CA 02286750 2003-03-26
~fITLE OF THE INVENTION
APPLICATION AND PROI)I,1C'TION fRCJCf~;SS t)F A CROUP OF
ANTICANCER C',OMfC>L~NOS
BACKGROUND OF 'THE INVENTION
field of the invention
The present invention relates to a group of compounds, i.e., specific branched-
chain fatty acids, and pharmaceutically acceptable salts and derivatives
thereof, with
significant anticancer activities, and rnethocls of treating cancer. The
invention also
rE:lates to a process of producing fermentation products containing said
specific branched-
chain fatty acids, using specific bacteria striiins, preferably in industrial
facilities.
Description of the Back rg_ound
Carcinoma is one of the most serious diseases threatening human's health and
life.
So far the predominant treatments to cancer patients are radiotherapy and
chemotherapy.
Both have certain toxicity or side effects on human while suppressing the
cancer cell
growth or killing cancer cells. Therefore extensive itwestigatio,vs have been
carried out in
order to find an effective anti-carcinogen with minimum side effects and
toxicity.
In 1987, when the inventor cultured K562 leukemia cell lines in the
laboratory,
cells in a culture flask were found completely disappeared 48 hours after
being
contaminated by a kind of rod bacteria. 'hhose rod bacteria we.rc then
intentionally
harvested and purified, and incubated. in the soybean media with appropriate
inorganic
salts. It was found in later animal studies that the fermentation solution
effectively
inhibited tumor growth with no toxicity or side effects. In the decade since
then,
thousands of cancer patients, including advanced stage cancer patients, have
been treated
with the oral liquid developed from this fermentation solutic7n. 'These
include leukemia,
tongue cancer, colorectal cancer, breast cmcer, prostate cancer and lung
cancer, gastric
cancer, hepatocarcinoma, melanocarcinoma, renal cancer, esophagus cancer and
pancreas
cancer patients. Most of them have responded to the oral liquid, such as
symptom
improvement, tumor shrinkage or even complete disappearance. Many of these pat
ants
are still alive today. The cases included patients in C_:hin~i, .~~upan.:
Korea, the United
States, and many other countries.
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In order to discover the active components in the fermentation solution that
play a
key role in killing cancer cells, persistent investigations have been carried
out for the last
ten years. In this period many books and papers were pul~lishc:d worldwide
trying to
explain the anticancer activity of this fermentation solutic>n. Mlost of these
reports
suggested that some soybean isoflavones (e.g. genistein, daidzein and saponin)
from the
soybean media contributed to the anticancer activities of'this fermentation
solution. On
tile other hand, some clinical trials indicated that the anticancer activities
of soybean
isoflavones were not great enough to explain the anticancer effects of the
fermentation
solution. The inventor has isolated many compounds fiom the fermentation
solution and
revealed that the anticancer activities of the solution were largely
contributed by 13-
methyltetradecanoic acid and 12-methyltetradecanoic acid. Further
investigations
discovered that other members of the family of branched-chain fatty acids and
their
derivatives, which were structurally characterised by terminal) y methyl
branch, also had
significant tumor-inhibition effects.
SUMMARY OF THE INVENTION
The present invention relates to a group of compounds, i.e., specific
terminally
branched-chain fatty acids, and pharmaceutically acceptable salts and
derivatives thereof,
with significant anticancer activities. and methods oFtreating cancer using
these
compounds. The comprehensive biochemical and morphological tests demonstrated
that
these activities are associated with induction of programmed cancer cell death
(apoptosis). Very importantly, the said specific branched-chain fatty acids do
not kill the
normal cells. In the animal studies, intraperitoneal injection of 13-
methyltetradecanoic
acid daily up to 800 mg/kg to mice did not reach the LL)5() level X50% lethal
dose). In
human clinical studies, six volunteers received ().6g --- 1.8g 13-
rnethyltetradecanoic acid
daily for one month without any side effects.
'the specific branched-chain fatty acids can be, hut are not limited to be,
obtained
by synthesis or by isolation from the said fermentation products,
Particularly, the present
invention relates to the fermentation products containing these specific
branched-chain
fatty acids, which have the capability of inhibiting the growth of'cancer
cells without any
toxic or side efff;cts, and the capability of aniiaging and immune boosting as
well. The
CA 02286750 2003-03-26
present invention also relates to a process of producing fermentation products
containing
the specific branched-chain fatty acids, using specif7c bacteria strains,
preferably in
industrial facilities.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 A and 1 B show the morphological changes of' K562 human leukemia
cells undergoing apoptosis using transmission electron microscope; A:
untreated; B:
treated with 13-methyltetradecanoic acid (f>0 pg/ml) for 4 hours.
Figures 2A and 2B show the morphological changes of SNU-423 human
hepatocellular carcinoma cells undergoing apoptosis under light microscope. A:
untreated; B: treated with 13-methyltetxadecanoic acid (6f) p,glml) for 24
hours.
Figures 3A and 3B show the morphological changes of SNU-I human gastric
carcinoma cell lines stained with H&E under light microscope. A: untreated; B:
treated
with 13-methyltetradecanoic acid (fi0 pg/ml) fiar 8 hours-
Figures 4A and 4B show the morphological changes of DU-145 human prostate
carcinoma cell lines stained with HOE under light microscope. A: untreated; B:
treated
with 13-methyltetradecanoic acid (60 ~g/ml) for 8 hours.
Figures 5A and SB show flow cytometric analysis of K562 huma.: leukemia cells.
A. untreated; B: treated with 13-methyltetradecanoic acid (;:30 E~glml) for 24
hours.
Figures 6A, 6B and 6C show flow cytometric analysis of MCF-7 human breast
adenocarcinoma cells. A: untreated; B: treated with 12-methyltetradecanoic
acid (60
pglml) for 4 hours; C: treated with 12-methyltetradecanoic acid (60 pglml) for
24 hours.
Figures 7A and 7B show flow cytometric analysis of normal human peripheral
blood lymphocytes (PBLs). A: untreated; B: treated with 13-methyltetradecanoic
acid (60
pg/ml) for 24 hours.
Figures 8A and 8B show detection of apoptotic SNIT-1 4ell lines added with
TL~1VEL-(TdT-mediated dUTP nick end labeling) reaction mixture under a
fluorescence
microscope. A: untreated; B: treated with l3~methyltetradecarloic acid (60
pglml) for 8
hours.
Figures 9A, 9B and 9C. show detection of apoptotic K-Sti2 cell lines added
with
peroxidase (POI)) and substrate under a light microscope. ~,,: untreated; B:
treated with
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13-methyltetradecanoic acid (60 ~g/ml) for 2 hours; C: treated with 13-
methyltetradecanoic acid (60 ~g/ml) for 4 hours.
Figures I OA and lOB show detection of apoptotic Hl 688 cell lines added with
POD and substrate under a light microscope. A: untreated; B: treated with 13-
methyltetradecanoic acid (60 ~glml) fox 8 hours.
Figures I 1 A and 1 1 B show detection of apoptUtic DU 14S cell lines added
with
POD and substrate under a light microscope. A: untreated; B: treated with 13-
methyltetradecanoic acid (60 pg/ml) for 8 hours.
Figures 12A and 12B show normal human PBI.s added with POD and substrate
under a light microscope. A: untreated; B: treated with 13-methyltetradecanoic
acid (60
pg/ml) for 8 hours.
Figure 13 shows DNA fragmentation gel electrophoresis of KS62 human
leukemia cells undergoing apoptosis, which were treated with l3-
methyltetradecanoic
acid (60 p.g/ml).
Figure 14 shows caspase target protein Lamin B cleavage in apoptotic SNU-423
human hepatocellular carcinoma cells treated with 13-methyltetradecanoic acid
(60
pg;/ml).
Figure 15 shows caspase target protein L,amin B cleavage in apoptotic K562
human leukemia cells treated with 1 ~~-methyltetradec:anoic: acid (60 p,g/m1).
Figure 16 shows caspase target pr°otein Rb hypophoshorylation and
cleavage in
apoptotic SNU-423 human hepatocellular carcinoma cells treated with 13-
methyltetradecanoic acid (60 p.g/ml).
Figure 17 shows caspase target protein Rb hypophoshorylation and cleavage in
apoptotic K562 human leukemia cells treated with 1 ~-rnethyltetradecanoic acid
(60
~rgiml).
Figure 18 shows comparison of the tumors removed from the mice of two treated
groups and control group of human prostate cancer 1~U 145 nude mice model.
Figure 19 shows comparison of the tumors removed from the mice of treated
group and control group of human hepatocellular carcinoma LC1-I)35 orthotopic
nude
mice model.
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~~~~:~~rt:~:r~ r~r-:~c~I~tP~t~tc~ra ~~~~ T'~~: I~~~.~r~rt,r~~r
l3eittniti~n oa ~,pe~etlis: 13r~:h~,c~-ch;~itt h~.~~ :'~~ids
Tlt~ ~~~n~ itt~'~~ti~~n ~'~lt~t~~ t~:~ s~~:ifiG itnc ~ITG~ht-tirt -ttunttc~l
;~ncl ~u~s;~~ ~r~ttc~I
tar i~i~,, ~~-itl~ :~i~ilG~c:~~ ti~.c~~~ acu~vt~~~, i-e., t~rr~titl I~~
tt~etl~~,=I-h~:.l~i~ct- d
~ntcis~- f tty a~id~. 'TI~~ prtzt irt~~ention alt includes any ~tnd all
~ieri~~~#ivof' tts~
fattty a~ic~.,~-. ~;~ I~t, as tl~ t~rsttiIl~' eth~~'I-I~raxtcl~~~, is~_f- ~~r
ttt~~ei~f- IEy t;iety
r~,mt~.uaw There fatty acids can be cl~aracac~raze~l lay th~-~ fc~rroula ~,,t-
~~C1I-I. ~~i'ter~in
r,:present..~ a, tidally eth'~el-f~-arrel~e~ iz~e~- ~;r attt~i~:r- f~,ity
gr~~xtp, lay tl~e' term
'"t~t~rtinall~- tn~hy4~br~tch~cl ice" ate "~~rn~ina~lv methyl-bt~ehe~
anteis~'"~ it fs
i~it~ that the ~~~h the EtE, ufit a~wvy fr't~m tt~ ~'l7t~H ,~ttp ha~~ tI
fitll~w~~in~: forttaulaef rea~e~:.tivel~'=
S
C='l~l~~ ~ C: t-l -- ~ t~'att~t a~a
~'~
C:H3 - CHZ - CH -- {anteiso fatty Ktcid).
The portion of the fatty group R~, other than the terminally-methyl branched
iso-
or anteiso-moiety, as described above, is not limited and may be saturated or
unsaturated,
linear or branched, for example.
An embodiment of the methyl-branched saturated fatty acids wherein the above
portion of the fatty group I2~, other than the terminally-methyl t~ranched iso-
or anteiso-
moiety is linear can be described by the formula (I)
H3C\
\CH (CH2)n--COOH {I~
H3C (GH2)m
In the above formula (I), m is 0 or l, and n is an integer. 'There is no lower
or
upper limit for n so long as the acid is a fatty acid. 'I~hus, n + m may range
as high as 96
or higher, with an upper limit of 46 being preferable. More pre;terably, n is
7 -- 16.
CA 02286750 2003-03-26
t ahe m~:'1=br~ant:lur~satur~~t~d itatt~~ aids ~ta~°~ the above
formula, except that
»is at 1~:~,.~t ?, aat 1~~,~t o~'H,-~ - CHt g,rt~ap ita ~~'if~~)~t i~ r~pl.~d
with a C°H.~=:~'H
l,r~.~t~,p.
The t~~ly ritethyl~br:hecl i.~ 1-a'Ikly ic~~ ~t~ ttt~ meth~lTb~het~ tur~tt
fatty ids ~~it~~ ~~~ a~,-l ~ =,_. ~. = ~ i~ tt~ ~_~ titular, aid lc~v
'"aso-~'~c°' in the pt~r~t m4~~nt'LOn. For ~xawpl~, 1 ~
TrrE~tl~~vt~trasiai~ arid is ~xpr~d
~..a "is~~-(.= t ~,. has tfay~eia
Chi
~~,,~'' ~'.'H - ~C~-I~) i - C'3HF
CHI
'Fl~~ rmunally m~h~~l-ltclan ~iw-t~tt~ ~i~-~ ~~hyl-tn~l~
turat~d fair aids ha~~in~ t ~.arl~ons a~~i n = :~ _::_ nT = 1 in the ar~~
furnzul~ and
knlwn z~s '*ant~iso~Cx'" in the txnnk inv~er~tion. for ~xa~npl. t?-
~n~ths~ltd~~aoi~
acld is ~~~rc~ as *ia~~t~i~,~t,_' 1 ~" a~cl l~~»tt~ula
CFI.
> CH - (CHZ) io - COON.
CH3-CHZ
An example of a terminally methyl-branched unsaturated iso-fatty acid of the
present invention is
CH3
> CH-(CHz)5-CT-I=CH~CHZ)~-(:OOH,
CH3
otherwise known as iso-17:1 ~ 9c, both in c,is and trans configurations.
The present invention also includes pharmaceutically acceptable salts of said
terminally methyl-branched iso- and anteisa- fatty acids, whicl~~ are obtained
by reaction
with inorganic bases, such as metal hydroxide, and have the ability to inhibit
cancer ~: ell
growth. Such compounds include R~COOM, wherein M stands for alkali metals,
such
sodium, potassium, ammonium, etc., and alkali-earth metals, s~~ch as
magnesium,
calcium and zinc.
The present invention also includes pharmaceutically acceptable lipoproteins
of
said terminally methyl-branched iso- and anteiso-fatty acids, which are
obtained by
CA 02286750 2003-03-26
conjugation with proteins, including pc>lypeptides and oligope,ptidss, and
have the ability
to inhibit cancer cell growth.
The present invention also includes pharmaceutically acceptable derivatives as
prodrugs other than lipoproteins, such as amides, eaters, etc., of ,aid
terminally mefhyl-
branched iso- and anteiso-fatty acids, which are obtained by reaction of the
fatty acid
with the corresponding amine, alcohol, etc. precursor, and have the ability to
inhibit
cancer cell growth and improve pharniacokinetic properties. ~uc.h derivatives
include,
but are not limited to, those that have the formula RoCU-A, where Ro is as
previously
defined, and A. represents one of the following groups'
1)
E ~ ~ COOK
wherein E can be O or NH; R represents H, Cl-13, e>r C?l~s etc.,
2)
R~
N\
Ry
wherein RI and Rz independently represent methyl or ethyl group; or R, and RZ
jointly represent tetra- or pentamethylene as
~n
N
t
wherein n = 0 - 2 .
