COMPOSITIONS A BASE D'INTERLEUKINE ET DE NUCLEOSIDES DE PYRIMIDINE

COMPOSITIONS OF INTERLEUKIN AND PYRIMIDINE NUCLEOSIDES

Abrégé français

L'invention porte sur une composition pharmaceutique synergique antitumorale, comprenant un volume efficace d'interleukine-12 et d'un dérivé de nucléoside de pyrimidine ainsi qu'un hydrate ou un solvate de celui-ci, converti en fluoro-uracil ou en un dérivé de ce dernier ainsi qu'un véhicule acceptable du point de vue pharmaceutique.

Abrégé anglais

A synergistic antitumor pharmaceutical composition comprising an effective
amount of interleukin-12 and a pyrimidine nucleoside derivative as well as a
hydrate or solvate thereof that is converted into fluorouracil or its
derivative, and a pharmaceutically acceptable carrier.

Revendications

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Claims
1. A synergistic antitumor pharmaceutical composition comprising
an effective amount of interleukin-12 and a pyrimidine nucleoside derivative
represented by the following formula (I) or
(II), respectively
<IMG> ( I ) <IMG> ( II )
wherein R1 is hydrogen or a radical which is easily hydrolyzable
under physiologicdl conditions; R2 is hydrogen, cyano,
fluorine,lower alkyl or lower alkylidene which may be substituted
with one or two fluorine atom(s), or OR1; and R3 is lower alkyl,
hydroxymethyl, or CH2OR1,
as well as hydrate or solvate thereof, and a pharmaceutically acceptable ~
2. The composition of claim 7, wherein the easily hydrolyzable radical
is R4CO-, R4OCO- or R4SCO-, wherein R4 is alkyl, cycloalkyl, aralkyl or aryl.
3. The composition of claim 2, wherein the alkyl, cycloalkyl, aralkyl or
aryl radical represented by R4 is a saturated, straight or branched
hydrocarbon radical twherein the number of carbon atoms in the longest
straight chain of this hydrocarbon radical ranges from three to seven], or a
radical of the formula (CH2)n-Y[in which n is an integer from 0 to 4, when Y
is cyclohexyl, or n is an integer from 2 to 4, when Y is lower alkoxy having 1
to 4 carbon atom(s) or phenyl].

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4. The composition of claim 1, wherein the pyrimidine nucleoside
derivative is selected from the group consisting of:
5'-deoxy-5-fluorouridine,
5-deoxy-5-fluorocytidine,
5'-deoxy-N4-(3,5-dimethoxybenzoly)-5-fluorocytidine,
5'-deoxy-N4-(3,5-dimethylbenzoly)-5-fluorocytidine,
5'-deoxy-N4-[(2,4-dichlorophenyl)acetyl]-5-fluorocytidine,
5'-deoxy-N4-(indol-2-ylacetyl)-5-fluorocytidine,
5'-deoxy-5-fLuoro-N4-(3,4,5-trimethylbenzoly)cytidine,
5'-deoxy-5-fluoro-N4-(propoxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N4-(hexyloxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N4-(isopentyloxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N4-(neopentyloxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N4-[(1,1,2-trimethylpropoxy)carbonyl]cytidine,
5'-deoxy-N4-[(3,3-dimethylbutoxy)carbonyl]-5-fluorocytidine,
5'-deoxy-5-fluoro-N4-[(1-isopropyl-2-methylpropoxy)carbonyl]cytidine,
5'-deoxy-N4-[(2-ethylbutyl)oxycarbonyl]-5-fluorocytidine,
N4-[(cyclohexylmethoxy)carbonyl]-5'-deoxy-5-fluorocytidine,
5'-deoxy-5-fluoro-N4-[(2-phenylethoxy)carbonyl]cytidine,
2',3'-di-O-acetyl-5'-deoxy-5-fluoro-N4-(propoxycarbonyl)cytidine,
2',3'-di-O-acetyl-N4-(butoxycarbonyl)-5'-deoxy-5-fluorocytidine,
2',3'-di-O-benzoyl-N4-(butoxycarbonyl)-5'-deoxy-5-fluorocytidine,
2',3'-di-O-acetyl-5'-deoxy-5-fluoro-N4-(pentyloxycarbonyl)cytidine,
2',3'-di-O-acetyl-5'-deoxy-5-fluoro-N4-(isopentyloxycarbonyl)cytidine,
2',3'-di-O-acetyl-5'-deoxy-5-fluoro-N4-(hexyloxycarbonyl)cytidine,
2',3'-di-O-acetyl-5'-deoxy-N4-[(2-ethylbutyl)oxycarbonyl]-5-fluorocytidine,
2',3'-di-O-acetyl-N4-[(cyclohexylmethoxy)carbonyl]-5'-deoxy-6-fluorocytidine,
2',3'-di-O-acetyl-5'-deoxy-5-fluoro-N4-[(2-phenylethoxy)carbonyl] cytidine,
5'-deoxy-5-fluoro-N4-(isobutoxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N4-[(2-propylpentyl)oxycarbonyl]cytidine,
5'-deoxy-N4-[(2-ethylhexyl)oxycarbonyl]-5'-fluorocytidine,
5'-deoxy-5-fluoro-N4-(heptyloxycarbonyl)cytidine,
N4-[(2-cyclohexylethoxy)carbonyl]-5'-deoxy-5-fluorocytidine,
N4-[(3-cyclohexylpropyl)oxycarbonyl]-5'-deoxy-5-fluorocytidine,
N4-(cyclohexyloxycarbonyl)-5'-deoxy-5-fluorocytidine,
5'-deoxy-5-fluoro-N4-[(3-phenylpropyl)oxycarbonyl] cytidine,
5'-deoxy-5-fluoro-N4-[(2-methoxyethoxy)carbonyl] cytidine,

