Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ANTI-TUMOR ACTIVITY OF VITAMIN E, CHOLESTEROL, TAXOL AND
BETULINIC ACID DERIVATIVES
DESCRIPTION
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention generally relates to the use of derivatives of Vitamin E
(tocopherol
and tocotrienol), cholesterol, taxol and betulinic acid as antitumor agents
for the treatment of and
prevention of cancers of the liver, lung, colon, prostate and breast as well
as melanomas and
leukemias.
Background Description
The antitumor effects of cx-tocopheryl hemisuccinate, free acid (TS) have been
reported in
numerous tumor cell types (1-S). Although it was previously believed that the
effects of this
compound were due to the enzymatic release of oc-tocopherol (Vitamin E) by
esterases in the cell,
recent studies have shown that the antitumor effects of TS are instead due to
the intact molecule
(4, 6). In support of the intact TS molecule as the active cytotoxic agent,
the non-hydrolyzable
ether derivative of TS, tocopheryoxybutyric acid (TSE) has also been shown to
possess antitumor
activity against leukemia (6) and breast cancer cells (10). Further, it has
been shown that the
antitumor effects of TS are not exhibited by unesterified oc-tocopherol (7). A
related compound,
cholesteryl hemisuccinate (CS) appears to have antitumor properties similar to
those observed for
TS (6, 8 and 9). However, the antitumor effects of CS and its ether derivative
cholesteryloxybutvric acid (CSE) have been tested in only marine leukemia
cells (6) and breast
cancer cells (10).
Though the administration of TS, TSE, CS and CSE have been shown to be
cytotoxic for
several tumor cell types such as leukemias and breast (hormone-dependent), the
activity of related
compounds in other tumor cell types is unknown and cannot be predicted from
previous studies.
For example, TS, TSE, CS or CSE treatments do not appear to adversely affect
some cell types
such as marine-derived bone marrow cells (6) and numerous other normal cell
types (2,3,13).
It is thus of interest to 1 ) identify and develop new derivatives of Vitamin
E, cholesterol,
taxol and betulinic acid which may be effective anti-tumor agents, and 2)
determine the range of
cancer types that can be treated with these derivatives.
SUMMARY
It is an object of this invention to provide a method of use of derivatives of
Vitamin E
(tocopherol and tocotrienol), cholesterol, taxol and betulinic acid as anti-
tumor agents. In
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particular, it is an object of this invention to provide a method of use as
anti-tumor agents for the
following derivatives: d-cc-Tocopheryl succinate tris salt. d-oc-Tocopheryl
methyl succinate, d-oc-
Tocopheryloxybutyric acid, d-8-Tocopheryl succinate, d-'y-Tocopheryl succinate
tris salt. d-oc-
Tocopheryl 2,2-dimethyl glutarate, d-cx-Tocopheryl 2,2-dimethyl succinate, d-
Tocopheryl y-
dimethyl aminobutvrate methiodide, d-oc-Tocopheryl 'y-dimethyl aminobutyrate,
d-cc-Tocopheryl
succinate polyethylene glycol 1000, tocotrienol rich fraction succinate ester,
tocotrienol rich
fraction succinate tris salt, d-cx-Tocopheryloxybutyric acid polyethylene
glycol 1000, 'y-
Tocotrienol succinate, cholesteryl succinate Iris salt, cholesteryl succinate
polyethylene glycol
1000, 'y-cholesteryloxybutyric acid tris salt, cholesterol hydrogen phthalate,
taxol monosuccinate
tris salt, taxol disuccinate tris salt, and betulinic acid succinate. The
derivatives of the present
invention may be used alone or in combination with one another, or with other
antitumor agents.
It is an object of the present invention to provide a method of use of the PEG
esters of
vitamin E, cholesterol, taxol and betulinic acid as antitumor agents.
In addition, it is an object of the present invention to provide a method of
use of the tris
salts of derivatives of tocopherol, cholesterol, taxol and betulinic acid as
antitumor agents.