3)
!'
N E
r~
wherein E can be O, NH, NClv3 or NCzHs.
4)
CHI
--H N
7
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7.~
---tits ~ ~ OH
i~~, 7~ ty'in~. ~ ) ~t~ u~:, ~ ~ gt~tt~r
1 ~l~
I!
R F~'
g~)in R i~ ~ ~icl~ cly~n c~f Limo ~~id~ and n ~ ~,1? c~~ ~.
'Tl~~ ~t ~s~eti~~u it~lid ~rmin~ly ~~~~1-~~lji~c~- ~r
ant~i~~.t~tty is~~, ~i~hor~in one hyd~g~n ii~t a wt.'1-l;- group is subs#ut~d
o~-ith a ~r~.~up X,
~h ~1, I, l~r, C3H or ~.1-1~ . i~ntl l~a~~ the abilit~,~ tt~ i~lttibi~t
~at~#:~r ~~I~ u~~t~,
l~:p~~~ of ~u~.h ~u,titutfatty acid_4 ha~r~ the fo~axula ~o~~l<Xtn:C,~t,~l-l,
wt~r~in R~ is
a'~ d~cfinl ~bc~~:. such ~ompos~nd~ irtolud~w
C'Hx
~"''~' ~vt='~~~~r~--~-'~lt)~~1-i
CH3
X
Wherein X stands for chlorine, bromine atorr~, c:~r hydro.x.y, amino group.
These
also include the corresponding enantiomers and racemates_
'The present invention also includes said temninally ot~etlnyl-branched iso-
and
anteiso- fatty acids with a double bond at thc,: following positioa~s, such as
2 and 3.
'The terminally methyl-branched iso- and anteiso- tatty acids of the present
invention can be obtained by, but not limited to, isolation from fermentation
or
incubation products using specific bacteria, or by chemical synthesis, or by
extraction
from natural materials.
s
. CA 02286750 2003-03-26
Having generally described this invention, a fixrther understanding can be
obtained by reference to certain specific: examples which are provided herein
for purposes
of illustration only and are not intended to be limiting unless otherwise
specified.
I. Demonstration of- Anticancer Activity .and Saf~~of, Specific Branched-chain
F.attX Acids
Example 1: Anticancer Activit In ~'itru
Samples:
iso-C 1 S, including extracted and synthesized.
The extracted iso-C15 was isolated by 1-1PLC" (high Performance Liquid
Chromatography) from the fermented solution (fermented using the specific
bacteria,
Stenotrophomonas maltoyhilia Q-can, and media and production process in
present
invention).
The synthesized iso-C15 was purchased from ~iigma Chemical Company (St.
Louis, MO.)
The other Specific branched-chain fatty acids tested include:
10-methylundecanoic acid (iso-C 12),
11-methyllauric acid {iso-C 13 ),
12-methyltridecanoic acid (iso-C 14),
11-methyltridecanoic acid (anteiso-C'14:),
12-methyltetradecanoic acid (anteiso-C~ 15),
14-methylpentadecanoic acid {iso-C I 6),
13-methylpentadecanoic acid (anteisc~-C' 16).
15-methylpalmitic acid (iso-C'17),
16-methylheptadecanoic acid {iso-C18),
15-methylheptadecanoic acid (anteisc~-C 18),
17-methylstearic acid (iso-C 19 ),
18-methylnonadecanoic acid (iso-C20).
All the samples above were purchased from Sigma C.'henucal Company.
Cell lines:
Human leukemia cell line I~5t72 and human gastric rancor cell line SGC7901.
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Methods:
MTT assay was performed tc> test the cytotoxicity. ~hhe K562 and SGC7901 cells
were maintained in exponential growth in RPMI 1640 medium supplemented with
15%
heat-inactivated newborn calf serum. 'hhe cc°lls were plated at a
density of 2 x 104
cells/100p1 medium/well into 96-well plate with medium containing samples in
five final
concentrations (7.5, 15, 30, 60 and 90 ~g/ml) for isa-C'.15 (either
synthesized or extracted)
and one final concentration (30~tg/m.l) lbr the others. The media in control
wells
contained no samples. The cells were incubated at 37°(; in a highly
humidified incubator
under 5% COZ atmosphere for 24 hours. The supernatant :vas removed by fast
inversion
of the plate. 20121 of Smg/ml M~fT solution were added into each well.
Incubation was
continued for 4 hours. DMSO 100p.1/well was added and the plate was vibrated
for 10
minutes. ~57pnm was read at the Immunoreader I3ioTek l?L.311 S.
The inhibition rate (°io) == i ~--- (mean Aa~~,"", in test wells i mean
AS~o~", in control
wells)
Results:
Table 1. Inhibitory rate (%) of synthesized iso-C.'15* on cell growth
Cell line90gg/ml 60pg/ml 30yg/ml l5p.g/ml 7.S~g/ml
K562 -. 85.3 83.1 ~_-._.__..__._...~_71..b...____ __'0.1 .__ X6.2
y __..... .
'
SGC7901 68.4 6 3 1 51).5 27.5
* the sample was dissolved with 10~~o ethanol
Table 2. Inhibitory rate (%) of extracted iso-C 15* on cell growth
Cell line 90ug/ml 60pg/ml 301.~g,'ml 15~g/ml 7.S~g/ml
K562 87.2 83.~~___~ 7?-,~ ._..__. -__ .._~ 1,2 _._ 27.1
SGC7901 68.8 62.1 51.2 28.1 -
* the sample was dissolved with 10% ethanol.
Table 3. Inhibitory rate (%) of Specific branched-chain ca~-baxylic acids*
on K562 cell growth
Sample iso-C12 iso-C13 iso-C14 iso-Cl6 isa-C17 isa-C18
to
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70.69 71.03 72,1 > 71.>8 70.79 68.39
Sample iso-C19 iso-C'.20 anteiso- anteisc>- anteiso- anteiso-
C~1'~ C'14 C16 C18
69.15 62.58 73.10 72_~~) 70.68 71.73
the concentration of branched-chain carboxylic aphids was 30pg/ml; the samples
were
dissolved with NaOI-1 solution to adjust to pH 7.>.
Example 2: Deterniination of IDS ID~~ and_IDy~,
Samples:
The extracted iso-C:15 was isolated by I~iPL,C.' (l~Iigh Performance Liquid
Chromatography) from the fermented solution (fermented using the specific
bacteria,
Stenotrophomona5 maltvphilia Q-can, and process in present invention, in~-a).
The
samples were prepared by dissolving thetas in NaOH solution (adjust to pH7.5)
and 0.5%
Tweeri'~80 (Sigma Chemical Company> St. Louis, MO).
Cell Lines:
All tumor ell lines were purchased from American 'type <'ulture Collection
(ATCC, Manassas, VU) and were cultured as recommendeGl by vendor, Human PBLs
were separated from whole blood of' h~:althy individuals lry using Ficoll-
Hypaque
gradients. They were maintained in suspension in RPMI 164() with 10'% plasma
from the
same individuals. All cell cultures were incubated in a C.OZ atmosphere (5%)
at 37°C.
Seven human tumor cell lines were studied. K-X62 human leukemia and SNU-1
human gastric carcinoma cell lines were cultured in suspension in RPMI 1640
supplemented with 10% heat-inactivated FBS. MC'F-7 human breast
adenocarcinoma,
DLJ- .145 human prostate carcinoma, '~NLJ-423 human hepatocellular carcinoma,
HCT 116
human colon carcinoma, and H1688 human small cell lung c:arc.inama cell lines
were
propagated as adherent cells in RPM1 1640 supplemented with 10% heat-
inactivated FBS
(fox SNU-423 and H1688), or in .McC'oy's Sa medium with 10°r~~ heat-
inactivated F'BS
(for HCT 116), or in minimum Eagle's mediGrna with 10°io heat-
inactivated F'BS (for
MC:F-7 and DU-145).
Methods:
irar~2-ma:-tc
11
CA 02286750 2003-03-26 . --
All cells in adherent culture were initiated at 5 x 104 cellsfwell in 96-well
rnicroplates and treated immediately with iso-(:15 at different concentrations
(0, 1.5, 3.0,
E~.0, 15.0, 30.0, and 60.0 p.g/ml) diluted with medium. E3oth untreated and
solvent (NaOH
and Tween 80) treated cells served as controls. 'fhe treated cells were
incubated for 48
hours at 37°C. After incubation, the supernatants were removed and the
cells were
trypsinized and collected prior to viability assessment by trypan blue dye
exclusion.
PBLs, K-562 and SNU-I cells in suspension culture were seeded in 96-well
microplates at a density of 5 x I04 cells>well for K-562 and SNt_t-1, and I x
105 cells/well
for PBLs. iso-C15 were diluted with medium to provide different concentrations
(0, 1.5,
3.0, 6.0, 15.0, 30.0, and 60.0 pg/ml). Both tmtreated and solvent (NaOH and
Tween 80)
treated cells served as controls. After incubation for 48 hours <~t
:37°C, cells were
collected directly from the wells for viability assessment.
The IDSO, IDES and ID9~ were detern~ined in cEuplicate in every set of
experiments,
and each experiment was repeated three times under identical c:.onditions.
ID$~, IDES and
ID9o were defined as the concentration of iso-C' 1 ~ reguired to kilE 50, 75
ox 90°:°,
respectively, of cells (compared with that in untreated cells) and computed
using
CalcuSyn for Windows software (Biosol't, Cambridge CJI~:) based on Median
Effect
method by Dr. T. C. Chou.
Results
The cytotoxic activity of iso-C 15 was quantified by determining IDSO, ID75
and
ID9o (pg/ml or laM) in several human hematological and solid tumor cell lines.
It is
indicated from Table 4 that iso-C15 was active in all tumor cell lines
studied. The
strongest cytotoxic activities were far MCF-7 human breast adenocarcinoma and
K-562
human leukemia. The activities were less fbr Ii 1688 human small cell lung
carcinoma
and HCT 116 human colon carcinoma cell lines. In contrast, is~~-C', l 5 is nit
toxic aLainst
normal human peripheral blood lymphocytes at concentrations lethal to tumor
cells.
Table 4. Cytotoxicity of iso-C'15 on human tumor and normal cells in vitro
cell line cell type 1D5,,a(Eig/ml) ID~,,(l~gJml) ID~o(Itg/ml)
MCF-7 breast carcinoma ~ ~ 10.()s~:()'9' ~ 15_~:>~)~1.28 25.49~1.6$
m
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K-S62 leukemia 11.451.82 2<'.274.6043.576.71
DL1145 prostate carcinoma1:3.98-12.1;4(1.435.7281.878.85
H1688 lung carcinoma 15.08-1.92 35.033.59 61.378.06
HCT-116 colon carcinoma 18.496.'~3 6 7.968.25108.6513.3
5
SNU-I gastric carcinoma2(:),77-2.474 1'.434.9580.4910.03
SNU-423 hepatocarcinoma 24.263.98 70..469.36120.7715.8
2
PBL normal human >400
lymphocytes
Example 3: In vitro Introduction of apoptosis in human tumor cell lines and
molecular pathway
Rea;~ents:
RPMI 1640, DMEM and McCoy culture medium, as well as Fetal and calf
bovine serums were purchased from Life Technologies (L,ong Island, New York).
Argarose for DNA gel electrophoresis was purchased li-orxr F1VIC, and
Acrylamide for
Western blot was from Bio-Rad. Antibodies against human c-rnyc, caspase 3,
caspase 8,
poly (ADP-ribose) polymerase (PARP;), lamins, p53 and retinoblastoma (Rb) were
from
Oncogene. Chemicals used in buffers and other reagents were taom Sigma (St.
Louis,
IvlO).
13-mehtyltetradecanoic acid (iso-C l :s) was chemically synthesized in
inventor's
laboratory, as described in Example 5, infra, (purity of 99.8%) and 12-
mEahyltetradecanoic acid (anteiso-C 15) purchased from Sigma were prepared by
dissolving in NaOH solution and then in 0.5"~° '1'ween 80 vt-~ith
pH'7.5.
Cell Culture:
Human cancer cell lines DU-14S (prostate cancer), KS62 (leukemia), HCT116
(colon cancer), 1-11688 (lung cancer), S1-.1N423 (hepatocaroin<>ma), MCF7
(breast cancer),
CRL-1687 (pancreatic cancer), and SUN-1 (gastric cancer) were obtained from
American
Type Culture Collection (A~I'C:C). 30m1 blood was collected ti-om a health
person and
normal peripheral mononuclear cells v~.rere separated by Ficoll separation
solution
1i
CA 02286750 2003-03-26
(Sigma). All cells were maintained is RPMI If~40, I)ME?M, or McCoy medium
supplemented with 10% fCS, 100mg/ml streptomycin arzcl 10(~w'ml penicillin.
Normal
human peripheral mononuclear cells, K562 and Sl.)N-I ware suspended cells.
After
spinning at 1,500 RPM for 5 min, supernatants were disch~~rged and cells were
reauspended and expended in fresh medium. The other tumor cell lines were
adherent
cc;lls and were dispersed with 0.05°,~~ trypsin / C1.01 ~ a I~D'1'A
I;Irvine Scientific., CA) for
expansion. Cells were seeded in T75 flasks at 2 x I O'' cellsfflask in culture
medium
supplemented with 10% fetal bovine serum and incubated overnight at
37°C with 5%
COz. The adherent cells attached to the plate were striped with disposable
cell scrapers
(fisher Scientific) after treated either with 1 °~~ iso-C.' 15 ,
anteisc>-C 15 or control solution
far 1, 2, 4, 8 and 24 hours and then combined with respective float cells.
Cells were then
prepared for flow cytometry analysis, irz .situ cell death detectican, DNA
fragmentation and
Western blot assay followed the preparation methods for each assay. Cell
pellets treated
for 2 and 4hr with either iso-C 15 or control were also stored at -70°C
for future studies of
gene regulation.
Methods:
The apoptosis (programmed cell death) of cancer cells induced by specific
branched-chain carboxylic acids was confirmed by: (a) morphc>logy, visualizing
morphological changes indicative o~~apoptosis; (b) flow cytometry, identifying
the cells
undergoing apaptosis and discriminating apoptosis from necrosis; (c) in situ
cell death
detection kit, POD, detecting apoptosis induced DNA strand breaks at single
cell level; (d)
gel electrophoresis assay, visualizing apoptc~tic DNA fi~agrnei~t~rtion.