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N4-(butoxycarbonyl)-5'-deoxy-5-fluorocytidine,
5'-deoxy-5-fluoro-N4-(pentyloxycarbonyl)cytidine,
2',2'-difluorodeoxycytidine,
5-fluoro-1-tetrahydrofuran-1-yluracil,
2'-deoxy-2'-methylidene-5-fluorocytidine,
2'-deoxy-2'-cyano-5-fluorocytidine
as well as hydrates or solvates thereof.
5. The composition any one of claims 1 to 4 wherein the weight ratio of
interleukin-12 to pyrimidine nucleoside is from about 1:100 to about 1:400,000.
6. The composition any one of claims 1 to 4, wherein the weight ratio of
interleukin-12 to pyrimidine nucleoside is from about 1:1,000 to about
1:10,000.
7. A synergistic antitumor pharmaceutical composition for the
treatment of colorectal cancer, brest cancer, stomach cancer, lung cancer,
cervical cancer, bladder cancer and other malignant diseases comprising
an effective amount of interleukin-12 and a pyrimidine nucleoside as well as
a hydrate or solvate thereof, that are converted into fluorouracil or other
active metabolite by pyrimidine nucleoside phosphorylase, and a
pharmaceutically acceptable carrier.
8. The composition of claim 7, wherein the pyrimidine nucleoside is an
uridine, cytidine or its derivative represented by the following formula (I) or
(II), respectively as defined in claim 2 as well as a hydrate or solvate thereof.
9. The composition of claim 7 wherein the easily hydrolyzable radical
is R4CO-, R4OCO- or R4SCO-, wherein R4 is alkyl, cycloalkyl, aralkyl or
aryl.
10. The composition of claim 9, wherein the alkyl, cycloalkyl, aralkyl
or aryl radical represented by R4 is a saturated, straight or branched
hydrocarbon radical [wherein the number of carbon atoms in the longest
straight chain of this hydrocarbon radical ranges from three to seven], or a
radical of the formula (CH2)n-Y[in which n is an integer from 0 to 4, when Y
is cyclohexyl, or n is an integer from 2 to 4, when Y is lower alkoxy having 1
to 4 carbon atom(s) or phenyl].

18
11. The composition of claim 8, wherein the pyrimidine nucleoside
derivative is selected from the compounds defined in claim 5.
12. The composition any one of claims 7 to 11, wherein the weight ratio
of interleukin-12 to pyrimidine nucleoside is from about 1:100 to about
1:400,000.
13. The composition any one of claims 7 to 11, wherein the weight ratio
of interleukin-12 to pyrimidine nucleoside is from about 1:1,000 to about
1:10,000.
14. A method of treating colorectal cancer, brest cancer, stomach
cancer, lung cancer, cervical cancer, bladder cancer and other malignant
diseases which comprises administering a synergistic antitumor
pharmaceutical composition comprising an effective amount of interleukin-12
and pyrimidine nucleoside or as well as hydrate or solvate thereof, that
are converted into fluorouracil or other active metabolite by pyrimidine
nucleoside phosphorylase, and a pharmaceutically acceptable carrier.
15. The method of claim 14, wherein the pyrimidine nucleoside is an
uridine, cytidine or its derivative represented by the following formula (I) or
(II), respectively, as defined in claim 2, as well as a hydrate or solvate
thereof.
16. The method of claim 15, wherein the easily hydrolyzable radical is
R4CO-, R4OCO- or R4SCO-, wherein R4 is alkyl, cycloalkyl, aralkyl or aryl.
17. The method of claim 16, wherein the alkyl, cycloalkyl, aralkyl or
aryl radical represented by R4 is a saturated, straight or branched
hydrocarbon radical [wherein the number of carbon atoms in the longest
straight chain of this hydrocarbon radical ranges from three to seven], or a
radical of the formula (CH2)n-Y [in which n is an integer from 0 to 4, when
Y is cyclohexyl, or n is an integer from 2 to 4, when Y is lower alkoxy having
1 to 4 carbon atom(s) or phenyl].