Further, it is an object of the present invention to provide five new
compounds which may
be utilized as antitumor agents in the practice of the methods of the present
invention: d-«-
tocopheryl 2,2-dimethylglutarate; d-cc--tocopheryl 2.2 dimethyglutarate tris
salt; d-cx-tocopheryl
(3-(N,N-dimethylamino)ethyl ether (free base); d-cx-tocopheryl ~3-(N,N-
dimethylamino)ethyl ether
oxalate salt; and d-tx-tocopheryl ~3-N,N-(dimethylamino)ethyl ether
methiodide.
ABBREVIATIONS
a-TS d-oc-Tocopheryl succinate
oc-TS-T d-cx-Tocopheryl succinate tris salt
cx-T-MS d-oc-Tocopheryl methyl succinate
TSE d-oc-Tocopheryloxybutyric acid
TSE-T d-oc-Tocopheryloxybutyric acid tris salt
8-TS d-b-Tocopheryi succinate
y-TS-T d-'y-Tocopheryl succinate tris salt
2,2-Dim-TG d-cx-Tocopheryl 2,2-dimethyl glutarate
2,2-Dim-TS d-oc-Tocopheryl 2,2-dimethyl succinate
T-DMAB-Q d-Tocopheryl 'y-dimethyl aminobutyrate methiodide
T-DMAB-T d-cc-Tocopheryl y-dimethyl aminobutyrate
T-DMAE ether d-a-Tocopheryl-(3-N,N-dimethylamino) ethyl
ether
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8-T d-8-Tocopherol
y-T d-'y-Tocopherol
T-P-Na, dl-cx-Tocopheryl phosphate disodium salt
TS-PEG d-oc-Tocopheryl succinate polyethylene glycol
1000
TSE-PEG d-oc-Tocopheryloxybutyric acid polyethylene
glycol 1000
PEG 1000 polyethylene glycol 1000
TRF Tocotrienol rich fraction
TRF-S Tocotrienol rich fraction succinate ester
TRF-S-T Tocotrienol rich fraction succinate tris salt
Rice-B-oil Rice bran oil tocol concentrate
Palm oil Palm oil tocol concentrate
'y-T3 y-Tocotrienol
'y-T3-S 'y-Tocotrienol succinate
y-T3-S-tris Y-Tocotrienol succinate tris salt
'y-T3 acetate Y-Tocotrienol acetate
CS-tris Cholesteryl succinate tris salt
CS-PEG Cholesteryl succinate polyethylene glycol
1000
y-CSE-tris y-cholesteryloxybutvric acid tris salt
Choles-h-p Cholesteryl hydrogen phthalate
Btl-acid Betulinic acid
Btl-acid succinateBetulinic acid succinate
Taxol-S-tris Taxol monosuccinate tris salt
Taxol-DS-tris Taxol disuccinate tris salt
DETAILED DESCRIPTION OF THE DRAWINGS
Figure lA-1F. Effect of indicated compounds on the growth of Hep3B liver
cells. Fig.lA: b-TS
(~) and CcTS (o); Fig.lB: TSE; Fig.lC: 'y-TS-tris; Fig.lD: cx-T-MS (~) and TS
(o); Fig.lE:
T-DMAB-Q; Fig.lF: b-T.
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Figure 2A-2F. Effect of indicated compounds on the growth of Hep3B liver
cells. Fig.2A: y-
T; Fig.2B: PEG 1000; Fig.2C: T-P-Na,; Fig.2D: TS-PEG; Fig.2E: TSE-PEG: Fig.2F:
TRF.
Figure 3A-3F. Effect of indicated compounds on the growth of Hep3B liver
cells. Fig.3A:
TRF-S-T; Fig.3B: TRF-S; Fig.3C: Rice bran oil; Fig.3D: Palm oil; Fig.3E: y-T3;
Fig.3F: y-
T3 succinate.
Figure 4A-4F. Effect of indicated compounds on the growth of Hep3B liver
cells. Fig.4A: y-
T3 acetate: Fig.4B: CS-tris (~) and CSE-Iris (o); Fig.4C: CS-PEG; Fig.4D:
Choles-h-p;
Fig.4E: Btl-acid succinate; Fig.4F: Btl-acid.
Figure 5A-SF. Effect of indicated compounds on the growth of Hep3B liver
cells. Fig.SA:
TS-tris; Fig.SB: TSE-tris; Fig.SC: CSE-tris; Fig.SD: 2,2-Dim-TS; Fig.SE: T-
DMAB-Q;
Fig.SF: T-DMAB-T.