The molecular mechanism of apoptosis induced by ~pecifle branched-chain
carboxylic acids was studied using Western blot. analysis.
A flow cytometer (FACScan t with Consort 30 software for gating analysis
(Becton Dickinson, San Jose, CA) was used, fI'he Apoptosis D~~tectiort kit
(R&D
Systems) was used to quantitatively determine the percentage of~ cells
undergoing
apoptosis by virtue of their ability to bind annexin V arad exclude propidium
iodide (PI).
Cells were washed in cold PBS twice and resuspended in binding buffer.
Fluorescent-
labeled annexin V and PI vt-ere added to the cells. 'hhe cells undergoing
apoptosis,
expressing phosphotidyiserine on the outer leaflet of~ cell membranes, would
bind annexin
~a
CA 02286750 2003-03-26
V . The cells in later stage of apoptosis or necrosis, with a compromised cell
membrane,
would allow PI to bind to the cellular DNA. 'fhe resulting; cells were
immediately
analyzed by flow cytometer eduipped with a single laser enxitting excitation
light at 488
rxm. The annexin V and Pl generated signals can be detected in signal detector
FLl and
FL2, respectively. 'hhree potential populations of cells can be presented in
FL 1 /F L?
pattern: live cells would not stain with either fluorochronxc (zorxe 3 ),
necrotic and later
apoptotic cells would stain with both tluorochromes (zone 2) while cells
undergoing
apoptosis would stain only with annexin V (zone 4).
In Situ Cell Death Detection Kit, POD (Manrxheirxx I3oel~ringer GmbH) was used
to detect the individual apoptotic cells. Cleavage of genomic DN.A during
apoptosis may
yield double-stranded, low molecular weight DNA ti-agments as well as single
strand
breaks in high molecular weight DNA. 'Those DNA strand breaks can be
identified by
Iaheling free 3'-~OH termini with modified nucleotides in an enzymatic
reaction. In this
kit terminal deoxynucleotidyl transfc;rase (TdT) is used to label free 3'-OH
ends in
genomic DNA with fluorescein-dUTP. 'fhe incorporated fluorescein is visualized
under
fluorescence microscope directly. The incorporated tluorescein can also bind
to anti-
fluorescein antibody POD and be detected by substrate reaction. Stained cells
can be
analyzed under light microscope.
T'he gel electrophoresis assay was used fear the detection of apoptosis-
specific
internucleosomal DNA degradation in these c;el(s. 'I'umox cell pullets,
treated with 1%
iso-C15 and controls, were lysed in 1 ml hypiatonic lysis buffer t l() mM
T'ris, pH 7.5, I
mNl EDTA, and 0.2% Triton x-100). ,After centrifuged at l~,Ot~(.i HPM for 20
min at 4°C,
the supernatants were transferred to new tubes and treated with IZNase and
proteinase I~
respectively. Supernatants were extracted with phenollchloroform twice, and
fragmented
DN.A was precipitated in ethanol. Samples were ulectrophoresed in a 1.5%
agarose gel in
1 x TAE buffer. The gel was st<~ined with ethidium bromide and destained with
distilled
watc~,r. The fragmented DNA was then visualized under L1V light.
For Western blot assay, each cell pellet collected from 1 °,fo iso-C:15
or control
treated cultures was lysed in 150 pl lysis buffE:r with (.).5°~°
NP-40, (>.5% deoxycholic acid
and 1mM PMSF. The cell lysates were mixed with equal volume 2 x l~aemmli
buffer and
boiled for 5 min before loaded into gel wells. Proteins were resolved in an
8°,~o SDS-
CA 02286750 2003-03-26
PAGE gel and transferred to nitrocellulose filter anembrane. T"le filters were
blocked
with PBS-T (PBS with 0.1% T'ween 20) containing 5°io nonfat dry milk
{Bio-Rad,
Richmond, CA) for 1 hr and then in cubated for 1 hr with proper dilution of
one primary
antibody in PBS-T containing 2% nonfat dry milk. The filters then were washed
in PBS-
T 5 min for 6 times and incubated with a 1:8000 dilution cgf HRP secondary
antibody in
PBS-T with 2°~o nonfat dry milk for 1 hr. After fi washes in PBS-T,
inunune complexes
were visualized on film using the E(~L nonradioactive detectican system
(Amersham,
Arlington Heights, IL). After detected with one primary antibc.ody, the filter
was striped
with 0.1 mM Tris pH 7.5 and 0.05 rr~T J3-metacapenanol at 50"C for 30 min. The
f lters
were washed in 300 ml PBS-T' buffer for 10 min twice before Mocking with PBS-T
with
5'.~o nonfat dry milk. The membranes were then reprobed with monoclonal mouse
anti-
hrunan (3-actin to determine the equal loading of protein for each well.
Results:
Morphological changes:
The apoptosis of cancer cells is morphologically characterized by cell
shrinkage,
chromatin condensation, nuclear fragmentation, intact cell membrane and
extensive
formation of membrane blebs and apoptotic bodies.
Figure 1 shows the morphological changes of K5C;2 leukemia cells undergoing
apoptosis using transmission electron microscope. Comparing to the untreated
intact cell
{Figure 1A), thc° cell treated with 13-methyltetradecanoic acid (,60
~glml) for 4 hours
(Figure 1 B) exhibits typical apoptotic feature, chromatin condensed into
dense masses
against the nuclear membrane, membrane intact: arid cell shrinkage.
Figures 2 - 4 illustrate the morphological changes of cancer cells undergoing
apoptosis in a light microscope. Cultured S~ItJ-423 human hepatocellular
carcinoma
cells treated with anteiso-C15 (60 pg/ml,) for 24 hours (Figure '?B) exhibited
cell volume
decrease due to shrinkage and bubbles inside; the cell, compared to untreated
control
(Fiigure 2A). Cultured SNtJ-1 human gastric carcinoma cell linc;s were treated
with
anteiso-C 15 (60 pg/ml) for 8 hours, and cellular morphology was evaluated in
preparations stained with H&E (Figure 3B). Cornhared to untreated control
(Figure 3A),
chromatin condensation and cytoplasmic granularity were noted. Cultured DU-145
human prostate carcinoma cell lines were: treated with iso-f 't ~ (60 p.g/ml)
for 8 hours,
r6
CA 02286750 2003-03-26
and cellular morphology was evaluated in preparations stained with H&E dye
(Figure
4B). Compared to untreated control (Figure 4A), membrane blc;bs were noted.
Flow Cytometry:
At least 104 cell events were analyzed. 'fhe f~L I IFL2 pattent of untreated
K562
hr.rman leukemia cells (Figure SA) revealed the e~p~:eted distril7ution of
cells in zone 3.
After treatment of K562 cells with iso-C.' 15 (30 pgiml) f<.~r 24 hours
(1~'igure SB), the
majority of the cells were undergoing apoptosis (-none 4, r'~nne.xio V
positive and PI
negative). 'The kinetic behavior of anteiso-C' I S in MCF-7 human breast
adenocarcinoma
cells was evidenced by Figure 6A, 6B and 6C, for treatment of MCF-7 cells with
anteiso-
C 15 (60 ~rg/ml) for 0, 4 and 24 hours, respectively. After treatment of
anteiso-C 15 for 4
hours, many cells were undergoing apoptosis (zone 4, Figure 613), while after
24 hours
the majority of cells had already died (later stage of apoptc.~sis, zone 2,
Figure 6C). The
flow cytometric analysis of untreated normal human PL3Ls (Figure 7A) and
treated PBLs
with iso-C15 (60 ~g/ml) for 24 hours (FigurL 7B) resulted in nearly identical
FL1/FL2
patterns (zone 3, viable and not undergoing apoptosis), revealing no
significant effects by
iso-C 15 on normal human lymphocytes.
In Situ Cell Death Detection:
Four human tumor cell lines, K-562 hurnarr leukemia, SNZ1-1 human gastric
carcinoma cell lines, MCF-7 human breast adenocarcinoma and 1-11688 human
small cell
lung carcinoma cell lines, as well as I~uman P13.L~s were treated with iso-C I
S (60 pg/ml).
SNU-I cells treated with iso-(' 15 for 8 hours were added with TC1N1?L-
reaction
mi;~ture and incubated 6U min at 37°C'.. After washing with 1'BS fur
three times, cell
morphology was analyzed directly under fluorescence microscopy. Several yellow
fluorescent spots of apoptotic cells were noted in cells treated ti>r 8 hours
(Figure 8B),
comparing to untreated ones (Figure 8A).
1-11688, K-562 and DC1145 human carscer ceIis and normal human PBLs were
added with POD and incubated 30 min at 37°C, washed three times with
PBS, then
reacted with substrate AEC and incubated for 10 min at room temperature. T'he
cells were
analyzed under light microscope. Comparing K-i62 leukemia ce ils untreated
(Figure 9A)
and treated for 2 and 4 hours (Figure OB and 9t=), it is found that some cells
started
apoptosis (stained red) 2 hours after treatment and the number of apoptotic
cells
CA 02286750 2003-03-26
increased with exposure time. 'Che apoptotic H1688 cancer cells (stained red)
were found
after 8 hours of treatment (Figure 1()I3) comparing try w~troated (1~igure
10A). Some
si:ained apoptotic DU145 cancer cells were shown 8 hours after treatment
(Figure l IB)
and no stained cells in untreated control (Figure 11 A). In contrast,
untreated and 8-hour
treated PBLs were almost the same (Figure 12:'~ and 12B), and few stained
apoptotic
cells were seen. It is evidenced that iso-C 15 induces apoptc~sis of cancer
cells but not
normal human cells.
DNA Fragmentation Gel Electrophoresis:
DNA fi°agmentation electrophoresis is one of most corrrmon applied
methods to
illustrate the apoptotic changes in experimental cells. Results fior K562
leukemia cell line
treated with iso-C15 (60 pglml) were shown in Figure 13. The lane of control
treated far
8 hours showed only DNA smear. ~t'he fragmented low rnolecirlar weight DNA
bands
wore seen at 2 hour and were prominent at 8 hour treated. 'fhe appearance of
an
oligonucleosomal ladder in treated cells indicated the break of double-
stranded DNA due
to apoptosis induced by iso-C:15.
Western Blot Analysis:
The Western blot analysis results (Figures 14 - 1 ~? are used as examples to
reveal
the signal transduction pathway for specific branched-chain carboxylic acid to
activate
apoptosis of cancer cells.
The cleavages of Lamin B, a caspase target protein, in apoptotic SNU-423 human
hepatocellular carcinoma cells and K562 human leukemia cells were shown in
Figure 14
and 15, respectively. The cells were treated with 1 °% control solution
and 1 % iso-C15 for
the: length of time indicated. Cell lysates were separated by SDS-PAGE. Lamin
B was
detected by immunoblotting with a monoclonal antibody. 'l"he cleaved 45 kDa
and 32
kDa products were shown in Figure 14, and the cleaved 45 kI)a products in F
figure 15.
The cleavage of caspase target protein Lamirr B suggested klre activation of
the caspase
cascade during apoptosis. The Western blot analysis of R.I3 protein in SNIJ-
423 and
K562 cells were shown in Figure 16 irnd 17, respectively. The r°esutts
showed that iso-
C 15 induced the change of hyperphosphorylrrted RB (pRB 12U/hyper) to
hypophosphorylated form (pRBI 15/hypo), and also induced the cleavage of full
length
t~
CA 02286750 2003-03-26 -
RB to pRB68 kDa fragment in Figures 16 and 17, and even slraaller pRB48 kDa
fragment
in Figure 16.
Example 4: Anticancer Actiyi~ In I'iycy and Humaln tc:~t
A. Determination of Ll:)5~
Materials and methods:
13-methyltetradecanoic acid (lso-C:1 ~) purchased t'irom sigma (St. Louis, MO)
was prepared in dissolving in NaOH solution and then in 0.35°ro Tween
80 with pH7.5.
ICR mice weighing 20.5 - 2:::.:ig of 1>oth sexes were treated with iso-C 1 S
i.p. qd x
3 in test groups and with solvent of same dose in control group. T'he doses
ranged from
to 800 mg/kg of iso-C 1 S and two mice were included il~. ~.acl~ dose group (
10 mg/kg.,
mg/kg, 40 mg/kg, 80 mg/kg, 160 mg/kg, 4md 8()l) mg/kg). 'I-'loe general
condition of
these mice were monitored daily for seven days.
Results:
No mice died after seven-day administration of iso-C:15 of dose up to $00
mg/kg.
It is shown that iso-C15 is basically root toxic to mice alld 50'io lethal
dose (LDso) was n.ot
determined.
B. Efficac~Evaluation of iso-C:15 in Orthotopic Nude Mice Model of Human
Prostate Carcinoma DU 14:>
Material and Methods:
13-mehtyltetradecanoic acid (iso-C1 ~) was chemically synthesized in
inventor's
laboratory, as described in Example 5, rnfi°cr, (purity of !)9.8%) was
prepared lby
dissolving in NaOH solution and then in ().35% ~I'ween 80 with pl-I7.5.
Total of 24 male athymic BA1_,B/c made mice between 4 and 5 w;,eks of age were
bred and maintained in specific pathogen free condition.
Human prostate carcinoma Dl_i 140 tu~rlco~ was implanted and maintained
subcutaneously in the flank of athymic nude mice. friar tc> orthotopic
implantation, the
tumor was harvested in log phase. ~I~l-u peripheral tumor tissue was collected
and minced
to shall pieces of one cubic millimeter each.
CA 02286750 2003-03-26
The mice were anesthetized for surgical orthotopic irnlylantation. A small
incision
was made along the midline of the :lower abdomen. rafter prc>lner exposure of
the bladder
and prostate, the capsule of the pr~~statG was opened a~ad three pieces of
DU145 tumor
fragments were inserted into the capsule. 'Che capsule was th~;n closed using
8-0 suture,
and the abdomen was closed using a 6-0 surgical suture.
The mice bearing orthotopic DC1145 were randomly divided into control and test
groups of eight mice each at the second day after tumor implantation. The iso-
C15
prepared above at doses 35 and 70 mg/kg and PBS were administered by gavage
once a
day in low dose and high dose test groups and control group respectively, for
43 days.
All the mice were sacrificed by CO~ inhalation at day-43 after the start of
treatment. The weights of primary tumors and bodies were measured. Tissue
samples of
the primary tumors were processed through standard procedures of hematoxylin
and
eosin staining for microscopic examination.
