Note: Descriptions are shown in the official language in which they were submitted.
~2~88191
. . .
Description
Method and Compositions for Ccn~atting Tutors
Technical yield
The present invention relates to a method and to
compositions for cohabiting timorous growths.
no specifically this invention relates to a method and
to cor~ositions which promote a reduction in the size of
established timorous growths.
Background Art
o'er than 50 years ago Otto ~arburg discovered that
malignant tumor cells ferment glucose to lactic acid much more
rapidly Han most normal cells, i.e. they depend upon
glycolysis for their primary source of energy or growth (the
"~1arb~urg effect"). (O. ~arburg, my Prime Cause and
Prevention of Cancer, Triltsch, Wurzbur~, Germany, 1967 pp.
6-16, Become. Z. 142, 317 (1923)). As used herein, the word "tumor"
refers to an abnormal mass of tissue arising without obvious cause from
cells of preexistent tissue and possessing no physiologic function. As
is discussed below, such tumors include benign, well-~ncapsulated
mammary tumors such as those characteristic of the female Sprague-Dawley
rat and well known to those skilled in the art, human breast cancer,
and other metastasizing cancers such as human bowel and colon cancers.
Recently, tacker et at have demonstrated high Autopsy
activity (AT ASP Pi) in tumor tissue and have proposed
that increased Autopsy was required for the high glycolytic
activity of tumor cells since AT hydrolysis to DO -I Pi is
known to be a rate-limiting step for glucose utilization
(Racker, E. and Spector, M. Science 213, 303 (1931)). Thus,
although the Racker et at finding support the '`~arburg effect"
the reason for the high rate of aerobic glycolysis in tumor
cells has not been planned
3L~2138~
Disclosure of Invention
It has now teen found that the Ruth of tumors can be
successfully ccm~atted by the adninist-r~tion to a
tu~,or-~earing animal. of relatively Llr(Je amo~mts of
L-carni.tine.
It has also been found that the growth of malignant
tumors in vitro can also be repressed by the presence of
L-canlitine.
In addition, it has been found that if the L-carnitine is
administered to the anin~l or used in in vitro situations in
combination tooth Sistine the repression and actual reversal
of tumor growth and formation is enhanced.
It is well known that L-carnitine is normally synthesized
in the human liver and kidney from Lawson and methionine; that
it is supplied to the heart, skeletal muscle and other tissues
via the blood stream; that its prior y fur~tion is to
facilitate mitochondrial fatty acid oxidation (Fritz, I
Fishily. Revs. 41, 52 (1961)). It is also Newton that the
fatty acid oxidation stunula-tion by carnitine is accomplished
through the joint action of the enzymes carnitine transfers
I, the carnitine translocator, and carnitine transfers II
because azalea Coy cannot pass across the inner mitochondrial
rn~nb.rane (Brerner, I., Trends in Biochemical Science, 2 207,
1977)).
Although the following theoretical explanation is not to
be considered binding, it was postulated that since tumor
cells require a high rate of glycolysis for their growth, and
that since carnitine starlights fatty acid oxidation, the
administration of high levels of carnitine might force tumor
cells to utilize fatty acids as an energy source which might
prevent glycolysis and have harmful effects on such cells. It
appeared that carnitine-stirnula-ted fatty acid metabolism might
be deleterious in possibly three ways.
1. If there is lowered ability to oxidize fatty acids
to COY and OWE, but normal earnitine trouncers
activity is present, increased activation of fatty
acids Woody lead to the accumulation of long chain
azalea carnit.ine and CQ~ esters of fatty acids (Chicano
to occur in ischemia - see shag, Lo et at, Arch.
Become. ifs. 187, 25, 1978). Such products
2~819
!
., .
have been shown to inhibit a number of mitochondrial
systems and could lyre mitoehondrial and cell
m~branes because of their powerful detergent -
action. (Snug, AWL., Texas Reports on Boo. and Med.
39f 409 tl979); Cell G et at, J. Blot. Chum.
256. 12740 (1981)~.
2. Since activation of fatty acids utilizes AT and
produces IMP + Pi Swahili Coax earnitine stimulated
fatty acid activation in tumor cells, where oxide-
lion was sled, old lead to lyrical AT levels,
diminished Autopsy formation of ASP + Pi, and less
glycolysis.
3. Carnitine-stim~la~ed fatty acid oxidation can
benefit normal cells (Snug, AWL. et at, Academic
Press, NY, NY, Carnitine Biosynthesis, Metabolism
and Functions, pp. 321-340 (1980)) by stimulating
their energy producing metabolism and production of
more AT, thereby making more energy available for
the synthesis of anti-tumor substances, e.g. inter-
freon. mix coupled with the known increased blood
flaw to timorous tissue and the increased uptake of
substrates characteristic of tumor cells, earnitine
when ac~ninistered in high dosage would, in effect,
tend to Salk out and destroy tumor cells.