Figure 6. Effect of DMAB-ether on the growth of Hep3B liver cells.
Figure 7A-7F. Effect of indicated compounds on the growth of Du145 prostate
cancer cells.
Fig.7A: aT-MS (o) and aTS (~); Fig.7B: TSE; Fig.7C: T-DMAB-Q; Fig.7D: PEG
1000;
Fig.7E: TS-PEG; Fig.7F: TSE-PEG.
Figure 8A-8F. Effect of indicated compounds on the growth of Du145 prostate
cancer cells.
Fig.BA: TRF; Fig.BB: TRF-S; Fig.BC: y-T3; Fig.BD: y-T3-S; Fig.BE: CS-tris;
Fig.BF: CS-
PEG.
Figure 9A-9D. Effect of indicated compounds on the growth of Du145 prostate
cancer cells.
Fig.9A: TS-tris; Fig.9B: 2,2-Dim-TS; Fig.9C: T-DMAB-T; Fig.9D: choles-h-p.
Figure l0A-lOF. Effect of indicated compounds on the growth of NIH-H69 small
cell lung
cancer cells. Fig.lOA: cc-T-MS; Fig.lOB: y-TS-tris; Fig.lOC: 8-TS; Fig.IOD:
ccTS; Fig.lOE:
TSE; Fig.lOF: PEG 1000.
Figure 11A-11F. Effect of indicated compounds on the growth of NIH-H69 small
cell lung
cancer cells. Fig.llA: 8-T; Fig.llB: T-P-Na,_; Fig.llC: y-T; Fig.llD: T-DMAB-
Q;
Fig.llE: TSE-PEG; Fig.llF: TS-PEG.
Figure 12A-12E. Effect of indicated compounds on the growth of NIH-H69 small
cell lung
cancer cells. Fig.l2A: TRF-S-T: Fig.l2B: TRF-S; Fig.l2C: TRF; Fig.l2D: Rice
bran oil;
Fig.l2E: Palm oil.
Figure 13A-13F. Effect of indicated compounds on the growth of NIH-H69 small
cell lung
cancer cells. Fig.l3A: y-T3; Fig.l3B: y-T3-S; Fig.l3C: y-T3 acetate; Fig.l3D:
CSE-tris;
Fig.l3E: CS-PEG; Fig.l3F: CS-tris.
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Figure 14A-14E. Effect of indicated compounds on the growth of NIH-H69 small
cell lung
cancer cells. Fig.l4A: TS-tris; Fig.l4B: TSE; Fig.l4C: T-DMAB-T; Fig.l4D:
choles-h-p;
Fig.l4E: 2,2-Dim-TS.
Figure 15A-15F. Effect of indicated compounds on the growth of HT29 colon
cancer cells.
Fig.lSA: a-T-MS; Fig.lSB: TS; Fig.lSC: 2,2-Dim-TG; Fig.lSD: TSE; Fig.lSE: PEG
1000;
Fig.lSF: TS-PEG.
Figure 16A-16E. Effect of indicated compounds on the growth of HT29 colon
cancer cells.
Fig.l6A:TRF (~) and TRF-S (o); Fig.l6B: y-T3 (~) and y-T3-S (o); Fig.l6C: CSE-
PEG;
Fig.l6D: CS-PEG; Fig.l6E: CS-tris.
Figure 17A-17F. Effect of indicated compounds on the growth of HT29 colon
cancer cells.
Fig.l7A: TS-tris; Fig.l7B: TSE-tris; Fig.l7C: CSE-tris; Fig.l7D: T-DMAB-Q
Fig.l7E:
T-DMAB-T; Fig.l7F: choles-h-p.
Figure 18. Effect of indicated compounds on the growth of HT29 colon cancer
cells. Fig.
18A: TSE-PEG; Fig. 18B: 2,2-Dim-TS.
Figure 19A-19E. Effect of indicated compounds on the growth of MCF-7 breast
cancer cells.
Fig.l9A: PEG-1000; Fig.l9B: cx-T-MS (~) and TS (o); Fig.l9C: TSE; Fig.l9D:
T12F-S (~)
and TRF (o); Fig.l9E: TSE-PEG (~) and TS-PEG (o).