The tumor inhibition rates ('I'IR} were determined by comparing the mean tumor
weight of the test groups (T } with that of tile control group (C') and
expressed as a (C -
T).~C percentage, and were analyzed by Student's test for statistical
significance.
Results:
Very promising antitumor efficacy was observed for iso-C15 at doses 35 mg~'kg
and 70 mg/kg in this nude mouse n:~odel of human prostate carcinoma DU145 with
the
tumor inhibition rates 54.8 % (p5; 0.051 and 84.b °r~ {p~~: 0.01 ) as
shown in 'fable 5.
Table 5. Efficacy of iso-C 15 on primary tumor and body weight
in nude mouse cnodc;l of human prostate carcixaoma DU145
Group Route No. of mice Mean t~.nnor weight (mg) TIR f,%) P
Control Oral ~~~.._~.__.___ .____ .____._......._.. _1 _C~~ ft. ~ ~ _._.
_....__....._~.. - ._
Low dose, 35 mg/kg Oral $193.25 54.8 0.042
High dose, 70 mg/kg Oral 8 I ti8.0U 84.fi 0.007
For comparison, all the primary tumors after removal fi~orn the nude mice are
shown in Figure 18, where it was note that the implanted tumor didn't grow up
in four
mice in high dose treatment group. Chore are no any signs of toxicity, as
judged by the
body weight curve and histology slides.
2~
CA 02286750 2003-03-26
C. Eff cacY Evaluation of iso-~ 1 S ire Orthotc.~ic Nude 1'ylice Model of
Human
Hepatocellular Carcinoma l~C.'I-D3S
Material and Methods:
13-mehtyltetradecanoic acid (iso-C 15) was cl~enlicall~r synthesized in
inventor's
laboratory, as described in Example 5, infra, {purity of !~9.8%), was prepared
by
dissolving in NaOH solution and then in 0.35% Tween 80 with pH7.5.
Total of 16 male and female athymic BALf3/c nude mice between 4 and 5 weeks
of age were bred and maintained in specific pathogen flee condition.
Human hepatocellular carcinoma I,CI-D35 :vas originally obtained from the
primary tumor of a 45-year-old female patient. Tile tumor was implanted and
maintained
subcutaneously in athymic nude mice. Prior to orthotopic implantation, the
tumor was
harvested in log phase. The periphf;ral tumor tissue ~r~as ~;oll~,Tcted and
minced to small
pieces of one cubic millimeter each.
The mice were anesthetized far surgical orthotopic implantation. A small
incision
was made along the midline of th~~ upper abdanlen. The left lobe of the; liver
was
exposed and a small incision was made on the liver surface. Two of the tumor
fragments
above were sutured into the incision using 8-C) suture. ~l-he ~udomen was then
closed
using a 6-0 surgicalsuture.
The mice bearing OrthOLOpI('. LC:I-D35 were randomly divided into control and
test groups of eight mice each at the second day after tumor implantation. The
iso-C 15
prepared above at dose 70 mg/kg and I'BS were adniinister~,%d bed- gavage once
a day in the
test and control group respectively, fir 40 days.
All the mice were sacrificed by CO Z inhalation al: day-40 after the start of
treatment. The weights of primary tumors alld bodies were measured. 7"issue
samples of
the primary tumors were processed through standard procedures of hematoxylin
and
eosin staining for microscopic examinatic>rl.
The tumor inhibition rates {'Ilk) were deternuilled by comparing the mean
tumor
weight of the test groups (T) with that of thv,~ control group (C l and
expressed as a (C -
T)/C percentage, and were analyzed by Student's toss for statistical
significance.
Results:
CA 02286750 2003-03-26
Very promising antiturrzor efficacy was observed for iso-C 1 S at dose ?0
mg/kg in
tl>is nude mouse model of human hepatocellular carcinonva LC.1-L)35 with a
tumor
inhibition rate 64.9 % (p< 0.01), as shown in ~Tablr: 6.
'liable 6. Efficacy o iso-C 15 on prinrazy tumor and body weight
in nude mouse model of human hepatocellular carcinoma LCI-D35
group dose route mi~-a No_ bodywei~htt~rmor weight 'I"IR
p
in. ! fl. . % fi. mean SD I,g) (%)
in
_
_ _ __~.~0,~ O.I a7 __
PBS ______ oral ..-._.j; p~.~ _ .__
._..._
i..~-3
iJ' j~_:5~
iso-CIS 70mg/kg oral fl I g 18.~3!21.7S0.r171 -~ 0Ø'>2
64.9 4.0086
Far comparison, all the primary tumors after removal from the nude mice are
shown in Figure 1.9, where it rvas note that the implanted tumor didn't grow
up in two
mice in treatment group. There are no any signs of toxicity, as judged by the
body
weight cw-ve and histology slides.
D, Efficac Evaluation of isc~~-C~1 S in Mouse Cervical Carcinoma L114 Model
Material and Methods:
13-mehtyltetradecanoic acid (iso-C15) was chemically synthesized in inventor's
laboratory, as described in Example 5, injiwa, (purit~,i of 99.H%) and
prf,par~°d in water
resulting suspension for intragastrical administration. Mouse c;ez-vical
carcinoma U~4 was
obtained from Pharmacology Reserrrch Bureau in Shanghai, an affiliation of
Chinese
Academy of Science. ICR strain mice were purchased fzorn Shanghai-Sipul-Bikai
Research Animals, Ltd, and maintained in breeding G;olorzy.
50 male ICR strain mice, weigling C9-22g, were ran<lornly divided into 5
groups
of 10 each. IJ~4 cervical carcinou~~z mass (about ~ mrn' each) was
transplanted
subcutaneously into the right armpit: c>f all animals following standard
procedure. 3 test
groups were given iso-C15 at 0.1, 0.'',, and 0.4 glkg intragastrically (i.g.)
daily for l 1 days.
The positive control group was given a single dose of~ cytoxari (('.TX, i.g.
(25 zng/kg) on
day 1. Negative control group was liven n~:>rrnal saline ~N.~~. } at 20 rnl/kg
daily for 11
days. On the 12th day, all mice were sacrifinwed and the tumor was isolated
and weighed.
The rate of inhibition of tumor growth w;rs c~rlculate~ and subject to t-test.
Rac,rltc~
3p « -.. ~ ,.
l l a ,~ a - t.W: ~ l~
CA 02286750 2003-03-26
Table 7. Inhibition of growth of mouse cervical carcinoma U» by iso-C 15
Body wE~ight Z~~lmor Inhibiti
C ~~~ x :~S0) w~:~ight
group mice on rate
X
-
d, a,
_ S0
'
N. S.
10 20. 7--0Ø l ~. oG0. 30
8 24. 9
0. 2ml/lOg
Iso-C15
10 2(). 8-i-0.2=I,. 7 1. ~~4--0. 25. 95
l + 1. 0 6~5~
0. lg/kg
S 10 20. 80. 24, 9"~. 1. i60. 4Ea640. 46
8 1
O
Zg/kt;
Iso-C15
10 20. 8 26. I -~~ 0,. ~~2 n4. 88
: 0. ~?. fi 0. 316
8
0. 4g/kg
CTX
10 20. 7 22. 7 1. 0. ~15 0. 83. 21
0. 8 I 186
25mg/k~;
_ , herecan.
aP<0.01, P compared
.001 to
the
control
group,
same
The results above indicated tha:rt administraticm of iso-C',15 at 0.1, 0.2,
and 0.4 g/ls:g
resulted in tumor inhibition rate of 25.95°ro, 40.46°/u, and
64.88°~~a, respectively. This
suggested .a dose-dependent inhibition of cervical carcinoma fJ 1.a growth in
vivo by iso-
Cls.
E, lfficacy Evaluation of iso-(~15 in Mouse Sarcoma 5~~~ Model
Material and Methods:
13-:mehtyltetradecanoic acid (iso-C15) was chemically :synthesized in
inventor's
laboratory, as described in Exarrrple 5, infra, (purity of 9~.8°rta)
and prepared in water
resulting suspension for intragastrical administration. Mouse sGrrcoma SiBQ
was obtained
from Pharnlacology Research Bureau irr Shanghai, an affiliation of Chinese
Academy of
Science. IC'.R strain mice were purchased fi-arn Shanghai-Sipul-I3ikai
Research Animals,
Ltd. and maintained in breeding colon~r~.
50 female ICR strain mice, weighing 19-22g, wr',re; r<~ndomly divide°d
into 5
groups of 10 each. S,BO sarcoma mass (about ? ao~m-' each] was transplanted as
described
above (D) into all a~~imals. Group separation and treatnrerzt were identical
to the above
~3
CA 02286750 2003-03-26
(D), except that treatment period u~as 10 days. On the 1 1 th d~~y, all mice
were sacrificed
and tumor weighs recorded. 'rhe tumor inhibition rate vvas tale Mated and
subject to t test.
Results:
Table 8. Inhibition of growth of mouse sarcoma S,~o by iso-C 15
Ii<adV we i ~;h 'Tumor
t Inhibiti
(g~ x SD) weight
group mice ors rate
(~;~ x~'
(%)
d~~
~D )
N. S. 10 20. 2 2~1. 8 1. 1. 700. 19
1. (1 2
~0. 2m1/lOg
S 10 2(). 2I. 25. 3l. ().83-~-0. 51.20
() 6 076
~~
lg/kg
Iso-C15
10 20. 2 25. 2 --1. 0. 71 0. 58. 49
-: 1. 6 0'76
CI
0. 2g/kg
Iso-C15
10 2().21.0 '~'S.4-I_wl.,~C~.41--0.10674. I6
0. 4g/kg
(~TX
10 20. 2 43. C~ 0. 39 -- 76. 90
1. 0 1. ~1 0. 086
a'.5mglkg __ _ __ _ _ __
~~
.
_ _ _
~hercon.
~'3P<0.01,
~<0.001,
compared-to
the corrtrcxl
group,
same
The results above indicated that administration c>f iso-C 15 at 0.1, 0.2, and
0.4 g/kg
resulted in tumor inhibition rate of 51.20''0, ~8.4~~°~0, and 71.16%,
respectir-ely. This
suggested a dose-dependent inhibition of sarcoma S a so growth irr r~ivo by
iso-C 1 S.
F, Life Span Extension EffeLts of iso~C.lS iri youse_I_iver Cancer~cites t
HAC Model
Material and Methods:
13-mehtyltetradecanoic acid I,iso-C 1 "i) was chemically synthesized in
inventor's
laboratory, as described in Example :s, infra, (,purity c:>f 99.8° o)
and prepared in water
resulting suspension for intragastrical administration. Mouse liver cancer
(ascites type)
HAC model was obtained from Pharmacology Research Bureau in Shanghai, an
affiliation of Chinese Academy of Science. 1CR strain mice were purchased from
Shanghai- Sipul-Bikai Research Animals, Ltd. and maintained in breeding
colony.
40 female ICR strain mice, weighing 18-21 g, r,vere randomly divided into 4
groups of 10 each. 0.? ml of H AC', cell suspension ( 10~ cellsfml) was
injected
2~~
CA 02286750 2003-03-26
inarap~ritorrk.-~all!a~ (i.~r.~ into all anin~,~ls. ~ tf:~st ~;r~.~ups
~~~t~r~: ~i~~~n is~.-~-~v ~ at ft.~"~ atx~ (1.5
ykg i.g. d~it~~' till cl~adl~. 'The p~.~~iti~~~ ~~~yr~t~:al ~r'~:i~ap
~~~~gi~'s.n ~->;l~r~:~u~~:ii 1~-T'~:l i:.~. ~t
2:i n~~?~'kota dad' t, i, aincl 5_ 'l~la~ t~i.~;gata~;~~ ~:c~,~trt~l g,r~up
~~a~ gi~~et~ 1"~l.S. at 2.0 n~l,rkg daily
as in tit gr'c~up5, 'l'h~ length of sur~w~,~al trf ~:h r~..~uap ~~v~~;
~G~ortd~d and rz-~t~ <3f ~xt~r~~ic~tt
of life ~~~w ~al~:dlart~~l ~ci sub,~ei-t to ~ t~.t.
Results,
1-able ~f ~.1~~ ~tl~,azs t71~ ~~"~-~,1 ~ c~tt 1~1~ s~ ~x ~rt~t~~n ~~l ll.r~~:
trt~~tt.
1 t5. /~ ~ A. "~" l~ .3 _. ~FyGi;T6 ~ 1. ~' t:.
~ ~,.#~e~. ~i~p ..~,~. ~~5:~"r~~l~rl
l~~'~~~~~:iC~ a '~
tr'~ ?~t :alt ~ t ~=a _'~
r~. ~.
iti 1~. ~~ I. '2 ~~. r~~l. ~ ~. ~'~' 1. ~$
i~. ~~ur~l;. 10
1 ~;~:~' t: 1.j
;. ltl 1!x.4 ~ .l. ~ '1~:. ~-~..,b :!~ 1':-~. a -~-~. 1~- ~.e,'~.;~h
1 ~ ~ ~: l sr
ltd 10. =1 alx l . 2 1~. ~.~h. ~l 1'l. ~1 °~' 1. t1° :l~, :~1
ll~ ~ft~:~~l~
_ ~-1j a ~~rn~.~' h~ 1 (1 1 ~, :~ 1. _~ _~ ~l_. ":'_~ 1 3'. ~'1 I _:~, ~ -~-1.
:fir .:g!~. ,'.~!
"1~=-1.(11'.1 ~ ~rctc.~ t~~ ~:~:~nt~il ~,np, same h~t~r~.
The results above indicated the effects of iso-C 15 on the life span extension
of
liver cancer (ascites type) HAC mice., that the extension rate was ~zp to
4.5.45% at dose
O.Sg/kg, (p<0.001).
G, Life Span Extension Effects of iso-C 15 in Mouse Leukemia P3$g Model
Material and Methods:
13-mehtyltetradecanoic acid (iso-C 1 ~) was chemically synthesized in
inventor's
laboratory, as described in Example 5, infi-ci, (purity of ~~3.~°%0)
and prepared in water
resulting suspension for intragastrical administration. Mouse leukemia P3gg
model was
obtained from Pharmacology Research Bureau in Shanghai, an affiliation of
Chinese
Academy of Science. DBA/2 strain mice were purchased tram Shanghai-Sipul-Bikai
Research Animals, Ltd. and maintained in breeding colony.