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18. The composition of claim 16, wherein the pyrimidine nucleoside
derivative is the one selected from the compounds defined in claim 5, as well
as a hydrate or solvate thereof.
19. The method any one of claims 14 to 18, wherein the weight ratio of
interleukin-12 to pyrimidien nucleoside is from about 1:100 to about 1:400,000.
20. The method any one of claims 14 to 18, wherein the weight ratio of
interleukin-12 to pyrimidine nucleoside is from about 1:1,000 to 1:10,000.
21. A kit for the treatment of colorectal cancer, breast cancer, stomach
cancer, lung cancer, cervical cancer, bladder cancer and other malignant
diseases which comprises as a first pharmaceutical composition containing
an effective amount of interleukin-12 and a pharmaceutically acceptable
carrier, and as a second pharmaceutical composition containing an effective
amount of a pyrimidine nucleoside derivative as well as a hydrate or solvate
thereof and a pharmaceutically acceptable carrier.
22 . The use of interleukin-12 and a pharmaceutically acceptable
carrier, and as a second pharmaceutical composition containing an effective
amount of a pyrimidine nucleoside derivative as well as a hydrate or solvate
thereof for the manufacture of pharmaceutical compositions as claimed in
claims 7 or 21.

Description

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Compositions of interleukin and pvrimidine nucleosides
i
This invention is concerned with a novel pharmaceutical composition.
More particularly, this invention is concerned with a synergistic antitumor
5 pharmaceutical composition comprising an effective amount of interleukin-
12 (IL-12) and a pyrimidine nucleoside, as well as a hydrate or solvate
thereof, that is converted into fluorouracil or its derivative, and pharma-
ceutically acceptable carrier, a synergistic antitumor pharmaceutical
composition for the treatment of various cancer and a method of treating
10 various cancers.
5'-Deoxy-6-fluorouridine (doxifluridine), a pyrimidine nucleoside, is
effective in the treatment of various m~lign~nt diseases. Doxifluridine is
converted into the active drug 5-FU by pyrimi~ine nucleoside phosphorylases
15 (PyNPase) in vivo, both thymidine and uridine phosphorylases. Therefore,
PyNPase is essential for the efficacy of doxifluridine. In fact, tumors with
very low levels of this enzyme were refractory to doxifluridine, and PyNPase
gene transfection made the tumors more susceptible to this drug. Now, it
has surprisingly been found that IL-12 up-regulates PyNPase activity in
20 tumor tissues and consequently enhances the antitumor activity of
doxifluridine. IL-12 also enhanced the activity of 5'-deoxy-~-fluoro-N4-(n-
pentylcarbonyl)cytidine (capecitabine), which generates doxifluridine and is
then converted to 5-FU by PyNPase. In contrast, IL-12 ~nh~n~ed the anti-
tumor activity of 6-FU to a lesser extent than the anti-tumor activity of
25 doxifluridine.
It has been reported that some infl~mm~tory cytokines, such as IL-la,
TNF-a and IFN-g up-regulate PyNPase activity in tumor cell cultures and
consequently enhance the susceptibility to doxifluridine (cf. Eda et al. Cancer
30 Chemother Pharmacol. (1993) 32:333-339, and Jpn. J. Cancer Res. 84, 341-
347, March 1993). These cytokines when given parenterally are distributed to
various normal tissues through the circulation and cause systemic side
effects, such as llu-like syndrome, leukopenia, hypotension, etc. In addition,