Figure 20A-20C. Effect of indicated compounds on the growth of MCF-7 breast
cancer
cells. Fig.20A: CS-tris; Fig.20B:CS-PEG; Fig.20C: y-T3-S (~) and y-T3 (o).
Figure 21A-21D. Effect of indicated compounds on the growth of MDA-231 breast
cancer
cells. Fig.2lA: TRF-tris; Fig.2lB: TRF; Fig.2lC: TS-tris; Fig.2lD: TS.
Figure 22A-2D. Effect of indicated compounds on the growth of MDA-231 breast
cancer
cells. Fig.22A: TSE; Fig.22B: TS-PEG; Fig.22C: 2.2-Dim-TS; Fig.22D: T-DMAB-T.
Figure 23A-23D. Effect of indicated compounds on the growth of HL-60 leukemia
cancer
cells. Fig.23A: y-T3 (~), y-T3-S (o), y-T3-S-tris (~) and y-T3-acetate (D);
Fig.23B: TRF
(~), T1ZF-S (o) and TRF'-S-T (1); Fig.23C: Choles-h-p; Fig.23D: T-DMAB-T.
Figure 24A-24D. Effect of indicated compounds on the growth of A375 cutaneous
melanoma
cancer cells. Fig.24A: cx-T-MS (~) and TS (o); Fig.24B: TSE; Fig.24C: TRF' (~)
and TRF-
S (o); Fig.24D: TS-PEG (~) and TSE-PEG (o).
Figure 25A-25C. Effect of indicated compounds on the growth of A375 cutaneous
melanoma
cancer cells. Fig.25A: CS-PEG; Fig.25B:CS-tris; Fig.25C: y-T3-S (~) and y-T3
(o).
Figure 26A-26D. Effect of indicated compounds on the growth of A375 cutaneous
melanoma
cancer cells. Fig.26A: TS-tris; Fig.26B: TSE-tris; Fig.26C: 2,2-Dim-TS;
Fig.26D: T-DMAB-
T.
-S-
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Figure 27A-27E. Effect of indicated compounds on the growth of OCMI ocular
melanoma
cancer cells. Fig.27A: cx-T-MS (o) and TS (1); Fig.27B: TSE; Fig.27C: 2,2-Dim-
TG;
Fig.27D: TS-PEG; Fig.27E: TSE-PEG.
Figure 28A-28F. Effect of indicated compounds on the growth of OCM 1 ocular
melanoma
cancer cells. Fig.28A: T-DMAB-Q; Fig.28B: PEG 1000; Fig.28C: 'y-T3-S; Fig.28D:
'y-T3;
Fig.28E: TRF; Fig.28F: TRF-S.
Figure 29A-29F. Effect of indicated compounds on the growth of OCMI ocular
melanoma
cancer cells. Fig.29A: 2,2-Dim-TS; Fig.29B: Btl-acid (~) and Btl-acid
succinate (o);
Fig.29C: T-DMAB-T; Fig.29D: CS-tris; Fig.29E: CS-PEG; Fig.29F: Choles-h-p.
Figure 30A-30D. Effect of indicated compounds on the growth of OCMI ocular
melanoma
cancer cells. Fig.30A: TS-tris; Fig.30B: TSE-tris; Fig.30C: T-DMAB-T; Fig.30D:
T-DMAB-Q.
Figure 31A-31C. Effect of indicated compounds on the growth of OCM1 ocular
melanoma
cancer cells. Fig.3lA: CSE-tris; Fig.3lB: choles-h-p; Fig.31C:2,2-Dim-TS.
Figure 32A-32D. Effect of indicated compounds on the growth of OCM 1 ocular
melanoma
cancer cells. Fig.32A: TS-tris suspension (~) and TS-tris pellet (o); Fig.32B:
Taxol (~),
Taxol 50xTS-tris (o) and Taxol 50xTS-tris pellet (~); Fig.32C: Taxol-S (~),
Taxol-S with
50xTS-tris (o) and Taxol-S with 50xTS-tris pellet (1); Fig.32D: Taxol-DS (~),
Taxol-DS
50xTS-tris (o), and Taxol-DS 50xTS-tris pellet (~).