50 male DBA/2 strain mice were randomly divided into 5 equivalent groups of
each based on body weights. 0.2 ml of P~~g cell suspension ( 10' cells/ml) was
injected
i.p. into all animals. 3 test groups were given S13A at 0. I , 0.4, and 0.4
g/kg i.g. daily till
2 <;
CA 02286750 2003-03-26
death. The positive control group was givc;n S-Fu i.g. at 2S axag/kg on day l
and 3. The
negative control group was given N.S, at '0 ml/kg daily as in test groups. The
le:zgth of
svzrvival of each group was recorded and raze of~extezu~iotx oi~lifc; span was
calculated and
subject to t test.
Results:
Table 10, 'Che effects of iso-C l S on life span extension o#° leukemia
P3~g mice
(3ody ~,veight R,3te
of
i,f, x _f~'~I) Survival
) Time life
group mice l,o't. (da,~rs., extension
X~SD)
fre-ttwat
t C % )
'f
T~E'<~
:
N. S. 0.. 2ml/lOg 10 <?1. 2'i. 0 & 0. 00
6:1. ~~ l ~ ?
5-Fu ;?5mg/kg 10 ~? 1. ~l 2~1. 3 15. Ei 56. 0
-~- .l . k3 ~: 1, -~- 2.
~:1 (.)
Iso-C15
22.0~l.li 2~'1.5:a:.1'3 12. F~_~-1. 1n 25. U
0. 1 g/kg
Iso-C1.5
0. 2g/kg 10 21. 6 _~-1. 4 Z:f. ~W-1. 0 13. 9 ~ 1. 06 39. 0
I so-C15 l0 ? 1. 6 :~ 1. 4 ?3. 8 :~t- I . 5 14. 4 :~ 0. 7h 4~1. 0
0. 4g/kg
'P<0.01,-X0.001, compared to the control group, same h~ereon.~
The results above indicated the effects of iso-C 15 on the life span extension
of
leukemia P3g8 mice, that the extension rate was up to 44°i~> at dose
0.4g/kg, (p<0.001 ).
H, Influence on Immune Functions
Material and Methods:
13-mehtyltetradeeanoie acid (iso-C 1 _'~) was chemically synthesized in
inventor's
laboratory, as described in Example 5, ir~~:r, (purity cof 9c).f3°a)
and prepared in water
resulting suspension for intragastrical administration. ICR strain mice were
purchased
from Shanghai-Sipul-Bikai Research Aniznal:a, Ltd. and maitrtained in breeding
colony.
( 1 ) Phagocytic function of reticuloendothelial system
50 female ICR strain mice, weighing 19-24g, wer a x-andomly divided into 5
groups of 10 each. One group was given N.S. at 20 mlikg i.g. as normal
control. Positive
control group was given CTX i.g. at :?s rmg/kg on day 1 arzca f>. A third
group was given
iso-C 1 S only at 0.4 g/kg i.g. 1"he remaining two group were givezu both C:TX
(2S mg/kg
26
CA 02286750 2003-03-26
on day 1 and 6) and iso-C 1 S (U.1 g~'kg and 0.4 glkg;, respectively). All
treatments except
C'rX were administered daily for 9 days. 3i~ minutes after the last
administration, 0.15m1
Yidege (1:10) was injected into the tail vein of each mou;~~:. 1 minute and 5
minutes after,
blood was drawn and 20 p1 serum was mixed with 0.1 '.'-a Na~t.'()~. OD68Q was
measured
and the clearance index (K) was calculated. 'Che results wore subject to t
test.
(2) Serum hemolysinogenesis
50 male ICR strain mice, weighing 20-23g, were rand.arnly divided into 5
groups
of 10 each. The method of administration in each group was the same as above
(phagocytic function). However, on the 6th day after administration, 0.2 ml
3:5 (VfV)
sheep red blood cell (RBC) suspension was injected i.p. into each mouse. 4
days later
(day 10), blood was drawn from all animals and serum was prepared and then
diluted 600
times. lml diluted serum was mixed with 0.5rn1 10"~o sheep RBC suspension.
N.S. was
used as blank control. All samples were incubated at 37"C fox 30 minutes, and
centrifuged (2000 rev/min) for 5 minutes. The supernatant was c~.~llected for
measurement
of ODS~o and HCSo was calculated. The results wore subject to .t test.
(3) Delayed hypersensitivity
50 female ICR strain mice, weighing 1$21 g, were randomly divided into 5
groups of 10 each. 'The method of administration in each groul7 was the same
as above
(phagocytic function). However, on the 5th day alter administration, each
mouse wa.s
injected with 50 ~.1 10 % sheep RBC suspension through tail vain. 4 days later
(day 9;1,
each mouse was injected intradermally with 20 pt 10~~4 sheep RI~C" suspension
or normal
saline into metatarus of left or right hind limbs, respectively. 'fhe digital
thickness of each
mouse was measured 24 hours later, arid the difference ira thickness between
left and right
digits was subject to t test analysis.
(4) Weights of immune organs
The mice were sacrificed after delayed hypersensitivity test and the thyrrm.s
and
pancreas was collected and weighed. Indices o9' each organ were calculated and
subject to
t test.
Results:
z~
CA 02286750 2003-03-26 ,
Table 1 I . o-C erum (:learancc~ Index co' C'.arbon
Effects of 1 S Grain in Normal and
is on
S
Inrrn une-comprorniseGl Mouse
_ _.__..._.._ ______...._.~~_..__....___..__..___~__~
__
P
Group No. h: _ _ . __ _ __ _
of" __-__._
~
(i.g.) mice Compared
(~-_S,[>) ! t'ornpared
_ ~ with (uroy with Gr_ oup2
N.S. 20 ml/kg 10 ().0741 =~-0 03? 1 _+ __.. _..._. .
__..__.__ _ ' 0_x01 __
CTX ZSmg/kg _'_, ~
l~ O~S9=x_0.0173 _.. ~_~.'~O1_ _
.
~~_
.....
Iso __ ._
C15 0.4g/kg ___ .
__ 10 ~ t~ OS < O.OOi
t~.()R4S ~::().(a33() ~
-
__._~__. __._ _._ ___.~__. _ ___ ____
' __ __
Iso-C 1 S 0.1
g/kg + 10 (?~.()4Z0-~ () 023(t ~ ~ (),()(:)1
< 0.001
CTX 25m __._ . _ _.. __ ____ . _l_ _.._ __..._ .
_ ~g __
Iso-C15 0.4glkg10 _ 0
+ ()O 1 < 0.()O1
~
0380
0.0530 ~::0
C:TX 25mg/kg '
'y
~'~
:
The results Table monstrated that iso-C 15 had no obvious
in 11 effect on the
de
clearance :index of Carbon grain in normal arouse (P~_3 =(:1.05). Moreover iso-
C15 could
improve the clearance of Carbon grain in (''f:K-treated mouse to a certain
extent: (both P; ,4
and P2,5 <0.01 ).
T able 12. Effects of iso-C 15 on Serum Hernolysincrgenc:sis in Normal and
Immune
compromised Mouse
Group No. of ~ IIC=;gin __~_.____ ..__. _P __ __~__
(i.g.) Mice ~ (.X ...~ SD) ~ Ooarpareci C~'ompared
_ _ with (:iruul~l with Group2
N.S. 20 ml/kg 10 ~~~~34~.44~._~:~?.4() ~._._~..._____.. __. < 0.1701
C1'X 25 mg/kg 10 ~2 '~ '' + -12C) _ (' (>(j 1 _ _-_~_ __
_d ~. ___._._. - __..__ _ _._.__ __ ____ _ _..__ _ __ ___ ___,
Iso-C15 0.4 g/kg 10 __~...-_-~1_~()~')~~().. . __._. '...~~.v~'._.__. ___~
_001
Iso-C 15 0.1 g/kg + ' y .,
CTX 25 mg/kg 10 ~ 0.40 ~ 1 00 ~ (1.-001 __. __ _ ___. 0.0i
_ _- _ _..___. _~_ _ . _....___._..___ ___ . ~ __ ._ .. . _ _. _
Iso-C',15 0.4 g/kg + 1C1 2:?.$0~ 3.60 ~ ~ ().00~ > 0.0~
(:TX 25 mg/kg ___.___.._.._ -
The .results in Table 12 showed that iso-(' 1 S had no obvious effects on
serum
hemolysinogenesis in either normal or immune-cornprornisecl me)use (P~,:,
P2,4, and PZ,s
?0.05). This suggested that iso-C 1 S doesn't evidently affect host lrurnoral
irnrnunity.
Table 13. Effects of iso-C.15 on de~ay~~d hyperseasitivit,y ~~f normal and
C'I~X-cc,mpr-omised mice
Groin No. _ Differc.ncc,yin tluicknes~ _~ ~ T _ _ ___ ~~_-
?8
CA 02286750 2003-03-26
(i.g.) of i between left and ( ComparedCompared
right
mice tai Group to Group
~
palm
(rnm ~ X'- Sl:~) c>ne two
~ ~~ ~
N. S. 20 ml/kg10 2.01: ~ 0.60 < 0.01
.. ___ .. _ .__ _ _ ,_ _.._ _ ___
._._. _. __ ______.__
._
..
_ 10- ___ E....__ _.___.
CTX 25 mglkg_ _ _ _ U:dl._
~ ..... _
__.___ _ ~~L_~_~().S()
____._. __
.
Iso-C15 0.4 10 1.93 ~0_70 ~ > 0.05 < 0.01
g/kg - _ __...._ _ _
_. _.._._. _ .._.
Iso-Cl 5 0. 1 ~ O~05 ~ 0.05
i g/kg ~ '4 _~ t). j()
2S mg/kg ~__ . _ ~ ..___ .._~__
+ CT x _. _ _.-._.__
_. _ _.___... _ _._
. __.__ ____ .._._
_....__
Iso-C15 0.4 10 1.53 ~f~0.5() ~ > O.OS => 0.05
g/kg
+ CTX 25 mg/kg _r_~..~...~__~_ ._~.__._____
Table 13 showed that iso-C 1 S had to the delayed
no inhibitory effect
hypersensitivity rmal and (.'TX-treated'flue values
of both no mice. of P~,3,
Pz,,r,
and P2,5
were all greater suggesting that there;o apparent
than 0.05, were n effects
of iso-C15
on
host cellular
immunity.
Table 14. Effect of iso-(J 15 on the indices of inuntrrzo organs of
both normal and Crl ~'K-treated rxric;e
Dmg Numbe Indices of thyrxrus Indices of
r (mg/1 Og pancreas (mg/lOg
(r'g' ~ of mice avoirdupois) avoirdupois)
N. S. 20 rxrl/kg 1« 3.;91-?-: s.<t.3r~" 4.472.~_0.906'
_ ~.I.X 25 mg/kg __~... _ _~i>___.. .. __.i.'~4~~,..pf~Y,:~_...__ _..
2,33+1.00
I so-C15 0.4 g/1<g ~.._-. 1~........ __._-3.4H1i-~-../ 17,r....__ _...
4,.0540.4?,Ot
ISO-c1;5 0.1 glkg + CTX 25 ' V to ~°?_573~c~.~43'' 2.411 ~-0.986
m~~ __.____~_...~ __.__.__.__.._____ ~__
Iso-cl 5 0.4 g/kg+ CTX 25 1« I ~.0~.3 ~-0.5~7~~~~ [ x_038~l.090
aP<0.01, °P<0.001, compared to the control group, same herc~On.
Results in Table 14 showed that there was no sigciifa~:arrt effect of iso-Cl.
5 on the
indices of immune Organs for normal mice (both thymus and pancreases, Pr,3
>0.05). As
to the mice that were treated by C'TX. these indices were increased after
combined
administration of iso-C15. The increase in the indices of thymus arrrong these
three
groups is statistically significant (;Pr,z acrd P2,a ~~0.00 i ). It is
suggested that iso-C 15 is
different from the anticancer compounds with cc»nmon clinical use, in that iso-
(_' 15 does
not 'inhibit frost immune function at thf;rapeutic dc:~sag~~.
29
~" CA 02286750 2003-03-26
I, Human Clinical Studies on.the Saf~t~a#; iso-C 1.5
Meterial and Method
Chemically synthesized iso-C 15 of ~~9.8% ptuity were conditioned in U.20g
capsule. Six healthy adult volunteers (4 male, ? fertrale) c>f average age
35.6 were
divided into three groups, and orally received iso-C I S trapsul~es for thirty
days. Low dose
group: one case, 0.6g daily; middle dose group: hvo cases, 1.:ZI~ daily; high
do se group.-
three cases, 1.8g daily.
The examinations were carried out before, durizig arrd after experiment,
including
physical examinations, blood and urine routine c:xaznizz<itic~ns, fc.tnction
of heart, liver and
kidney, x:-ray radioscopy of lung and subjective symptom.
Results
The effects of iso-C 15 on blood routine and platelet shown in Table 7
indicated
significant increase of white blood cells {WIC.) and granuloc;yrtL (GRAM),
while no
significant changes of red blood cells {I~13C), homeglobin (I--If~) and
platelet (PL'r). 110
abnormality was observed from alanine aminotransferase (~1I,I~1 and blood urea
nitrogen
(BLJN) for all subjects, as shown in 'I"able 8. Nn abnorntality wars observed
on heart and
lung from electrocardiogram (EKG) and ?x-ray radioscopy, anti zno abnormality
on urine
routine as well.
Table 15, Effects of iso-C I S on blood routine ar7d platelet
~: Sfa
Iterrt Case ~ ~~for~, test After test p
'
WB(~ 6 5.800.9y x 10?-I~y-_._.--.---7. ~00.710.0072
~
GRAM 6 3.20 ~0. 's9x 3.80_+0.$9 0.0311
l ON
RBC'. 6 ~1.64~0.4(ix I0'z/t.4.620.69 0.8719
HB 6 ;22.00li).40~/I: iwa8.0016.90 0.1839
PLT 6 ;~03.0~33.3X109~'1_X32.052.1
~ 0.0809
1
Table 16. Effects of iso-C 1 S on liver and kidney functions
h ~ SD
Item Case ___ l3efor~ test- . . _'..~' - _..~~~r te5t _ _ __.__
ALT ti 15.17~8.7()llrL 15.00-.+v 10. 3 0.4398
BUN fi 6.69~2.1~lrnmol~'L, I t'a.4''7f1.?"~> 0.2850
CA 02286750 2003-03-26
It indicated wider safety range of 1 3-methyltetrad~car~c>ic acid for oral
administration (0.6g - 1.8g daily for one month) without any t~~xicity
effects. It also
showed the improvement of white blood cells and graraulocye t~n~l immune
boosting.