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these cytokines distributed to normal tissues as well as tumor tissues would
enh~nce PyNPase activity there and make both the normal and tumor
tissues more susceptible to doxifluridine. Therefore, these cytokines would
enh~nce both the efficacy and toxicity of doxifluridine when given in
5 combin~t,ion IL-12 given parenterally, howevel, induced much higher
levels of IFN-g in tumor tissues than in normal tissues. The~efole, IL-12
given parenterally enh~nces PyNPase activity preferentially in tllmor
tissues without c~ ing IFN-g-associated systemic side effects.
In a ~lef~lled embodiment of the present invention, the pyrimidine
nucleoside is an uridine, cytidine or its derivative represented by the
~ following formula (I) or (II), respectively
HNR1 0
N ~ F H N J~ F
R3 N (I) R3 N (II)
YY YY
R10 R2 R1O R2
wherein Rl is hydrogen or an radical which is easily hydrolyzable
under physiological conditions; R2 is hydrogen, cyano,
fluorine,lower alkyl or lower alkylidene which may be substituted
with one or two fluorine atom(s), or ORl; and R3 is lower alkyl,
hyllro~y~ethyl, or CH20Rl,
as well as a hydrate or solvate thereo~
r.erelled r~lic~l~ which are easily hydrolyzable under physiological
conditions of Rl in the above formulae (I) and (II) are R4Co-, R40Co- or
R4SCo-
wherein R4 is alkyl, cycloalkyl, aralkyl or aryl.
Furthermore, ~lefell~d alkyl, cycloalkyl, aralkyl or aryl radical
represented by R4 are a saturated, straight or branched hydrocarbon radical
[wherein the number of carbon atoms in the longest straight ch~in of this
hydrocarbon radical ranges from three to seven], or a radical of the formula
(CH2)n-Y[in which n is an integer from O to 4, when Y is cyclohexyl, or n is

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an integer from 2 to 4, when Y is lower alkoxy having 1 to 4 carbon atom(s) or
phenyl] .
In the above, the term "a saturated, straight or branched hydrocarbon
5 radical ~wherein the number of carbon atoms in the longest straight chain of
this hydrocarbon radical ranges from three to seven]" preferably signifies
n-propyl, 1-isol.lo~yl-2-methylpropyl, 1,1,2-trimethylpropyl, n-butyl, isobutyl,2-ethylbutyl, 3,3-dimethylbutyl, n-pentyl, isopentyl, neopentyl, 2-propylpentyl,n-hexyl, 2-ethylhexyl, n-heptyl, allyl, 2-buten-1-yl, 3-buten-1-yl, 3-penten-1-yl,
10 4-penten-1-yl, 3-hexen-1-yl, 4-hexen-1-yl, 5 hexen-1-yl, and the like.
The terIn "a radical of the formula (CH2)n-Y lin which n is an integer
from O to 4, when Y is a cyclohexyl radical, or n is an integer from 2 to 4,
when Y is a lower alkoxy radical having from 1 to 4 carbon atom(s) or a
~5 phenyl radical]" ~.efelably .cigini~es cyclohexyl, cyclohexylmethyl,
2-cyclohexylethyl, 3-cyclohexylpropyl, 4-cyclohexylbutyl, 2-methoxyethyl, 2-
ethoxyethyl, 2-propoxyethyl, 3-metho~LylJLo~yl, 3-ethu~y~Io~yl, 4-methoxy-
butyl, 4-etho~LyL.ulyl, phenethyl, 3-phenylpropyl, 4-phenylbutyl, and the like.
Preferred pyrimilline nucleoside for the present invention are:
6'-deoxy-5-lluorouridine,
5-deoxy-5-fluorocytidine,
6'-deoxy-N4-(3 ,5-dimethoxybenzoly)-5-fluorocytidine,
25 5'-deoxy-N4-(3,5-dimethylbenzoly)-5-fluorocytidine,
B'-deoxy-N4- [(2,4-dichlorophenyl)acetyl] -5-fluorocytidine,
5'-deoxy-N4-(indol-2-ylacetyl)-5-fluorocytidine,
5'-deoxy-5-fluoro-N4-(3,4,5-trimethylbenzoly)cytidine,
5'-deoxy-5-fluoro-N4-(propoxycarbonyl)cytidine,
30 5'-deoxy-5-fluoro-N4-(hexyloxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N4-(isopentyloxycarbonyl)cytidine,
6'-deoxy-5-fluoro-N4-(neopentyloxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N4-[(1,1,2-trimethylpropoxy)carbonyl]cytidine,
5'-deoxy-N4- [(3 ,3-dimethylbutoxy)carbonyl] -5-fluorocytidine,
35 5'-deoxy-5-fluoro-N4-[(l-isopropyi-2-methylpropoxy)carbonyl]cytidine,
5'-deoxy-N4- [(2-ethylbutyl)oxycarbonyl] -5-fluorocytidine,
N4- [(cyclohexylmethoxy)carbonyl] -5'-deoxy-5-fluorocytidine,
5'-deoxy-5-fluoro-N4- [(2-phenylethoxy)carbonyl] cytidine,

