CA2286750C - Application and production process of a group of anticancer compounds - Google Patents

Abstract

A group of specific branched-chain fatty acids, with significant anticancer effects on human and animals; methods of making using either chemical synthesis or biosynthesis methods; and methods of treating cancer.

Description

~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.

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 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 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.

~~~~:~~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.

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.

An example of a terminally methyl-branched unsaturated iso-fatty acid of the present invention is > CH-(CHz)5-CT-I=CH~CHZ)~-(:OOH, 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 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.~
---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 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.

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 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, 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 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$~, IDES and ID9o were defined as the concentration of iso-C' 1 ~ reguired to kilE 50, 75 ox 90°:°, respectively, of cells (compared with that in untreated cells) and computed using CalcuSyn for Windows software (Biosol't, Cambridge CJI~:) based on Median Effect method by Dr. T. C. Chou.
Results The cytotoxic activity of iso-C 15 was quantified by determining IDSO, ID75 and ID9o (pg/ml or laM) in several human hematological and solid tumor cell lines.
It is indicated from Table 4 that iso-C15 was active in all tumor cell lines studied. The strongest cytotoxic activities were far MCF-7 human breast adenocarcinoma and human leukemia. The activities were less fbr Ii 1688 human small cell lung carcinoma and HCT 116 human colon carcinoma cell lines. In contrast, is~~-C', l 5 is nit toxic aLainst normal human peripheral blood lymphocytes at concentrations lethal to tumor cells.
Table 4. Cytotoxicity of iso-C'15 on human tumor and normal cells in vitro cell line cell type 1D5,,a(Eig/ml) ID~,,(l~gJml) ID~o(Itg/ml) MCF-7 breast carcinoma ~ ~ 10.()s~:()'9' ~ 15_~:>~)~1.28 25.49~1.6$
m 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 SNU-I gastric carcinoma2(:),77-2.474 1'.434.9580.4910.03 SNU-423 hepatocarcinoma 24.263.98 70..469.36120.7715.8 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 (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 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-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 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 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 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 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~

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.

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-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~

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:

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~

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 O
Zg/kt;

Iso-C15 10 20. 8 26. I -~~ 0,. ~~2 n4. 88 : 0. ~?. fi 0. 316 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 (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~~

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 <;

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 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 ~

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 (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~~.

~" 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 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 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-Materials and method:

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

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.

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 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.

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 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.

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 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.

~~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 _ (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 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 +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 . 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 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 ~