J, Chemoprevention of _7.12-di~net~lb~nr~a~_s~nthracene (DMBA -induced
mammar~carcino~enesis in rats by iso-C 15
Materials and Method:
13-methyltetradecanoic acid (iso-C1 S) was prepared by dissolving in NaOH
solution and then in 0.35% T'ween 8() with pH7.5.
Fifty female Sprague-Dawley rates were maintained 0n laboratory chow. At 50
days of age, rats were given a single dose (Stag) of I)MB,~, (Sigma Chemical
Co., St.
Louis, MO) via an intragastric tube. Seven days after IaML3A administration,
the rats
were randomly divided into two groups of 2.~~. I:aclr rat in control group was
given 0.5 ml
of 0.35% Tween 80 five times a week via an intragastric tube throughout the
experiment,
while each in test group was given 0.5 ml iso-C I 5 solution of concentration
0.'7% five
times a week. Food and water were available ad Iibitutn. l w~r~ty weeks after
DMBA
administration, all rats were scarified, and all palpable tumc3rs 'sere
removed.
Results:
Table 17 Effects of iso-(::l S on tumorigc;nesis of IIMBA-induced
marrunary cancer tn rats
group case No. of rats tumor No. No. of tumors /
No. of of mean
with incidence tumors tumor tumor~bearing rat
tumor
tumors pcr weight (g)
rat
control 25 21 ~84% -.. ~,~._ __,-y, _.....__ .: 5.~2.q
l +.~ __~ 2.45.8
7....
iso-C15 25 3 12°r 1 (:1.2~4~ 1 1.3~0.5 0.8-1.3
It is shown that iso-C 15 significantly reduced the incidence of mammary
cancer.
iso-C 15 has offered prevention in experimental rnatmnary carcinogenesis. It
caused
slower tumor growth, though it could not achieve a Complete, prevention.
K, Chemo~prevention of ultraviolet I3 ray (UVI3~ induced skin cancer by iso-
C15
Materials and method:
31
CA 02286750 2003-03-26
13-methyltetradecanoic acid (iso-C 15) was prepared by dissolving in NaOH
solution and then in 0.8% Twecn 8t) with pH7.S, with rescllting concentration
10 %.
forty female SKH-1 hairless, mice were divided into cc.~ntrol and test groups,
with
20 in each. The mice in both groups were treated topicallyr once 'with 51.2,
pg of DMBA
dissolved in 200 p1 acetone per mouse to achieve tumor initiation. One week
later (day
8), animals in test group were applied tc>pica:llly with 200 1.1l i:~o-C'15
per application pe:r
mouse per day. Control group received 200 p1 Tween 8() per application per
mouse per
day. Thirty minutes later, the animals in both groups 4vere E:xpc>sed to UVB
(290-320
nm) radiation at the dose of 180 m3icm~ per day to achieve UVB radiation-
induced tumor
promotion. The iso-C 15 or vehicle treatments followed by t.JVB irradiation
were
performed twice a week up to 30 weeks from the start of Li VB exposure. The
animals
were evaluated for tumor incidence rt the end of 30 weeks.
REaults:
The results assessed the protective effect of iso-~', I S during tumor
promotion
stage. At the termination of the c:xperilnent, the animals in iso-$:' l5-
treated group showed
a 40% reduction in tumor incidence. ~1 he iso-C; I ~-treated group also showed
90%
reduction of tumor volume comparinf; to vehicle-treated group.
The branched-chain fatty acid:! can be administered in the form of liquid,
powder,
tablet, capsule, injection or encapsulated with liposome, to be delivered
bypassing
digestive tract, or directly into bloodstream. They also can be u:;ed as
topical drug for
skin cancer or other skin diseases.
II. Production Process of Specific Branched-drain ljatty Acids
The present invention includes the production me hods ol' the said specific
branched-chain fatty acids with anticancer activities.
The specific branched-chain fatty acids of present invention can be isolated
from
natural resources occurring including, but nc>t limited to, tll~ organisms
containing the
specific branched-chain fatty acids.
The specific branched-chain fatty acid;> of present inv~nti~~rn can also be
synthesized by chemical or biological m~tl~ad:~,. The classical Kcslbe's
synthesis methods
.~ L
CA 02286750 2003-03-26
of branched-chain fatty acids are well known and a ,pacific example of a
method for
electrosynthesis of 13-methyltetradecanoic acid is provided in the example
below. The
biosynthesis methods of specific branched-chain fatty ~~cids related to
present invention
are fermentation or incubation process using specific bacteria strains
containing high
percentage of specific branched-chain fatty acids in their cellular lipids.
The production
process of fermentation solution containing specific branched-chain fatty
acids and
having anticancer functions is provided iry the examples below .
Example 5: Electrolytical Syn~hesis of 13-Methyltetradecanoic Acid
13-methyltetradecanoic acid is synthesized electrolytically from isovaleric
acid
and methyl hydrogen dodecanedioate; in methanolic solution, based on k:olbe
electrolysis.
Dimethyl dodecanedioate was prepared from dodecanediaic acid by esterification
with 5 pacts v/w methanol containing; 5% w/v conce~~trated sulfuric acid. The
dimethyl
ester, after purification by vacuum fractional distillation, was converted to
the mono-ester
using the theoretical quantity of kalixarra hydroxide in anhydrous methanol.
Thc: methyl
hydrogen dodecan-1,12- dioate was purified by distillation.
The electrolytic coupling reaction was carried out with mono-ester and a 2-
fold
molar excess of isovaleric acid dissolved in methanol c«ntainins~ sodium
methoxide,
using 2 x 10 cm' platinum electrodes. 'fhe reaction nnixture waa stirred and
maintained at
50"C by water cooling until the solution going alkaline. Flectrodo polarity
was reversed
every 30 min to prevent the built-up of deposits an the electrode surfaces.
After electrolysis the reaction mixture was cooled tc~ room temperature and
the
by-:products, dimethyl docosanedioat~:, which. precipitated was retrroved by
filtration.
The filtrate was acidified with acetic acid and the methanol rerncyved by
rotary
evaporation under reduced pressure. ' (he crude methyl 13-methyltetradecanoate
was
purified by fractional distillation. Finally the methyl ester was loydrolyzed
by refluxing
with excess 10% wlv sodium hydroxide: in etl~anollw~~ter (S(:1:50~ vlv). After
cooling andl
acidification, the free acid was extracted with diethyl ether and purified by
vacuum
distillation.
33
CA 02286750 2003-03-26
The purified 13-methyltetradecanoic acids or its derivatives are reprocessed
in
various formulations, including liquid, powcaer, capsule, tablet, injection,
cream, or
encapsulated with liposome.
E~cample 6: Process of Making a Ferrn~,ntation solutions C'ontainin~g Specific
Branched-chain Fatty Acids
A process for making a fermentation solution containing a high percentage of
specific branched-chain fatty acids is exemplified below.
The starter cultures grow at as slant agar medium for 24 hours first, then are
inoculated onto the liquid medium in the culture flask and cultured on the
incubator
shaker for 24 hours. Next, the liquid ~:ultures in the flask are inoculated
onto seeding tank
in the inoculating rate of 0.1-0.5% (w/w). ~l"he cultures, after fermenting in
seeding tank
for 24 hours, are replaced onto production fe~mentors tc> ferrx~ont f'or 48
hours, with
aseptic airflow passing the mass. Generally, the magnification ration from
seeding tank to
production ferment<~r is about ten. The incubation conditions area aeration
rate of 1: 0.6-
1.2 (mass/air) v/v in, agitation speed of 180-260 rpm, and the tern~perature
of 28-38° C.
After the incubation is finished, the resulting culture~ solution is
autoclaved at
100° C for 30 minutes, and then the harvested solution can he
pKsc;kaged and autoclaved at
120°C. The resulting product in this way is the oral nutritional liquid
with anticancer and
salutary functions for human use.
Alternative products can be obtained by different procedure including, but not
limited to, the method below. After the incubation is finished, add
appropriate amount of
hydrochloric acid to the resulting culture solution to lower tlm pl I to 3-4,
and autoclave it
at 100°C for 30 minutes and centrifuge the i;ooled solution finally.
The resulting
supernatant, in which soy saponir~ is prcsdominant crrnrposition, can be used
to
manufacture the nutrient drink with variou:w tastes. Then add ;game volume of
95°ro
aqueous ethyl alcohol and same volume of 2 N NaC~lv to the resulting
precipitate, agitate
and heat at 100°C. After cooling and centrifuging, while collect the
resulting supernatant
for late use., add same volume of 1 I~ fIC'l t~;o th a remainder precipitate
and heat for 5
minutes at 80~C. After cooling anc.~ centrifuging, collect thca resulting
supernatant.
Combine two fractions of supernatant:; together and adjust the pE-t to 9.0,
which is the
.; 4
CA 02286750 2003-03-26
concentrated oral nutritional liquid product containing various I~ranched-
chain fatty acids,
and soy isoflavons such as saponin, ciaidzin, genistein, ~~rltl other
anticamer substances.
The another procedure is that after the irrcubatiofn I~inish~~.d, directly
atomize and
dry the solution into powder product, and encapsulate the powder into capsule
or make
tablets.
The specific branched-chain ratty acids can be isolated firam the fermented
solution using well-known method in the art for isolating ~clhala.~r fatty
acid. The isolated
active branched-chain fatty acids are reprocessed in various formulations. The
formulations of the present invention comprise at least one specific branched-
chain fatty
acid or its pharmaceutically acceptable salt including sodium salt.
~fhe°.y can be: contained
in an ampoule for injection or transfusion, e:;pecially for advarl~:e stage
cancer patients.
They may also be mixed with a carrif:r, or diluted by a carrier, czr unclosed
or
encapsulated by a digestible carrier is the fo~~m c~f a capsule, sachet,
cachet, paper or other
container or by a disposable container such as m ampoule. ~~~. carrier or
diluent rnay be a
solid, semi-solid or liquid material, which serves as ~~ vehniclo, excipient
or medium for
the active therapeutic substance.
In the process described above, soy: bean media are used. 'fhe components are
listed by weight with water making up the reminder. Proper amount of trace
elements
necessary for human body in addition to said nutritional c~c~nnporrents are
added.
Soybean medium
Soybean 5-10
or soybean milk or bean cake (by :soybean wt.) 5-15
Yeast extract 0.02-0.5
or yeast powder 0.02-0.5
Ca~C:o3 t_i.OS-0.25 %
KI-12PO4 0,02-0.10
Mgso~ c) 01-o.os r
NaCI 0.01-0.04
Na,MoO.~ ~.0-30ppm
Zn;~O.~ 2.5-1 Sppm
Co~',~ 1 Z :~.()-20ppm
3.
CA 02286750 2003-03-26
In addition to the bacteria which have been identified as containing high
p~°rcentage o.f branched-chain fatty acids in their cellular lipids,
such as the genus
Si'enotrphomortas, Xartthomonas, Flavohacierit~m, (."apna~~vtoEahczga,
Altermonas,
C'ytophage, Bacillus, C:.'hryseobcrcterittrrr. Fmpdobacaer, Aureb~acterium,
SphinggobactErrium, .Staphylococcu.s, ,~zotohacter and P.scaudorrtonas, the
bacteria of
present invention also include all other bacteria strains containing branched-
chain fatty
acids.
The products in the forms of oral liquid, capsule, tablets or injections,
produced
using said bacteria and the media, with a production process designated by
present
invention, have anticancer functions and other nutritional effects for human
and animals.
III Anticancer and Other F~.metion;~ of the_ ~~~enr~entation Solutions
C;ontainin~
Specific Branched-chain Fa~Acids
'The said fermentation solutions are produced using specific bacteria strains,
which contain high percentage of branched-chain fatty acids in their cellular
lipids, and
soybean media and process designed by present inventiUn. ~hhe fermentation
solution
contains various specific branched-chain fatty acids with significant
anticancer activity,
and other nutritional compc>sites from soybean media and bacteria metabolite.
':fo
demonstrate their anticancer, antiagir7g, irnrnune boc>stirrg arid other
functions, the
following animal experiments and clinical txials are presenl:ed. .1s an
example of said
fermentation solutions, in following experiments th a fermentation solution,
named Q-can
Oral Liquid, was used.
Q-can Oral Liquid was produced using ,ftenotrophontonas maltophilia strain Q-
car. as production strain, while using the soybean medium above and production
procedure designated by present invention. The par°allel animal
experiments and clinical
trials were also conducted using its atomized capsule product. 'I'he same
conclusions
were obtained as for Q-can oral liquid product.
The production strain, Stenotrophomonas maltophidaa Q-can, has all the
identifying characteristics of the sample on deposit with American Type
Culture
'~ 'Cr~d~-nark
y t
CA 02286750 2003-03-26
Collection, 10801 University Boulevard, Manassas, VA 20110-2209, and assigned
ATC'C 202105. The bacterial characteristics was identified by ATCC as
following:
The cellular morphology is motile, non-sparing, Gram negative, and aerobic
rods.
The colony morphology is following: Colonies at 24 hours on ATCC medium #3
{nutrient agar) were L -90% circular (approximately I rnm in diameter) with
entire
margins and convex elevation, rough surfaec:, semi-translucent, light beige
color; II, -10%
small circular (< 1 mm in diameter), entire margins, convex elevation, semi-
translucent,
smooth surface and darker than I. Same chaaracteristics were found when grown
on
ATCC medium #18 (T-soy agar), #44 (8H1 agar) and #260 (Sheep blood agar). Both
colonies were characterized and found to be the same.
The cellular fatty acid composition of Stenotrophomonas maltophiliu Q-can is
following:
Fatty Acid ~;°i~ o~~ total)
strai hg t-chain acid * branched-chain acid
10:0 0.48 i l I :E) 3.21
14:0 3.14 i 13 a) 0.50
15:0 0.33 i i 5:1) 39.34
16:0 5.52 a 15 :0 ?.44
16:1 15.9_5 ii5:1 1.02
hydroxy acid i 16:0 0_88
30H-10:0 0.12 i t?:0 3.68
30H-i11:0 1.51 i19:(~ 0.33
30H-i12:0 2.68
30H-i 13:0 3.57
20H- 13:0 0.29
* The number to left of colon refers ber of carbon atoms,
to the num the number
to right refers to the number of double
bans.
** i = iso fatty acids, a = anteiso
fatty acids.
Since the fatty acid composition of
bacaeria is influenced by biosynthesis
conditions including temperature and may be considered as
pH, the siata above a typical
value.