Detailed Description of the Invention
Example 1
In the follc~ing Examples the I~earnitine was
achninistered as tile free base (inner salt). The D-carnitine
was also ac~ninistered as the free base.
Female Sprac~le-Dawley rats which spontaneously develop a
benign ~el1-encapsulated menu tumor were utilized (the
tumors were considered to be classic fibroadenomas composed of
whorls and bundles of dense co~lacJen). The Sprague-Dawley mammary
tumor is a standard model for human breast cancer. Six animals with
tumors of various sizes were selected for each group to be
~22~8~g
subjected to treatment. ~11 animals were maintained on a high
fat diet.
One group of six animals was injected imp. with 3 gm/kilo
of L-c~rnitine twice per day. A second set ox six animals
received a like dose of D-carnitine. third set of six
animals received 3 gmlkilo of L-carnitine and 1 gm~kilo of
Sistine twice per day. The injections in each case were
continued for 25 days with the tumor size being evaluated on
days 8, 15, 19, 22 and 25. The animals Tory observed for an
additional 17 days and the tumors evaluated or size on the
Thea and ~3rd days.
Results are presented in the Table below.
TABLE 1
Average Tumor Size: (inches)
; L,carnitLne
Day Control L,carnitine L,cysteine
1 1.5 1.65 1.0
8 1.42 1.45 0.96
1.42 1.45 0.96
19 1.54 1.3 0.79
22 1.54 1.3 0.79
1.66 1.4 0.75
Average tumor change:
0.16 (0.25) (0.25)
Percent of change:
+10.66% -15.15% -25.0%
Treatment discontinued day 25:
36 2.45 1.4 0.75
43 2.5 1.5 0.79
Tutor change day 1-43
1.0 (0.15) (0.21)
Percent of change:
+G6.6~, -9.09Q -21.0%
i~;288~9
It is evident from the above data that carnitine when
administered alone or in combination with Sistine was
effective to reduce Thor size.
It is also to be noted that at an injection rate (imp.)
of 3 gms./kil,o of body weight, plasma levels of L-carni-tine of
greater than 15~M were observed. Since the half life of
carnitine in plasma is only about 30 minutes, it is apparent
that concentrations of carnitine of loom, as shown in the
following Examples can be of clinical importance anti
particularly since in Example 1 two injections of carn;tine at
3 gm~s/kilo of body weigh-t were given daily to the rats over a
long period of time with no observable harmful effects.
3L22881g
.
sample 2
Female Sprague-Da~ley rats with -the mammary terror as in
Example 1 were injected respectively with L,carnitine and
D-carnitine. Roy control rat received injections of an
isotonic saline solution ~0.15 molar). The It and D-carnitine
were injected imp. in the amounts specified in Example 1 the
D-carnitine was injected in the skim amount as the
L-carnitine). The injections were continued on a daily basis
for 28 days. Misorient of the tumors was made just prior to
the beginning of the injection regimen and at the end of the
28-day time period with the results shown in Table 2.
TABLE 2
Tumor Size
Ray Original 28-Day
Control 3 x 2.5 in. 8.5 x 6.07 in.
L~carnitine injected 1.5 x 1.0 in. 3.00 in.
2.2 x 2.5 in.
D-carnitine injected 4.0 x 4.0 in. 6.57 in.
Visual inspection of the dissected fibroadenomas from the
rats having received I.-carnitine revealed that the tumors were
very soft, brownish in color and without the lobular growth
pattern always observed in the dissected fibroadenomas from
the control rats. In addition the fibroadenomas from the
LrcarTIitine-treated rats skewed exceptionally high levels of
both free and esterified carnitine. Moreover, pathological
examination of the tumors from such treated rats showed that
in three out of four rats the tenor tissue was dead.
J~xa~
ale effects of various concentrations of It and D-carni-
tine were evaluated in their effects upon the proliferation oEVAC~ 5 human bowel cancer cell line in tissue culture in
accordance with standard procedures (Symposium - (Ixok)
Progress in Clinical and Biological Research, Vol. 48,
"Cloning of Tlurnan Tumor Stern Cells", edited by S. E. Sallnon,
Publisher - Arthur R. Loss Inc., Now York, pp. 332-338). The
~8~3~L9 Jo
L- and D-carni-tine was added to the growth Madeline to achieve
the concentrations indicated in 'fable 3. Results obtained are
Schick in Table 3.