~

MMC 2.0 i.p. Qd x 6/6 22.4122.00.33+0.271.49 <0.0 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 ~)-can1.0 ml/kgp.o. Qd x 6/6 21.9122.01.1.~z00.448.23 <0.0 Q- 2.Oml/kgp.o. Qd x 6/6 21.6/21.71:).57-+0.351.28 <0.0 can 14 .

"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 _ IVfMC 2.0 i.p. Qd x 6/6 21.1 /21.11:).30+0.373.51 <0.0 mg/kg 3 1 Q-can 0.5 ml/kgp.o. Qd x 6I6 21.9122. 1).90-+-0.521.58 <0.0 14 >

Q-can 1.0 ml/kgp.o. Qd x 6/6 '?2.0f.21.60.66+0.442.47 <0.0 Q-can 2.0 ml/kg p.o. Qd x 14 6/6~ ??.3/?0.3 i).51+0. 3 55.45 <0.0 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.

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 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 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.

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 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 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 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.

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.

Claims (38)

1. The use of at least one terminally methyl-branched iso- or anteiso-unsaturated fatty acid, or a pharmaceutically acceptable salt or derivative thereof, for treating cancer, said fatty acid having a R0COOH, wherein R0 represents a terminally methyl-branched iso- or anteiso- unsaturated fatty group, said R0 other than the terminally- methyl-branched iso- or anteiso-moiety is linear or branched.

2. The use of claim 1, wherein the terminally methyl-branched iso- or anteiso- unsaturated fatty acid has a formula as set in formula 1:
wherein m is 0 or 1, and n is an integer between 7 and 16 inclusive, and at least one of CH2-CH2 group in (CH2)n is replaced with a CH=CH group.

3. The use of claim 1, wherein said fatty acid is obtained by isolation from fermentation or incubation products using a bacterial strain containing said branched-chain fatty acid.

4. The use of claim 3, wherein said bacterial strain is from a genus selected from the group consisting of Stenotrophomonas, Xanthomonas, Flavobacterium, Capnocytophaga, Altermonas, Cytophage, Bacillus, Chryseobacterium, Empdobacater, Aurebacterium, Sphinggobacterium, Staphylococcus, Azotobacter and Pseudomonas.

5. The use of claim 4, wherein said bacterial strain is Stenotrophomonas maltophilia.

6. The use of claim 5, wherein said bacterial strain is assigned ATCC
202105.

7. The use of claim 1, wherein the fatty acid is obtained by chemical synthesis.

8. The use of claim 1, wherein the fatty acid is obtained by extraction from natural materials.

9. The use of claim 1, wherein the fatty acid is 15-methylhexadecenoic acid (iso 17:1 .omega.9c).

10. The use of claim 1, wherein the cancer is selected from the group consisting of leukemia, tongue cancer, colorectal cancer, breast cancer, prostate cancer, lung cancer, gastric cancer, hepatocarcinoma, melanocarcinoma, renal cancer, esophagus cancer, pancreas cancer and skin cancer.

11. The use of claim 1, wherein the fatty acid is administered as part of a fermentation product also containing a nutritive medium.

12. The use of claim 11, wherein the nutritive medium comprises a soybean medium.

13. The use of claim 12, wherein the soybean medium has the following formula:
Soybean 5-10 %;
or soybeanmilk or bears cake (by soybean wt.) 5-15 %;
Yeast extract 0.02-0.5 %;
or yeast powder 0.02-0.5 %;
CaCO3 0.05-0.25 %;

K2HPO4 0.02-0.10 %;
MgSO4 0.01-0.05 %;
NaCl 0.01-0.04 %;
Na2MoO4 5.0-30ppm;
ZnSO4 2.5-15 ppm;
CoCL2 5.0-20 ppm.

14. The use of claim 11, wherein the fermentation product is obtained from a culture of Stenotrophomonas maltophilia assigned ATCC 202105.

15. The use of claim 1, wherein the fatty acid, or a pharmaceutically acceptable salt or derivative thereof, is formulated in liquid form, powder form, a capsule, a tablet, for injection, in encapsulated liposome, and topical formulation applied as a cream, ointment, or lotion.

16. Use of at least one terminally methyl-branched iso- or anteiso- fatty acid, or a pharmaceutically acceptable salt or derivative thereof, or a pharmaceutically acceptable lipoprotein thereof; for the manufacture of a medicament for alleviating toxicity and side effects on cancer patients undergoing chemotheraphy or radiotherapy.

17. Use of at least one terminally methyl-branched iso- or anteiso- fatty acid, or a pharmaceutically acceptable salt or derivative thereof; for treating a skin disease.

18. A method of making a terminally methyl-branched iso- or anteiso-fatty acid, or a mixture thereof, which comprises the step of culturing a bacterial strain containing said fatty acid to form a fermentation solution containing said fatty acid, and then isolating said fatty acid from the fermentation solution.