37
CA 02286750 2003-03-26
The typical fatty acid contents in 500 ml of Q-~~;ac~ oral liquid are
following:
straight-chain acid branched-chain
acid
10:02.0-2.7 mg i11:(.1 11.2-15.4
mg
12:02.9 - 4.0 mg i 15:0 I 145.8mg
06.0
-
14:013.0- 17.7 mg il~sa) 3.1 --4.3
mg
15:02.8 - 3.8 mg i 1'x:0 12.4 - 17.Omg
16:0251.7 - 346. lmg i 19:0 2.2 - 3.0
mg
17:02.9-4.0 mg a15:0 23.4-32.1
mg
18:075,6 -- 104.Orng
20:05.5 - 7.6mg
12:14.3 - 5.9 mg hytarox, acid ,
16:121.0 - 28.9mg; 3t)1-I-i 11:0 6.3 8.6
- rng
18:1488.8 -- 672.Omg. 30E-I-12:0 12.0 16.5
- mg
18:2825.9- 1135.6mg :~~C)1i-i13:0 13.2 l8.lmg
--
A. Animal Studies
Example 7: Acute I'oxicit~r 'felt of Q-can Oral Liguid
Materials and Methods:
Q-can oral liquid; ICR mice weighing 20.5-22.Sg.
Based on the preliminary test which could not determine 50% lethal dose
(LD50),
twenty ICR rraice (half each sex), weighing 20.5-22.Sg in fasting, were given
intragastrically with the most endurable capacity of 3 ml Q-can oral liquid
per mouse for
fou~.r times within 24 hours (6a.m. l Ua.m., 4p.m., and 10p.rn.. ?
Results:
It was observed that all the tested mice were less active five minutes after
every
adrriinistration, and returned to normal abo~.zt one hour later. '1"hree mice
suffered from
diarrhea one or two days after the administration, but none of tested mice
died within the
following seven days. After the course of treatment, the tested mice were
sacrificed and
dissected. Visual observation showed no abnornlality in internal organs. This
limited test
3
CA 02286750 2003-03-26
indicated that Q-can oral liquid had no taxis effects, even when large doses
were taken
a<:utely. Based on the conversion a body surface area, this dose corresponds
to 4642 ml
Q-can oral liquid per day for an adult weigluing 70kg.
Example 8: Subacute 'toxicity Test of Q-can Oral Liquid
Materials and Methods:
Q-can oral liquid; Kunming mice weighing 22-24g.
Twenty-four mice (half' each sex,) were randomly divided into control group
and
test group, and were administrated intragastrically with the r~arrnal saline
in control group
and Q-can oral liquid in test group at dosage: of 0.$ ml per day for 21 days.
On the 22nd
day, two mice randomly selected front each group were scarified; the paraffin
sections of
their viscera were made for microscopic examination.
Results:
No pathologic changes in internal organs were found by either visual
observation
or observation under microscope. The remaining ten mice in each group were
further
observed for seven additional days. No mice died in this ohservation period.
This test
showed that intragastrical administration of (~-can oral liquid for 21
consecutive days did
not result in toxicity or pathologic changes in mice.
Example 9: Long-term TUXICIty I"est of (~>-can Oral l:.iquid
Material and Methods:
Q-can oral liquid; Forty male and forty female Sprague-1)awley rats weighing
60~0.75g were supplied by Sino-English Joiryt ~entur~ed Shanghai Sipure-Bikai
Experimental Animal Co. Ltd.
Eighty mice were randomly divided into four groups: high-dose group (20m1/kg
Q-c;~n oral liquid); mid-dose group (1()mhkg Q-can oral liquid); low-dose
group (Sml/kg
Q-can oral liquid) and control group ( ( Otnl/kg normal saline). The samples
were given
intragastrically once a day for three months. C>ver the course of experiment,
behavior,
app~aite, gastrointestinal reaction and body weight of~ the; rats were
recorded. The index of
blood routine, blood platelet, electrocardiogram, liver function and renal
function were
mea;~ured. ~fhe tested rats were sacrificed and dissected after three-month
administration.
39
CA 02286750 2003-03-26
~~isual and ,pathological examinations were made for their main organs
including heart,
liver, spleen, lung, kidney, stomach, jejunum and brain.
Results:
C_Tenerally speaking, the tested r ats were wel l, r~o abnormal behavior, no
gastrointestinal reaction, good appetite. The curve ot~ weight increase of
test group was
similar to the control group (p>0.05). The electrocardi«grarph examination
result was
normal. The hematology (including blood routine and blood platelet) was not
statistically
different between the test group and control group 1;p=~(!.()S). 7 he liver
function (including
A,LT and TTT) and the renal function ( including 13L.11~1 arad Cr) showed no
obvious
changes either (p>0.05). Although the creatinine of tire test group was a bit
higher, it was
still in the normal range. The pathological section ex; urination of the main
organs
showed that the cell structure and lristomorphology in test group were not
obviously
different from those in the control group. It is c: c>ncludecl that Q-can oral
liquid can be
used safely, based on the fact that continuous admirristrraticarr had no
toxicity reactions.
Example 10: Effects of Q can Oral Liquid on I'rolon~ing Life-span of Fruit Fly
Method:
The fruit flies of both sexes were divided into control and test groups. In
making
forage fed to flies, water was used in control group, ~:wlnile 2%,
10°ro and 20%
concentrations of Q-can Oral liquid were used in test groups, respectively.
The numbers
oil dead fruit flies were counted every day till the last cm~ died. The mean
life-span (mls)
and the maximum life-span (MLS) were calculated.
Results:
Table 18. Effects on mean and maximum life-spGuu of fruit flies
mls (d, M~SD) MLS (d, M~SD)
group ~ male -a_~_____ fernal~-.--~--_..____..___..._._ morale female
control 31.99.7 26.99.6 40.i)~-4.3 43.04.9
(46) (47) ~ 0 ~
Q-can 30.8fi.5 31.8a-9.S *' 39. S~:2.1 63._50.7
2%
(45) (481 ~0J [1]
Q-can 3 8. 9 13 3 5 . 9 12 64. ~i-:2.1 61.04.2
10% .3 * .4 * *
(4S) (-17)
Q-can 50.815.8 ** 45.5l4.fi ** 64.~i~2.1 64.52.1
20% ~
ao
CA 02286750 2003-03-26
_ (44) (47) 1 [ 12] [4]
( ) case number, [ ] residual number of flies, comparing with control, * p <:
0.025,
*'~ p < 0.001
Q-can Oral Liquid significantly , increased the mean and maximum life-span of
fruit flies, suggesting the antiaging function.
Example 11: Effects of Q-can t:)ral I,yid on I-le tic and Cerebral
Lipoperoxide
Method:
Forty Balb/C mice of both sexes were randomly divided into control and three
test
groups. The mice in test groups were supplied with (~-catn Oral Liquid instead
of
drinking water for four weeks, and then were starved for 24 hours. The
malondiadehyde
(NiDA) contents were measured to determine the hepatic and cerebral
lipoperoxide
(LPO) levels.
Results:
Table 19. Effect of Q-can on hepatic and cerebral hPO level
(MDA nmol/g, X~SD)
male I female
group ~ case ~ liver I brain ~ liver ~ brain
control 10 75.85.50 1()fi.8--:2.67'~4.84.93 108.84.10
~
Q-can 30% 10 56.32.20* 83.04.83* X5.03.13* 87.05.13*
~
Q~-can 10 63.2-X2.30* ~)2.~~-3.27*6(7.83.07* 94.03.39*~
20% ~
Q-can 10% 10 62.23.33* 96.73.83* 61.02.27* 98.82.50*~
~
cor~nparing with control: * p<0.001, camparsng botweer~ C~1-can '20% and 30%:
° p<0.01
Q-can Oral Liquid significantly decrease the hepatic and cerebral LPO levels,
suggesting its antiaging function.
Example 12: Enhancement of the Effectiveness_af C'hemotherapeutic D
Materials and Methods:
Q-can oral liquid; mouse liver cancer HAC cell line; male Kunming mice
weighing 20 - 25g; commercial cyclophosphamide (C',P).
Forty mice were randomly divided into ~ groups. In three test groups, 36%, 60%
and 100% Q-can oral liquids (diluted with water) were given, respectively,
while water
~t
CA 02286750 2003-03-26
was given in both control groups. C)n the 8th duy, 0.2m1 I-lAC cancer cell
suspensions
(;L 07 /ml) were injected into the abdominal cavities of each nac~use under
aseptic condition.
Cm the 1st, 3rd and Sth days after irzject..ion, C'P (50zng~'kgj was injected
i.p. into the mice
in all test groups and positive control group. From the 9th day after
injection, normal
feeding was resumed as before the fist. Date of death of e;ae,h zr ouse was
recorded, and
the average life span and the rake of' increase in life span were calculated.
The rate of
increase in life span (ILS%) is defined as following:
Life span of test group - life span af~ control ~ro~ ,: 100%
Life span of control group
Results:
The results below (Table 6) showed the enhancement of anticancer effect of
chemotherapeutic drug, CP, by combining treatment with Q-can oral liquid. The
average
life span of mice in control group (without drug) was only 10.63~1.03 days,
while that in
positive control group (only taking CP) was 13.06~3.03 days, with IhS of
22.86%. The
combination of Q-can oral liquid (with dosage of 60°i~ and 1
()0° o) and CP increased the
effectiveness of CP, as evidenced by increased average IL.,S and increased
numbers of
mice that survived over 17 days (60°.o prolonged). Therefore the ILS
rate by CP treatment
ha., been increased 56.78% and 143.86°~o by combination with 60% and
100% Q-can oral
liquid, respectively. The difference r~~as statistically significant.
Table 20. Q-can oral liquid enhanced the effectiveness of C'.P for liver
cancer
group drug life span rrwice No prolonged days ILS% p*
( > 17d)
1 ___..___ 10.63+1.03 0 __._____ ____
:? 36% Q-can + 12.38-2.3'7 1 1.75+2.05 16.46 <0.10
C'. P
a 60%Q-can 14.44+3.54 '? 3.94_+-2.87 35,84 <0.02
+C'.P
~E 100%Q-can 16.56--_3.9(, (~ 5.94;-x;,2.96 55.75 <0.01
42
CA 02286750 2003-03-26
+CP
CP 13.06+3.03 1 2.4~4~+-2.58 22.86 <0.05
* compared with group 1 (control group)~.___....__.__. _-_. _._-____
Example 13. Tumor Inhibition Effects on Mouse. L,ewis Lun~Tumor
Materials and Methods:
Concentrated Q-can oral liquid (containing specific branched-chain carboxylic
acids 36 mg/ml.); Female Fl mice (C',S7/B1 and DBA/2) weighing 18 -- 22 g;
L,ewis
mouse lung tumor.
The mice of test group were administrated with coace:ntrated Q-can oral liquid
at
1 rng/kg for 10 days before transplantation of L.ewis mouse lung tumor. All
the mice were
transplanted subcutaneously in the subaxillary region with a piece of L,ewis
tumor of
approximately 2 mm in diameter. The treatments of intraperitoneal injection of
chemotherapy drug CT X in 30 mg/kg were liven once a day alter transplantation
over 8-
da:y period for positive control group. The treatments fc>r normal control
group were daily
injection of normal saline for 8 days. The administration of concentrated Q-
can oral
liquid for test group was initiated l 0 days before tumor transplantation and
continued for
another 8 days. Finally all the animals were sacrificed by spinal elongation.
Tumors were
excised and body and tumor weights were recorded.
Results:
The significant inhibition effect of Q-can oral liquid was shown in the test
data
(Ta.ble 7.) Although the inhibition rate of positive control group
(chemotherapy drug
CTX i.p. injection) was higher than test group, it is noticed that pliability
of° the positive
control (70 %, only 8-day period) was lower than test group (10() °~o,
18 - 28 days),
implying toxicity of CTX. As oral administration is expected to be less
effective than
intraperitoneal injection, change in route of administration or increase in
dosage should
enhance the tumor inhibitory rate of Q-can oral liquid.
Table ? 1. Effects of Q-can Oral Liquid on Mouse L.,~~wis Lung Carcinoma
Xenograft
Implanted into ~ubcutanzous Area o#' Nude Mice
group dose route mice No, roc3~_weigtrt tumor weigl;yt TIR (%) p
43
. CA 02286750 2003-03-26
I11. Ill. ~ Itleal'1 ' ~1J ~g~
/ fl fl.
__.._ 1.p. 12/ _....- ._._. __._j _.~~ ~.~1,~6
11-.._..._~ 1.2'22.5__~.__._ _ _T__ _.~ _
_..._.
CTx 30mg,/kg 1.p. 10 / 21.2120.30. 71 =t ~).3b 62.6 ~ 0.01
7
Q-can 1 ml~'kg p.o.10 /10 20.9/21.91.1 1 .- {j.46 41.6 ~ 0.05
Example 14. Tumor Inhibition Effects on 1~~luman (;:iastric Adenocarcinoma SGG
7~~01 Xenografted into Nude Mice
Material and Methods:
Female Balb/c-nu/nu athymia mice, b weeks old, weighing 18-22g, housed in
specific pathogen free (SPF) condition throughout the course of experiment;
concentrated
Q-can oral liquid contains 36 mg/ml Specific branched-chain carboxylic acids;
the drug
used for positive control mitornycin C: (MMC) was con nnercially available
from Kyowa
Hakko Kogyo Co., Ltd., Japan.
Human gastric adenocarcinoma SGC-7901 xenografit was established and
maintained. For the experiment, the xenograft fragments of diaameter of about
2mm were
inoculated subcutaneously into the right subaxillary regions c~f nude mice.
The animals
were randomly divided into five groups hve days after inoculation. NS and MMC
(2mg/kg) were given once a day 1.p. in normal and positive; control groups,
respectively,
while concentrated Q-can oral liquid was given in test groups once a day p.o.,
starting on
the: same day, at doses of O.Smllkg, l.Omlikg, and 2.Omllkg respectively, for
14 days.
Experiment was terminated 20 days post-implantation, and mice were sacrificed
by spinal
elongation. Tumors were removed and the weights of treated versus control
tumors were
compared. Inhibition rate was calculated. 'Ifie expe.ritnent was repeated
once.
Results:
The results below indicated that Q-can oral liquid at doses of 0.5, 1.0 and
2.Om1/kg given p.o. once a day for 14 days after tumczr inoculation offered
antitumor
activity against human gastric adenocarcinoma SCiC'-7901 xenograft with no
marked
toxicity. The tumor inhibition rate increased with dosage of oral
administration. The
obvious shrinkage of the tumors was observed.