TIE 3
Treatment mount of Growth* Effect
_
None (control) 95
L-carnitine 5mM 26 73% inhibition
lam I 58
O.lr~l 52 45%
loD-carnitine firm 92 No inhibition
lr~l 95 No inhibition
O.lmM 113 19% stimulation
*Growth is measured by the nunnery of
colonies which appear.
It has also been found that in in vitro studies with
tumor cells, the presence of long chain fatty acids, e.g.
palmitic, rustic oleic, Starkey, in the Greek medium
appears to enhance the inhibitory effect of L-carnitine on the
proliferation of tumor cells as is Chicano in the following
Examples.
Example 4
A single cell suspension of Scholl cells, a colon tumor
cell line, was prepared; the varying concentrators of both D-
and L-carnitine specified in Table 4 were added to portions of
this suspension. Lowe respective portions were then putted inn
soft agrees ion welt. trays and inc~lba-tecl to l~mlit clarity of
the sightless. 'Ire wells were Isolated for each treat~llent. The
results were tallied my continue tulle colonies grown in each
well and averayirly the three replicates. The results are
Chicano byway
lZ~38~9
TABLE 4
-
Grc~th of Control
Ave. Grc~th (I Colonies in Treatment)
Treatment (# of Colonies) Colonies in Control)
1. Control 152 -
2,10 no ~-carnitine294 183%
3.1 nil D-carnitine231 143~
4.O.lmM D-carnitine245 152%
5.10 L-carnitine 97 60%
6.1 my L-carnitine 190 118%
7. 0.1 no L-carnitine 182 113%
8. Control 170 - -
Average Growth represents the average of the actual
number of colonies counted in the three wells of each
treatment. % Growth of control represents how such growth
took place in the carnitine treated trials in relation to the
control trowels. 'this was calculated by dividing the overage
Grcx~th of each treatment by 161 (the average growth of the two
controls).
It is apparent -from the foregoing data that D-carnitine
appears to stimulate growth at all concentrations shcx~7n while
L-carnitine exhibits a concentration dependent decrease in
kenning efficiency with increasing amount of L-carnitine.
Example 5
A suspension of SKYE, cellulose was prepared as in Example 4.
The varying concentrations of It and D-carnit;.ne cud pal.mitie
acid and combinations of L- old D-carnitine with palmitic acid
specified in Table 5 were added to portions of the cell sup-
pension card plated in soft agrees in well trays in accord-
ante with the procedure in Example 4. The results obtained,
expressed as % inhibition of growth over the average of the
control values Sheehan in Table 4, are set forth yin -the table
' ' below.
Jo i 88~9
additions to Cell % Inhibition
Suspension of Growth
1. L-carnitirle Norm 30
2. L-carnitine lo 25
3. I.-carnitine (O.lr~M) 10
4. L-carnitine (loom) + O.Olr~M Palmitic acid (plates lost
5. L-carnitine lam + 0.01 Mel Palmitic acid 65
6. L-carnitine Old O.Olrn~ Punish acid 45
7. L-carnitine (O.OlmM) + Oilier Palmitic acid 20
8. D-carnit;ne (Lowry) slight stymie-
lotion of
growth
9. D-carnitine (lr~l) O
10. D-carnitine (Oilier) O
- 11. D-carnitine (Lomb O.OlmM Palrnitic acid 10
12. D-carnitine in + O.Olm~ Palmitic acid 10
13. Palmi-tic acid (O.OlmM) 15
14. Palmitic acid (0.05m~) 65
It is evident from the foregoing data that L-carnitine
exhibits an anti-proliferative effect toward the SEIKO, colon
tumor cell line and that such effect is enhanced by
combination of the L-carnitine with palrnitic acid.
Utilizing the skim cell line as in Exar~?le 5, nude mice
were injected subdermally with the viable tumor cells (it is
well known in the art that such injection induces the growth
of skin lesions on the nude mice). Immediately altar the
subdural injection the control mice were injected with saline
solution while the test nice were injected with L-carrlitille in
an anoint equal to 3 Scholl of booty White.
It Wakeless observed tl-lLIt on those nice reccivirl~ the saline
injection the expected slain lcsiolls row in the neural fashion
whereas those injected with l,-carnitine habited no skin
lessons.
In general, amounts of carnitine frock 1 to about 5 trams
par kilo of a n~nal'x weiclht can be awful used for the
Sue
purpose of the present invention and where Sistine is used in
conjunction, the amount of eysteine can range from about 0.5
to about 1.5 grams per kilo of a mammal's weight. When a
fatty acid is used in conjunction with L-carnitine amounts can
be used which will provide concentration of from Abbott 0.4~1
to about 1.2 my in the blood of the animal. mounts of fatty
acid in excess of that which will provide concentrations in
excess of 1.2 my should be avoided because of the potential
toxic effects which may be associated with such greater
lo concentrations.