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2',3'-di-O-acetyl-5'-deoxy-5-fluoro-N4-(propoxycarbonyl)cytidine,
2',3'-di-O-acetyl-N4-(butoxycarbonyl)-6'-deoxy-5-fluorocytidine,
2',3'-di-O-benzoyl-N4-(butoxycarbonyl)-6'-deoxy-5-fluorocytidine,
2',3'-di-O-acetyl-5'-deoxy-5-fluoro-N4-(pentyloxycarbonyl)cytidine,
5 2',3'-di-O-acetyl-~'-deoxy-6-fluoro-N4-(isopentyloxycarbonyl)cytidine,
2',3'-di-O-acetyl-6'-deoxy-6-fluoro-N4-(hexyloxycarbonyl)cytidine,
2',3'-di-O-acetyl-6'-deoxy-N4- [(2-ethylbutyl)oxycarbonyl] -5-fluorocytidine,
2',3'-di-O-acetyl-N4-[(cyclohexylmethoxy)carbonyl] -6'-deoxy-5-fluorocytidine,
2',3'-di-O-acetyl-5'-deoxy-5-fluoro-N4-[(2-phenylethoxy)carbonyl]cytidine,
10 5'-deoxy-5-fluoro-N4-(isobutoxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N4-[(2-propylpentyl)oxycarbonyl]cytidine,
5'-deoxy-N4-[(2-ethylhexyl)oxycarbonyl]-5'-fluorocytidine,
5'-deoxy-5-fluoro-N4-(heptyloxycarbonyl)cytidine,
N4-[(2-cyclohexylethoxy)carbonyl]-6'-deoxy-6-fluorocytidine,
15 N4-[(3-cyclohexylpropyl)oxycarbonyl]-5'-deoxy-6-fluorocytidine,
N4-(cyclohexyloxycarbonyl)-6'-deoxy-6-fluorocytidine,
5'-deoxy-5-fluoro-N4- [(3-phenylpropyl)oxycarbonyl] cytidine,
5'-deoxy-5-fluoro-N4-[(2-methoxyethoxy)carbonyl]cytidine,
N4-(butoxycarbonyl)-5'-deoxy-5-fluorocytidine
20 5'-deoxy-5-fluoro-N4-(pentyloxycarbonyl)cytidine,
2',2'-difluorodeoxycytidine,
5-fluoro-1-tetrahyL Or~ an-1-yluracil,
2'-deoxy-2'-methylidene-5-fluorocytidine,
2'-deoxy-2'-cyano-6-fluorocytidine and
25 as well as hydrate or solvate thereo~
The above mentioned specific compounds are described in U.S. patent
Nos. 4,071,680 and 4,966,891, European Patent Nos. 602290-A1, K. Takenuki
et al. J. Med. Chem. 31, 1063 (1988), K. Y~magami et al. Cancer Research 61,
30 2319 (1991) and A. Matsuda et al. J. Med. ~hem. 34, 2917 (1991), respectively,
and those compounds can be produced according to the method described in
the respect*e references or the analogous method thereof.
IL-12 is a heterodimeric cytokine which is produced by antigen
35 presenting cells and serves as a pivotal regulator of T and NK cell function
(cf. Stern, A.S. et al. Proc. Natl. Acad. Sci. USA. (1990) 87, 6808-6812 and
Kobayashi, M. et al. J. Exp. Med. (1989) 170, 827-845). Biological activities
associated with I~12 include its ability to ~?nh~nce the lytic activity of