The results from two tests (test 1 and test II):
4~t
, CA 02286750 2003-03-26 .
Table 22. Effects of Q-can oral liquid on human gastric acierrocarcinoma
xenograft
SGC-7901 implanted into subcutaneous areas of nude mice
Test I
Grou dosage routeschedulMice Body' 'humor Inhibitiop
wt.
p a In./I~i.Iivl~i. wt. n
XSD, .~o
g
NS --- ~ i.p. Qd x 12/12 21.9/.>3.31.17+0.4---
i 4 ~
~
5
MMC 2.0 i.p. Qd x 6/6 22.4122.00.33+0.271.49 <0.0
14
mg/kg 4 1
C>-can0.5 ml/kgp.o. Qd x 616 22.0/2 1).80+0.431.19 >0.0
14 2.5
2 S
~)-can1.0 ml/kgp.o. Qd x 6/6 21.9122.01.1.~z00.448.23 <0.0
14
5
Q- 2.Oml/kgp.o. Qd x 6/6 21.6/21.71:).57-+0.351.28 <0.0
can 14 .
5 5
"test II
CJrou dosage routeschedulMice Body wt. Tumor Inhibitiop
p a In./Fi.Ir>/I'i. wt. n
-SD, 4>
g
NS __- i,p. ~d x 12/1? ~ '1.6/23.51.15+0.3_~._
l~l _
0
IVfMC 2.0 i.p. Qd x 6/6 21.1 /21.11:).30+0.373.51 <0.0
14
mg/kg 3 1
Q-can 0.5 ml/kgp.o. Qd x 6I6 21.9122. 1).90-+-0.521.58 <0.0
14 >
9 5
Q-can 1.0 ml/kgp.o. Qd x 6/6 '?2.0f.21.60.66+0.442.47 <0.0
14
5
4S
CA 02286750 2003-03-26
Q-can 2.0 ml/kg p.o. Qd x 14 6/6~ ??.3/?0.3 i).51+0. 3 55.45 <0.0
7 1
B. Clinical Trials
Example 15. Clinical Trial on Effects of Q-can tJral Liquid on Supplementary
Treatment of Cancer
Methods:
The clinical trial on the effects of Q-caxr oral liquid as supplementary
treatment of
carncer was carried out by the cooperation caf five hospitals in China. 333
cases of cancer
patients were involved and were randomly divided into two groups, chemotherapy
and
radiotherapy. The chemotherapy group in~:luded a control subgroup, which only
took
chemotherapy and contained 131 cases, and a tort subgroup, which combined
chemotherapy with Q-can oral liquid and contained 136 cases. The types of
cancer
involved included gastric, hepatic, esophageal, colon, pulmonary and mammary
cancers,
which were distributed similarly and comparably in the two subgroups (p>0.1 ).
The
radiotherapy group included a control subgroup (radiotherapy only, 32 cases)
and a test
subgroup (combination of radiotherapy and Q-can oral liqui~.i, 34 cases). The
types of
cancers involved included nasopharyngeal and laryngeal cancers, which were
distributed
similarly and comparably in the two subgroups (p'0,1 ). Meanwhile, sex and age
distribution of cancer patients in test and control subgroups was comparable
(p>0.1 ).
The dosage of Q-can oral liquid for two test subgroups was 80 ml x 2 per day
and
it was given for two months.
The clinical observations and records were proceeded daily and filled in the
unified observation forms. 'fhe changes of tl~e deficiency syndrome, symptoms,
blood
routine plus platelet counts, the toxic reaction of chemotherapy or
radiotherapy, and the
side effects of Q-can oral liquid were recorded weekly. The cardiac, hepatic
and renal
functions, the living quality and the tumor size were exanaineGi or analyzed
monthly. 'The
serum, albumin and globulin, cellular immune functions (lymphocyte
transforniation, NK
cell and subgroup composition of' '1'-lymphocytes) and the lrumoral immunity
were
determined before and after the clinival trials.
CA 02286750 2003-03-26
Results:
A. Effects on clinical symptoms
Four classes of therapeutic effects on deficiency syndrc>rne were defined as:
Si nificant effect -- the symptoms of the dcfic;ienc,y syndrome disappeared or
got
a significant favorable turn at the er~d of therapy;
Improvement -- the symptoms got a favorable tur~r at tl°ie end of
therapy;
Stability -- the symptoms remained unchanged;
No effect -- the symptoms became worse at the end of therapy.
In chemotherapy group, the effectiveness rate of test subgroup was 67.46%
(significant effect plus improvement), which was signifieantly higher than
that of control
subgroup (40.60%), p<0.01. In radiotherapy group, the e:ff~cti ~leness rate of
test subgroup
was significantly higher than that of control subgroup, p~: ).OS based c>n
Ridit analysis.
Table 23. Symptom changes in cheanotl~~erapy group
symptom subgrou case mitigation stability aggravation p
p case (%) case (~o)
appetite Test 72 ~49(68.Ot~)~.J..______...i~(25~t~0~....._._S(6.94) _-
<0.01
Control 81 18(2'2.22 3 3(4(1.74)30(37.04)
)
weakness Test 90 56(6i?.22.)28(31.11) 6(16.67) <0.01
control 70 13(13.57) 29(4I.43) 28(40.00)
Table 24. Weight change in chemotlmrapy group
subgroup case increase ~~~ stability ~ decrease p
case (°i°) ease (°ro) case (%)
test 136 63(46.32) ~.-33(24.27) _.~_.~~~29.41) <0.01
control 131 20(15.27) 35(26.72) ~6{58.01;)
y * increase and decrease were defined < s more than t:).SICg changes of body
weight,
and intermediate was stability.
B. Effect on immune system
CA 02286750 2003-03-26
Table 2S. Cellular irrununity changes in chemotherapy group
item subgroup ease Pre-treat host-treat p
(X~..~I~)~r~(:Xy~SD),~o
LTT _ test -__-..~~.~.5.~~+~ __Sh. yB+B.SS- <0.01
.- .__._ ()~._
*
control 75 SS.8s+8.8'T4~3.~11+12.2
1
CD3 test 3U ~~:1.5_~+4.554_~.47--S.iO<0.01
control 30 45.47+~.Sf~38.57+4.50
CDQ test 30 44.0'7+4.604_i.10+5.13<0.01
control 30 42.60+5.20 38.27+S.fi2
NK cell test 1 S 9.60+S. 1 ~.00+4.23<0.01
l I
control 14 12.96-+-4.311).804.00
l
* LTT: Lymphocyte Transformation 'I"est
Table 26. Humoral immunity changes in chemotherapy/radiotherapy groups
Item group subgroup case Pre-treat fast-treatp
~gjL) (X~_SL7)ar~,(;5;-+.-~D)plo
I~;G chemotherapy! tests..__-_._71_..._~ __...I - <0.01
__ () ~t0+4 I.925.06
~._. -_
control 7S 11.90y4.3 11.054.99
IgA chemotherapytest 72 1.63+0.67 I .73 ~-1.32<0.01
control 7S I.6S~0,76 1.380.76
radiotherapytest 34 1.74 + '~ , 3 <0.01
I , 31 92.18
control 31 2.0 7~ 1.880.80
I .03
IgVI chemotherapytest 71 1.24-+~C~.S~)1.551.05 <0.01
control 7S 1.53-0.78 1.30-+-0.73
The cellular and humoral immunity was enhanced in the test subgroup of
combining chemotherapy and (;~-can oral liduid. 'fhe cone;entration of IgA
increased in
the test subgroup of combining radiotherapy <~nd Q-can oral liquid.
48
CA 02286750 2003-03-26
C. Effects
on chemotherapeutie
toxic reaction
Table toxic reaction
27. Effects of blood
on syStelIl
item subgroup case ~ Pre-trecatPost-treat p
(?t:+S1)) (:5~-+ SD)
WBC(x10 test ~~ .. 3'~ ____~_~-74+.~.'?.1_.__.___.._.5.450.86<0.01
) ___.
control 3(~ ~.2~)+0.8~ 4.45+0.80
Neutrophil test 3ti 3.'20+().8?3.66-+-0.69 <0.01
cell
control 30 3.'72+0,58 3.09+U.45
Hb (g/L) test 30 94.63+18,0096.89+16.08 <0.01
control 30 103.67+ 99.20+ 11.63
13.2
4
platelet(x10~test 3(i 140.304.811160.034.36 <0.01
/L)
control 30 I >7.33~3.~2 145.53-1--5.33
The blood routine and platelet quantity in test subgroup dropped less than
those in
control subgroup. This indicated that Q-can oral liquid can prevent the
hemogram
decrease caused by chemotherapy. Meanwhile, Q-can oral liquid was effective on
the
patients whose WBC and Hb were lower than normal before el~emotherapy.
'Table 28. Effects on hepatic function of chemotherapy group
SGPT (n~nal/L, X+-S D
subgrou case pre-treat past-treat p
P
!test 89 ---___ 460,06+2~.3.~-__.______....__ __...__ __.30.11+245.01 -__
<0.05
control 84 261.=17+ l 9 I .23 284.00+217.30
Table 29. Effects on serum protein of chemotherapy
item subgrou case pre-treat (g/L, X=~-ST)) x~c~st~~treat (g/L, X+SD) p
CA 02286750 2003-03-26
p
total test l .01 __ ~ a 3 I + 1 ().I~ ~ ___.. _____ _____ __ X7.47-+5.99 <0.01
protein
control 103 65.64+6,53 64.20+6.07
albumin test 107 38.785.65 39.135.26 <0.01
control 102 39.44+4.74 38.18+5.24
SGPT decreased and serum total protein izzcreasecl in the test subgroup of
combining chemotherapy and Q-can oral liquid. T'lze results showed that Q-can
oral
liquid could alleviate the damage of hepatic; functions caused by chemotherapy
and
promote protein synthesis, thus protect the liver.
Table 30. Effects on renal functions of chemotherapy group
case Blood urea vitro en BlUOd creative ~,nmol/L~
group nmol/L case Pre-treat post- p
Pre-treat post-treat treat
test 111 5.13+2.95 110 9'7.15~30.64 <0.0
4.95+1. 33 97.99+-23.46 1
control 10U 4.26+1.03 91) 8~).'~8~'2.13
5.04+ 1.42 I 07. () 8+.=1 I .27
Blood urea nitrogen and creative decreased in test grol.Gp, which indicated
that Q-
can oral liquid could alleviate the damage of renal function caused by
chemotherapy.
In summary, compared with the chemotherapy only treatment of 131 cases of
cancer patients, the results of combinational treatment with Q-can oral liquid
showed
m~~rkedly enhanced therapeutical ef'tects with statistical significance. These
effects
included amelioration of the deficiency syndrome, irnprctvement of the
appetite,
weakness, living quality and immune ~unctic>ns, v~itigati~>n o1' the degree of
leucopenic
action induced by chemotherapy, alleviation of the lc>w leukocyte count and
the
hemoglobin concentration which decreased after t:rec~trn~.zit, 4~.nc:1
protection of the hepatic
~o
CA 02286750 2003-03-26
and the renal functions. In coyparison with the r:~diotherapy only treatment,
the
amelioration of the deficiency syndrome and increase of the serum 1gG level
were faund
in cancer patients, who were treated by combinatic.m cat" r~a~:lic~therapy
with Q-can oral
liquid. Q-can oral liquid had no toxic effects on the blood, heart, liver and
kidney. Thus,
~!-can oral liquid can be used as a supplementary tlaerapecrtic 4ugent for
cancer patients.
Example 16. Clinical Observation for '~S Cases of American Prostate Cancer
Patients Treated b -can Oral Liguid
The effect of Q-can oral liquid on PSA levels was tested for 8-18 weeks
(average
14 weeks) in two hospitals in E1SA., whc;re ;an integrative approach to treat
prostate cancer
was applied. Patients were not on radiotherapy, chemotherapy, or hormonal
treatment
during the recording period and followed a customized nutritional protocol. At
a daily
dosage of 250m1 concentrated Q-can oral liquid (containing ~OOmg specific
branched-
chain carboxylic acids), assay of PSA level was made for all patients. The
average drop
in PSA level was noted. It is also found that drops in 1"~A level of the
patients who had
higher pre-treat PSA level was more significant than those of the patients who
had lower
pre-treat PSA level.
Table 31. The effects of Q-can oral liquid on PSA level {mg/ml)
Case Pre-treat (mean ~ 5D) ~ Post-treat (mean ~ p
number 'ilk)
__ _--__._~.____...___..__..___.__-
____..____._.__.._......_...___...____.._._..._.__~-_..~,
35 10.22 ~ 10.72 7.45 ~ 6.06 < 0.01
Example 17: Industrial Process for Making~Fermentation Liquid
This example describes one method of industrial production of Q-can oral
liquid
in a more detailed manner.
Medium composition is: soybean 40kg (milling to milk and removing residue),
KH2P04 200mg, CaC03 2008, yeast extract 160g, MgSO~ 80g, NaCI 80g, Na2MoOd
l0ppm. ZnSO4 lOppm, CoCl2 5ppm., NaI-INO.~ 2ppm, :Yoybean oil (as antifoam
addition)
4kg, and add water to 400kg totally.
CA 02286750 2003-03-26
The above media is put into a seeding tank wand lead steam 120°C for 30
minutes,
than cooled to 30°C. Onto seeding tank are inoculated 3kg liquid
cultures, which were
cultured on the incubator shaker at 30°C for 24 ho~us. heryraent~~tion
proceeds in the
seeding tank for 24 hours, 30°C temperature, 2C)0 rpm agitation speed,
and 1:1 (v/v min)
aeration rate. After confirming no infection under cnicrosc.ope, it is then
transferred into a
ton production fermentor for 48 hours, where col~lp~~red to that in seeding
tank before,
the media is ten times of quantity and same percentage of ci~mposition, and
the same
parameters of temperature, agitation speed and aeration rate are used. When
fermentation
is finished and no infection is confirmed under microscope, the temperature is
increased
to 100°C to autoclave for 30 minutes. ~fhe cooled solution can be
packaged and packaged
fermented solution is again autoclaved at 118°C' for 4~ minutes. This
is a semi-finished
product waiting for quality inspection and final package as the Q-can oral
liquid product.
Every description in the above specification c>f <~ m~anerical range and of a
genus
is intended to inherently include a description of all possible values and
subranges within
thc: range, and all possible species arid subgenues within tb.e gtr~ues,
respectively.
