisting of:
(1) (a) pyruvate selected. from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured mammalian cells; and (2) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b) an antioxidant; and (B) providing an anticancer medicament having cytotoxic properties; and (C) administering the cytoprotective composition from step (A) and the medicament from step (B) concurrently to mammalian cells to protect the mammalian cells from the medicament having cytotoxic properties.

25. A method for selectively protecting non-cancerous mammalian cells in the presence of cancerous mammalian cells from an anticancer medicament having cytotoxic properties which comprises the steps of:
(A) providing a cytoprotective composition in an immediate release form selected from the group consisting of:

(1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid , and mixtures thereof;
(b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured mammalian cells; and (2) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b) an antioxidant; and (B) providing an anticancer medicament having cytotoxic properties in timed-release form; and (C) administering the cytoprotective composition from step (A) and the medicament from step (B) concurrently to mammalian cells to selectively protect non-cancerous mammalian cells in the presence of cancerous mammalian cells from the anticancer medicament having cytotoxic properties;
wherein the cytoprotective composition is released substantially immediately and the anticancer medicament is released after a period of time sufficient such that the cancerous cells have substantially metabolized the cytoprotective composition and the non-cancerous cells have not substantially metabolized the cytoprotective composition.

26. A method for selectively protecting non-cancerous mammalian cells in the presence of cancerous mammalian cells from an anticancer medicament having cytotoxic properties which comprises the steps of:
(A) administering to mammalian cells a cytoprotective composition to prevent and reduce injury to the mammalian cells selected from the group consisting of:
(1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured mammalian cells; and (2) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b) an antioxidant; and (B) waiting a period of time sufficient such that the cancerous cells have substantially metabolized the cytoprotective composition and the non-cancerous cells have not substantially metabolized the cytoprotective composition; and (C) administering the cytotoxic anticancer medicament to the mammalian cells to treat the cancerous cells which are unprotected by the cytoprotective composition and the non-cancerous cells which are protected by the cytoprotective composition to thereby increase the therapeutic effect of the anticancer medicament.

27. A cytoprotective pharmaceutical composition which comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of a cytoprotective composition for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties, wherein the cytoprotective composition is selected from the group consisting of:
(1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured mammalian cells; and

WO 93/16690 PCT/US93/00260 (2) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b) an antioxidant, 23. The cytoprotective pharmaceutical composition according to claim 27, further comprising a medicament having cytotoxic properties.

29. The cytoprotective pharmaceutical composition according to claim 28, wherein the cytoprotective composition is in immediate release form and the medicament having cytotoxic properties is in sustained release form.