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natural ~illertlymphokine activated killer cells, to induce the secretion of
inte.r~:lo.l-g (IFN-g) by both resting and activated T and NK cells, to
stimulate the proliferation of activated T and NK cells, to facilitate cytotoxicT lymphocyte responses and to play a critical and unique role in promoting
5 Th-1 type cytokine responses, thereby facilitating cell-mediated immunity
(c~ Brunda, M. J. J. Leukocyte Biol. (1994) ~5, 280-288 and Taniguchi, G.
Blood (1994) 84,4008-4027).
IL-12, both human type and murine l,ype, is composed of two dis7l1fide-
10 bonded ~ly~op.otein subllnits a~..Jx;..l~tely 35 KDa and 40 KDa in size.
cDNAs encoding each subunit of IL-12 have been cloned and coexpressed in
Chinese Hamster Ovary (CHO) cells to yield the secreted, bioactive,
hetero~imeric lympho7~ine (Gubler, U. et al. Proc. Nat~. Acad. Sc~. USA.
(1991) 88, 4143-4147 and Sr-h~Rnh~l7t~ D.S. et al. J. Immunol. (1992) 148, 3433-
15 3440) A clone of tr~n.qfected CHO cells secreting recomhin~nt IL-12 was
selected. Recnmhin~nt IL-12 was purified from the culture supernatant of
CHO cells grown in a serum-free medium, by ion P~h~n~e and gel filtration
chromatography.
The pharmaceutical compostion of the present invention can be
~r7mini~qtered in any form, for example, tablets, pills, suppositories,
capsules, granules, powders, etc. or emulsions. The pharmaceutical
composition of the present invention are especially suitable for
intramuscular, subcutaneous, or intravenous a~mini.~tration.
Pharmaceutically acceptable carriers and excipients useful in formulating
the pharmaceutical composition of the present invention are those
commonly used. Pharmaceutically acceptable materials can be an organic
or inorganic inert carrier material suitable for enteral, percutaneous or
parenteral ~(lmini.qtration such as water, gelatine, gum arabic, lactose,
starch, m~gnecium stearate, talc, vegetable oils, polyalkylene glycols and
petrolel~m jelly. The pharmaceutical composition provided by the present
invention can be ~lmini.~tered orally, e.g. in form of tablets, capsules, pills,powders, granules, solutions, syrupsj suspensions or elixirs. The
~lmini.~:tration can also be carried out parenterally, e.g. in form of sterile
solutions, suspensions or emulsions; or locally, e.g. in form of solutions,
suspensions, salves, powders or aerosols. The pharmaceutical composition
can be sterilized and/or can contain further adjuvants such as preserving,

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st~hili~in~, setting, emulsifying agents, flavor-improving, salts for variation
of the osmotic pressure or substances acting as buffers.
The synergistic antitumor pharmaceutical composition of the present
5 invention comprises a single pharmaceutical composition as well as a kit of
pharmaceutical compositions each cont~ining the individual active
ingredient in a desirable dosage for_. thus, the present invention is also
concerned with a kit for the treatment of colorectal cancer, breast cancer,
sto~n~h cancer, lung cancer, cervical cancer, bladder cancer and other
10 m~lign~nt diseases which comprises as a first pharmaceutical composition
cont~ining an effective amount of IL-12 and a pharmaceutically acceptable
carrier, and as a second pharmaceutical composition cont~ining an effective
~mount of pyrimidine nucleoside derivative, as well as hydrate or solvate
thereof and a pharmaceutically acceptable carrier.
Dosage ranges for the pharmaceutical composition of the present
invention can easily be determined by one .qkille~l in the art, and depend on
the route of ~lmini~ctration~ the age, weight and condition of the patient and
the particular disease to be treated. In the case of oral, rectal or parenteral
mini~tration for adults, an a~.uxi..~te range from about 0.05
mg/body/day to about 500 mg/body/day of IL-12, and about 50 mg/body/day to
about 20,000 mg/body/day of pyrimidine nucleoside generally range from
about 1:100 to about 1:400,000. A weight ratio from about 1:1,000 to about
1:10,000 is preferred. Rectal ~mini~tration and intravenous injection are
25 ~ere. . ed routes of ~lmini.ctration of the pharmaceutical composition
according to the present invention.
The pharmaceutical compositions of the present invention are useful
for the treatment of colorectal cancer, breast cancer, stomach cancer, lung
30 cancer, cervicial cancer, bladder cancer, and other m~lign~nt diseases and
the like.
The synergistic antitumor activity of the pharmaceutical composition
of the present invention is evident from the tests described hereinafter.
35 (1) Up-regulation of the enzyme for the activation of doxifluridine and 5'-
deoxy-5-fluoro-N4-(n-pentyloxycarbonyl)cytidine (capecitabine) by mIL-12.