Figure 33A-33E. Compounds representing new compositions of matter. 33A: 2,2-
Dim-TG;
33B: 2,2-Dim-TG-tris; 33C: T-DMAE-ether; 33D: T-DMAE-ether oxalate; 33E: T-
DMAE-
Q-ether methiodide.
DETAILED DESCRIPTION OF
PREFERRED EMBODIMENTS OF THE INVENTION
The current studies were undertaken in order to investigate the anti-tumor
potential of
several derivatives of Vitamin E, cholesterol, taxol and betulinic acid. In
the Examples which
follow, we demonstrate that the compounds of the present invention possess
potent anti-tumor
activity when tested against a wide variety of human cancer types. The
compounds were
tested against cancerous cell lines derived from liver, lung, colon, prostate,
breast. cutaneous
melanoma, ocular melanoma and leukemia. Though many compounds display similar
antitumor activity in most of the human tumor cell lines tested, there are
several exceptions.
For instance, most of the derivatives tested were less potent in killing
hormone-
dependent breast tumor cell line MCF-7 as compared with other tumor cell
types. In addition,
the methyl derivatives of TS (cxT-MS and 2,2-Dim-TS) were especially active
cvtotoxic
agents in the human lung tumor cell line, NIH-H-69. Furthermore, in contrast
to most tumor
cell types, human leukemia cells (HL-60) seemed insensitive to the toxic
effects of tocopherol
ester amines (e.g. T-TDAB-T). Interestingly, the Tris salts of TSE appeared
dramatically more
potent than TSE (free acid) with respect to cytotoxic action against tumor
cells derived from
the colon and liver. In contrast, the tris salt of TSE did not improve the
antitumor activity of
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TSE towards human melanoma cells. The succinate ester of betulinic acid was a
potent
antitumor agent for cutaneous melanomas but not for other tumor cell types.
Our findings
suggest that the active antitumor agents studied in these experiments are
probably active
against most tumor cell lines. Overall. the tocopherol amine containing
compounds (e.g. T-
TDAB-T, T-TDAB-Q, and T-TDAE-ether), the PEG ester compounds. the taxol
succinate
Tris compounds and choles-h-p were the most potent compounds in terms of
cytotoxic
abilities toward human tumor cell lines derived from liver, lung, colon,
prostate. breast,
cutaneous melanoma, ocular melanoma and leukemia. However. there is also some
basis for
selectivity in that certain compounds may prove advantageous with respect to
the treatment of
certain types of cancer. For each tumor cell line, specific examples of
compounds that are the
most effective in inhibiting specific tumor cell growth are summarized in each
Example.
It is of interest to note that by replacing the succinic acid (a 4 carbon
straight chain
dicarboxylic acid) group on CS with phthalic acid (a 1,2 benzene dicarboxylic
acid) the
resulting compound, cholesterol hydrogen phthalate, is a more potent cytotoxic
agent against
all of the tumor types tested. Thus, we conclude that the attachment of a ring
structure
(benzene) that contains free carboxylic acids to the cholesterol molecule
results in a compound
that has a free carboxylic acid and has potent antitumor activity. Based on
our previous studies
with CS and its ether derivative CSE (which shows antitumor activity identical
to CS), we
predict that the attachment of a monocarboxylic acid benzene molecule to
cholesterol through
an ether linkage would also result in a cholesterol phthalate ether compound
that would have
potent antitumor activity, as well as be non-hydrolyzable, which is important
for oral
absorption. It is also predicted from our antitumor data with TS and TSE (the
ether form of
TS), which showed that these compounds have antitumor activity similar to CS
and CSE, that
phthalate esters and ethers of tocopherols, tocotrienols, taxols, and
betulinic acids will also
have excellent antitumor activity. Similarly, since our antitumor data with
tocopherol amines
(e.g. T-DMAB-T and T-DMAB-Q) show that when the carboxylic acid on the 4
carbon side
chain of TS is replaced with a tertiary or quaternary amine, an improvement in
terms of
antitumor activity is observed. These data suggest that the replacement of the
carboxylic acid
on phthalate esters and ethers with an amine will also improve the antitumor
activity of these
compounds.
