Note: Descriptions are shown in the official language in which they were submitted.
W093/16690 PCT/US93~00260 ~-
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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
4 --
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
- 21 -
~ / 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
WO93/16690 PCT/VS93/00260
~ 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
- 46 -
~ ~' (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
WO93/16690 PCT/US93/00260
~ 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
5 0
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,
WO93/166~0 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.
, -- ~