19. A method of making a fermentation solution containing at least one terminally methyl-branched iso- or anteiso- fatty acid, said method comprising the step of culturing a bacterial strain containing said fatty acid in a nutritive medium to form a fermentation solution containing said fatty acid.

20. The method of claim 19, wherein said bacterial strain is from a genus selected from the group consisting of Stenotrophomonas, Xanthomonas, Flavobacterium, Capnocytophaga, Altermonas, Cytophage, Bacillus, Chryseobacterium, Empdobacter, Aurebacterum, Sphinggobacterium, Staphylococcus, Azotobacter and Pseudomonas.

21. The method of claim 20, wherein the bacterial strain is Stenotrophomonas maltophilia.

22. The method of claim 21, wherein said bacterial strain is assigned ATCC 202105.

23. A composition for treating or preventing cancer, or for treating skin disease, or for providing an anti-ageing effect, or for providing immune boosting, said composition comprising an effective amount of at least one terminally methyl-branched iso- or anteiso- unsaturated fatty acid as defined in claim 1, or a pharmaceutically acceptable salt or derivative thereof, and a pharmaceutically acceptable carrier.

24. The composition of claim 23, wherein the composition is formulated as a liquid, a powder, a capsule, a tablet, for injection, as an encapsulated liposome, or for topical application as a cream, ointment, or lotion.

25. Use of at least one terminally methyl-branched iso- or anteiso-unsaturated fatty acid as defined in claim 1, or a pharmaceutically acceptable salt or derivative thereof, or a fermentation solution containing said fatty acid, for immune boosting.

26. Use of at least one terminally methyl-branched iso- or anteiso-unsaturated fatty acid as defined in claim 1, or a pharmaceutically acceptable salt or derivative thereof, or a fermentation solution containing said fatty acid, for prolonging ageing.

27. Use of at least one terminally methyl-branched iso- or anteiso-unsaturated fatty acid as defined in claim 1, or a pharmaceutically acceptable salt or derivative thereof, or a fermentation solution containing said fatty acid, for cancer prevention.

28. The use of a fermentation product containing a nutritive medium, which contains at least one terminally methyl-branched iso- or anteiso- fatty acid, or a pharmaceutically acceptable salt or derivative thereof, for treating cancer, said fatty acid having a formula as set in formula 1:

wherein m is 0 or 1, and n is an integer between 7 and 16 inclusive and wherein said fatty acid is obtained from a bacterial strain containing said branched-chain fatty acid.

29. The use of claim 28, wherein said bacterial strain is from a genus selected from the group consisting of Stenotrophomonas, Xanthomonas, Flavobacterium, Capnocytophagu, Altermonas, Cytophage, Bacillus, Chryseobacterium, Empdobacter, Aurebacterium, Sphinggobacterium, Staphylococcus, Azotobacter and Pseudomonas.

30. The use of claim 29, wherein said bacterial strain is Stenotrophomonas maltophilia.

31. The use of claim 30, wherein said bacterial strain is assigned ATCC
202105.

32. The use of claim 29, wherein the nutritive medium comprises a soybean medium.

33. The use of claim 32, wherein the soybean medium has the following formula:
Soybean 5-10 %;

or soybeanmilk or bean cake (by soybean wt.) 5-15 %;

Yeast extract 0.02-0.5%;

or yeast powder 0.02-0.5%;

CaCO3 0.05-0.25%;

K2HPO4 0.02-0.10%;

MgSO4 0.01-0.05%;

NaCl 0.01-0.04%;

Na2MoO4 5.0-30ppm;

ZnSO4 2.5-15 ppm;

CoCL2 5.0-20 ppm;

34. Use of at least one terminally methyl-branched iso- or anteiso-fatty acid, or a pharmaceutically acceptable salt or derivative thereof, or a fermentation solution containing said fatty acid. for immune boosting.

35. Use of at least one terminally methyl-branched iso- or anteiso-fatty acid, or a pharmaceutically acceptable salt or derivative thereof, or a fermentation solution containing said fatty acid, for prolonging ageing.

36. Use of at least one terminally methyl-branched iso- or anteiso-fatty acid, or a pharmaceutically acceptable salt or derivative thereof, or a fermentation solution containing said fatty acid, for cancer prevention.

37. A soybean medium-based product comprising one or more terminally methyl-branched iso- or anteiso-fatty acids and a soybean medium.

38. The soybean medium-based product of claim 37, wherein the soybean medium has the following formula:

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%

CaCO3 0.05-0.25%

KH2PO4 0.02-0.10%

MgSO4 0.01-0.05%

NaCl 0.01-0.04%

Na2MoO4 5.0-30ppm ZnSO4 2.5-1.5ppm CoCl2 5.0-20ppm.