Vitamin E is a generic term that includes, in nature, eight substances. d-cx-,
d-~3-, d-'y-,
d-8-tocopherol and d-cx-. d-~i-, d-'y-, d-8-tocotrienol. Succinate esters for
each of these
substances can be made. We investigated whether the combination of several of
these
tocopherol and tocotrienol succinate esters would have antitumor activity. To
accomplish this,
a succinate ester of TRF was prepared (TRF-S) in the form of both a free acid
and a tris salt.
TRF-S is a mixture of vitamin E compounds containing the succinate ester of d-
oc- tocopherol
(28%) and d-oc- (35%), d-'y-(22%), and d-b-tocotrienol (16%). The presence of
the succinate
ester of each of these vitamin E derivatives in the TRF-S preparation was
confirmed by HPLC
analysis using base hydrolysis. Our findings as shown in the examples below
indicate that a
mixture of tocopherol and tocotrienol esters (as, for example, TRF-S-tris or
TRF-S free acid)
are potent antitumor agents.
Implementation of the claimed invention will generally involve identifying
patients
suffering from tumors and administering the compounds in an acceptable form by
an
appropriate route. The dosage to be administered may vary, depending on the
age, gender,
weight and overall health status of the individual patient, as well as the
nature of the cancer
itself. The exact dosage will thus be determined on a case by case basis by
the attending
physician or other appropriate professional, but will generally be in the
range of 1 to 100
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mgikg of body weight. Alternatively. the compound may be administered
prophylactically to
patients identified as being at risk for the development of cancer, either
due. for example, to a
genetic predisposition or as a result of exposure to a carcinogenic substance.
In a preferred embodiment of the invention. the compounds may be administered
by
injection either intravenously or parenterally. Injectable preparations. for
example, sterile
injectable aqueous or oleaginous suspensions may be formulated according to
the known art
using suitable dispensing or wetting agents and suspending agents. The sterile
injectable
preparation can also be a sterile injectable solution or suspension in a
nontoxic parenterally
acceptable diluent or solvent. for example, as a solution in 1,3-butanediol.
Alternatively, the compounds may be administered by inhalation of an aerosol.
This
method has the advantage of delivering the antitumor compound directly to the
lungs where it
could, for example. provide protection against carcinogens such as those found
in cigarette
smoke and atmospheric pollutants, or effectively kill cancer cells located at
this site. Those
skilled in the art will recognize that a variety of inhalers appropriate for
the practice of the
invention are available. including those with various dose metering chambers,
various plastic
actuators and mouthpieces, and various aerosol holding chambers (e.g. spacer
and reservoir
devices) so that an appropriate dose of the Vitamin E compound can be
delivered. Also,
several non-ozone depleting (non-chlorofluorocarbon) propellants, such as
various
hydrofluoroalkanes (e.g. HFA 134a and HFA 227) are available. For
administration via
inhalation, the dose may be less than for other methods and will vary
according to the exact
delivery technology that is employed.
Administration may also be achieved transdermally using a patch impregnated
with the
compound, by ocular administration (eye drops), sublingual administration,
nasal spray
administration and rectal administration (suppository).
Administration may also be oral. In the case of oral administration,
absorption of those
compounds which are susceptible to inactivation by digestive enzymes may be
accomplished
by coating the compounds with an impermeable polymer membrane that is not
susceptible to
the action of digestive enzymes (duodenal esterases) or is biodegraded very
slowly. In
addition, amino acid polymers such as polylysine could be used. Impermeable
polymer films
would be degraded by microflora found in the colon. Thus, the compound would
be released
in a part of the intestine devoid of secreted digestive enzymes. However, it
will be readily
understood by those of skill in the art that other methods for preventing the
hydrolysis of the
compounds and promoting their absorption following oral administration can
also be used in
the practice of the present invention. For oral administration, the compounds
may be
administered in any of several forms, including tablets. pills, powders,
lozenges, sachets,
elixirs, suspensions, emulsions, solutions, syrups, aerosol, soft or hard
gelatin capsules, or
sterile packaged powders.
The compounds administered in according to the methods of the present
invention may
be administered as a composition which also includes a pharmaceutically
acceptable carrier.