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Six weeks old female C57BL/6 mice or male BALB/c mice were
inoculated with A755 m~mm:~y adenocarcinoma (2 x 105 cells) or with
Meth A fibrosarcoma (5 x 105 cells), respectively. The mice were given s.c.
mouse IL-12 (mIL-12) at 0.1 mg/mouse or vehicle (0.1 mg/ml of mouse
5 serum albumin dissolved in phosphate-buffered saline) daily for 7 days
~laL l,illg at day 7 and day 8, respectively, after the tumor inoculation. One
day thereafter, PyNPase activity in the tumor tissues was measured as
described in Eda et al. Cancer Chemother. Pharmacol. 1993; 32:333. Mouse
IFN-g (m IFN-g) levels in the tumor tissue were also measured by a
10 commercially available ELISA system (Intertest g, Genzyme).
As Table 1 shows, mIL-12 enh~nred PyNPase activity 11.9 fold in A755
tumors and 2.4 fold in Meth A tumors. This is probably the result of the up-
regnl~tion of mIFN-g, which is an up-regulator of PyNPase.
~5
Table 1
Up-regulation of PyNPase and mIFN-g by mIL-12
PyNPase mIFN-g
Tumor Model A~mini.qtration Activity Levels
(,ug 5-FU/mg/hr)(ng/g tissue)
A756 m.qmmZ3ry ca. vehicle 4.4+4.6 a) <2.0
mIL-12 52.3+28.8 *46.8+11.7 *
Meth A fibrosarcoma vehicle 21.4+2.6 <1.1
mIL-12 51.7+9.4 * 6.7+~.1 *
* Statistically .~ignific~nt diLre~eLlce from the vehicle groups (p<0.0~, Student
t-test).
35 a) Mean + SD of 6 mice.
2) Selective induction of mIFN-g production by mIL-12 in the tumor
tissue.

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Six weeks old female C57BL/6 mice were inoculated with A755
m~mm~ry adenocarcinoma (2 x 10~ cells). The mice were given s.c. mIL-12
at 0.1 mgtmouse or vehicle (0.1 mg/ml of mouse serum albumin dissolved in
5 phosphate-burre~ed saline) daily for 7 days starting at day 6 after the tumor
inoculation. One day thereafter, mIFN-g levels in the serum and tissue
homogenates of tumor and other organs were measured by ELISA system as
mentioned above.
-
As Table 2 shows, mIL-12 greatly increased mIFN-g levels in tu~nors
as compared with those in normal organs. The tumor tissue level of mIFN-g
is 3 to 7 fold higher than those of normal tissues so far ~mined and 50 fold
higher than those in the circulation. These results suggest that mIL-12 up-
regulates PyNPase selectively in the tumor tissue, through the up-regulation
~5 of mIFN-g production by IL-12 selectively in tumor tissues.

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Table 2
Selective induction of mIFN-g production by mIL-12 in tumor tissues
.
mIFN-g levels (ng/g tissue) a)_
Organs treatment of the mice
vehicle mIL-12
tllmor 3.2 + 0.9 32.7 + 9.8 *
serum 0.27 + 0.05 0.66 + 0.22 *
small intestine <2.5 5.3 +4.0
large intestine 2.8 + 0.8 4.3 + 0.5 *
spleen 2.9 + 0.5 9.3 + 1.9 *
liver 2.1 + 1.0 4.2 + 0.6 *
kidney 5.1 + 1.1 7.5 + 1.4 *
thymus b) 6.2 10.3
*; Significantly higher than vehicle group (p<0.05).
a); Mean + SD. n=4 and 5 in vehicle and mIL-12 group,
respectively, with an exception of thymus.
b); Values obtained from a comhine~l homogenate of 4 thymuses.
(3) Antitumor effects of combination of doxifluridine or capecitabine and
IL-12
30 1) A755 m~n~m~ry adenocarcinoma model
A755 (2 x 105 cells) was inoculated s.c. into female C57BL/6 mice. The
mice were given mIL-12 (0.03 ,ug/mouse, s.c.), doxifluridine (1.5 mmol/kg,
p.o.) and their comhin~tion, daily for 4 weeks, starting from day 9 after the
tumor inoculation.
Doxifluridine as a single agent did not show act*ity in tumor ~ ~ ow lh
inhibition because A755 tumor has only low levels of PyNPase, whereas

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mIL-12 was effective (Table 3). mIL-12 in cnmhin~t.ion with doxifluridine
was much more effective than mIL-12 alone and regressed the tumor.
The combination effect was more obvious when the survival period
5 was observed (Table 4). Doxifluridine was not effective either in prolongationof the ~u. ~ival period, and IL-12 slightly prolonged the survival period. In
contrast, IL-12 and doxifluridine in combination prolonged the survival
period much longer than IL-12 alone and some mice were cured (3/5).
Table 3
Antitllmor activity of doxifluridine, mIL-12 and their comhin~tio~ in
A755 m~mm~ry adenocarcinoma model.
Treatment Tumor Volume (mm3) % Tllmor Growth
on day 25 a) vs. Control vs. mIL-12 alone
Control 8969 + 4159 b) 100
doxifluridine 8380 + 2907 93
mIL-12 2399 + 1567 c) 22 100
doxilluridine 216+ 187 c) d) 4 - 16
~ mIL-12
a) Tumor volllme of 26 days after tumor inoculation was indicated, since
thereafter death of mice in the control group were observed because of large
tumor burden. The tumor volume on day 9 when the treatment initiated was
513 + 300 mm3.
b) Mean + SD
c) Significantly different from the control group. p < 0.05
d) Significantly different from the mIL-12 group. p < 0.05

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Table 4
Survival of mice treated with doxifluridine, mIL-12 and their
combin~tion in A755 m::~mm::~ry adenocarcinoma model
r
TreatmentMedian Survival Increase in Survivors
Days (range) Life Span % onDay 153
Control 28 (23 - 42) 0 0/6
doxifluridine29 (22 - 36) 4 0/5
mIL-12 49 (43 55) a) 75 0/5
~5
doxifluridine
+ mIL-12>153 (51 - >153) a) b) >446 3/5
a) Significantly dirre~ el~t from the control group. p < 0.05
b) Significantly different from the mIL-12 group. p < 0.05
2) Meth A fibrosarcoma model
Meth A fibrosarcoma (5 x 105 cells) was inoculated s.c. into male
BALB/c mice. The mice were given doxifluridine (0.5 mmol/kg, p.o.),
capecitabine (1.0 mmoVkg, p.o.), 5-fluorouracil (0.075 mmol/kg, p.o.), mIL-12
(0.03 ~Lg/mouse, s.c.), and their combination, daily for 3 weeks, starting from
30 day 8 after the tumor inoculation.
Three fluo~o~y~ ddines or mIL-12 as single agents showed moderate
activity in tumor growth inhibition, at the doses employed. mIL-12 in
combination with either doxifluridine or capecitabine showed more potent
35 antitumor activity than either drug alone (p < 0.05). On the other hand,
mIL-12 and 5-fluorouracil in combination was only slightly more effective
than either drug alone (not statistically significant).

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Table 5
Antitumor activity of three fluoropyrimidines (doxifluridine,
capecitabine, 5-fluorouracil), mIL-12 and their combination in Meth A
fibrosarcoma model.
Treatment Tumor Volume (mm3) % Tumor Growth
on day 29 a) vs. Control vs. mIL-12
Control 10489 + 2054 b) 100
doxifluridine 5170+ 887 c) 48
capecitabine 4941_1397C) 46
~5 5-fluorouracil 6168+ 530 c) 58
mIL-12 4584_ 1198 c) 42 100
mIL-12 + doxifluridine 19l2-l322cde) 16 39
mIL-12 + capecitabine 2199+ 132gcde) 19 45
mIL-12 + 5-fluorouracil 3603_1786 ce) 32 76
a) Tumor volume of 29 days after tumor inoculation was indicated. The
mean tumor volume on day 8 when the treatment initiated was 230 mm3.
b) Mean_ SD
25 c) Significantly different from the control group. p < 0.06
d) Significantly different from the mIL-12 alone group. p < 0.06
e) Significantly different from the corresponding fluoropyrimidine alone
group. p < 0.06
The following examples illustrate a pharmaceutical preparation of the
present invention and do not limit the scope of the present invention.

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Example 1
An injectable solution cont~ining the following ingredients was
~, manufactured in a conventional manner:
S doxifluridine 1000 mg
hIL-12 50 ,ug
NaCl 41.4 mg
NaH2PO4 16.2 mg
Na2HPO4 36.7 mg
polysorbate 80 4 mg
adjust pH 7.0 with 1.2 N HCl or 1 N NaOH
adjust total volume of 20 ml with distilled water for injection
~5 Example 2
An injectable solution each cont~ining the following ingredients was
manufactured in a conventional m:inn~r:
capecitabine 100 mg
hIL-12 50 ,ug
NaCl 718.2 mg
NaH2PO4 81 mg
Na2HP04 188 mg
polysorbate 80 20 mg
adjust pH 7.0 with 1.2 N HCl or 1 N NaOH
adjust total volume of 100 ml with distilled water for injection

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Ex~mple 3
A kit having the following components A and B for treatment of colorectal
cancer was manufactured in a conventional manner:
Component A
(granule for oral a~mini.ctration)
capecitabine 150 mg
hyL o~y~ o~ylmethyl cellulose 29104.5 mg
crystalline cellulose 14.7 mg
croscarmellose sodium (Ac-Di-Sol)6.0 mg
magnesium stearate 1.8 mg
coating agent 3.0 mg
Total 180 mg
Component B
(sterile solution for injection)
hIL-12 0.2-20 mg
NaCl 116 mg
NAH2PO4 62.2 mg
Na2HPO4 115.8 mg
polysorbate 80 4 mg
adjust pH to 7.0 with 1.2 N HCl or lN NaOH
adjust total volume to 20 ml with water for injection