The compounds may be mixed with a carrier, or diluted by a carrier, or
enclosed within a
carrier. When the carrier is a diluent, it may be a solid, semisolid or liquid
material which acts
as a vehicle, excipient or medium for the compound. Some examples of suitable
carriers,
excipients and diluents include lactose, dextrose, sucrose. sorbitol,
mannitol, starches, gum
acacia, calcium phosphates, alginate, tragacanth, gelatin. calcium silicate,
microcrystalline
cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose,
methyl and
propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The
formulations can also
include lubricating agents, wetting agents, emulsifying agents, preservatives,
and sweetening
_g_
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or flavoring agents.
The compounds can be administered in the pure form or in a pharmaceutically
acceptable formulation including suitable elixirs, binders, and the like or as
pharmaceutically
acceptable salts or other derivatives. It should be understood that the
pharmaceutically
acceptable formulations and salts include liquid and solid materials
conventionally utilized to
prepare injectable dosage forms and solid dosage forms such as tablets and
capsules. Water
may be used for the preparation of injectable compositions which may also
include
conventional buffers and agents to render the injectable composition isotonic.
Solid diluents
and excipients include lactose, starch, coatings and the like. Preservatives
such as methyl
paraben or benzalkium chloride may also be used. Depending on the formulation.
it is
expected that the active composition will consist of 1-99% of the composition
and the
vehicular ''carner'' will constitute 1-99% of the composition.
In particular, one advantage of the present invention involves the oral
administration of
TSE, 2,2-Dim-TS, 2.2-Dim-TG and T-DMAE ether. Previous to the practice of the
present
invention, it was not possible to effectively administer known derivatives of
Vitamin E orally
due to the fact that they were enzymatically hydrolyzed by esterases in vivo.
In contrast, based
on their structure, the derivatives TSE, 2.2-Dim-TS, 2,2-Dim-TG and T-DMAE
ether are
non-hydrolyzable and thus high levels of bioavailability are maintained until
they are taken up
by cells (see Example 9).
One aspect of the present invention contemplates administering to a patient a
compound by the methods of the present invention in combination with
administering to the
patient an acceptable chemotherapeutic agent or combination of agents. As used
herein
"chemotherapeutic agent'' means any chemical agent or drug used in
chemotherapy treatment
which selectively affects tumor cells, including but not limited to such
agents as taxanes (e.g.
taxol), adriamycin, amscrine, etoposide, cisplatinum, vincristine, vinblastine
and methotrexate.
Other such agents are well-known in the art. It is anticipated that the
compounds of the
present invention may be administered either in combination with or separately
from the
chemotherapeutic agent(s). The dose will vary depending on a variety of
factors including the
route of administration, choice of chemotherapeutic agent, etc.
Another aspect of the present invention is to administer compounds by the
methods of
the present invention in combination with treating the patient by exposing the
patient to
ionizing radiation. The protocols for traditional radiation therapy, e.g. y-
radiation. are
known, readily available, and routinely practiced by those of skill in the
art. These include
established protocols for the administration of drugs in combination with
radiation therapy
[Wobst et al. (1998) Ann.Oncol. 9. 951-962]. The dose of ionizing radiation
will vary
depending on a variety of factors including intensity, source of radiation,
etc.
Another aspect of the present invention is the administration of compounds by
the
methods of the present invention in order to prevent metastasis of cancer. For
example, the
compounds may be administered following the surgical removal of a tumor. The
compound
may also be administered prophylactically to patients identified as being at
risk for the
development of cancer.
The compounds which are administered in the practice of the present invention
may be
administered singly (i.e. only one derivative compound administered at.a time)
or in
combination (i.e. more than one derivative compound administered at a time).
Numerous studies have shown that the compounds utilized in the practice of the
present invention appear to be safe when administered in vivo. In fact,
numerous studies
clearly demonstrate that the in vivo and in vitro administration of tris salts
and PEG esters of
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TS and other vitamin E and cholesterol derivatives protect tissue, cells and
subcellular
fractions from toxic insults (8, 9,1 l, 13, 14). Fariss et al. (6) have shown
that the growth of
normal bone marrow cells (cells in vivo that are usually sensitive to the
toxic effects of cancer
chemotherapeutic drugs) isolated from mice are not inhibited by treatment with
TS, CS, TSE
and CSE at concentrations up to 200~tM. Other investigators have also shown
that TS
administration does not adversely affect normal cells (2, 3, 13) in contrast
to its effect on
tumor cells. Furthermore, after the administration of the antitumor agents TS-
T. TS-PEG,
and TSE-T to rats at a single dose of 0.19 mmol/kg (approx. 100 mg/kg), ip,
the tissue
histopathology (liver. lung, heart, brain and kidney) was normal 24 hours
following
administration.
In another aspect, the present invention provides five novel compounds(see
Figure 33),
namely
1 ) d-cx-tocopheryl 2,2-dimethylglutarate
2) d-cx-tocopheryl 2,2 dimethyglutarate TRIS salt
3) d-oc-tocopheryl (3-(N,N-dimethylamino)ethyl ether (free base)
4) d-cx-tocopheryl ~3-(N,N-dimethylamino)ethyl ether oxalate salt, and
5) d-a-tocopheryl ~3-N,N-(dimethylamino)ethyl ether methiodide.
The compounds may be used in the practice of the present invention as
antitumor
agents. Details of the syntheses of the five new compounds are given in
Examples 10-14
below. Those of skill in the art will recognize that certain modifications of
these compounds
may be made in order to, for example, optimize bioavailability, solubility,
potency, and the
like, or for any other reason. All such modifications of these compounds and
the derivative
compounds which result from such modifications are intended to be encompassed
by the
present application.
The following examples illustrate the use of the derivatives of Vitamin E,
cholesterol,
taxol and betulinic acid as antitumor agents. The details of the examples
should not be
construed to unduly limit this invention.
EXAMPLES
Materials
The human cells lines used in the present investigation and their origins are
as follows:
Hep 3B (liver); NIH-H69 (lung); HT29 (colon); Du145 (prostate); MCF7 and MD-
231
(breast); A375 (cutaneous melanoma); OCM1 (ocular melanoma); HL60 (leukemia).
All cell
types except OCM1 are available from the American Type Culture Collection
(ATCC). OCM1
cells were obtained from Dr. June Kan-Mitchell at the U.of California, San
Diego.
The anti-tumor agents of the present invention, except as noted, were
synthesized by
Dr. J. Doyle Smith. Virginia Commonwealth Univ., Richmond, VA. The succinate
esters,
PEG esters, butyrate ethers and the tris salts were prepared by procedures
similar to those
described in U.S. Patent 5,610,180 to Fariss.
T-DMAB-T was prepared as follows: In a flask equipped with a magnetic stirrer
and
reflux condenser was placed 5~3mg (3.3mM) of'y-(dimethylamino)butyric acid and
2 ml (18.9
mM) of POC13. The mixture was stirred and heated at 93°C for 1 hour to
give a clear,
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colorless solution. The excess POC13 was removed under reduced pressure with
warming to
give an oil, which is the crude 'y-(dimethylamino)butyryl chloride. To this
was added 1.02g of
d-cx-tocopherol and 3 ml of CHC13. The mixture was stirred and warmed to
53°C for 4 days.
This mixture was cooled and then treated with 3g of ice, 6m1 of 5% Na,C03
solution, and 6m1
of CHC13 to form an emulsion. After cooling in the refrigerator overnight, the
layers separated
and the CHC13 was removed. The water layer was again extracted several times
with CHCl3
and the combined extracts were dried over MgSO~. This extract, which contained
the crude
product, was chromatographed using a column of silica gel and CHC13/methanol.
The desired
product was isolated as a pale, yellow liquid; 968 mg (75%). IR (thin film)
Absorption at 1757
cm'' shows the presence of an ester. TLC (CHCI~/methanol, 9:1 ) Rf = 0.54;
single spot.
T-DMAB-Q was prepared as follows: to a cooled vial containing 336mg (0.618mM)
of d-oc-tocopheryl ~-(dimethylamino) butyrate was added l.Sg (a large excess)
of methyl
iodide and 1 ml of CHCl3. The vial was stoppered and the reaction was allowed
to proceed at
room temperature for 24 hours. Reduced pressure was used to remove the
solvents and form
the crude product; 345mg (81%). After multiple recrystallizations using
acetone/ethyl acetate ,
tan crystals were obtained; 222mg (52%); mp 165-167°C. Anal
(C36H64N03I) CHN. TLC
(CHCl3/methanol, 9:1 ), Rf = 0.22, single spot.
The exceptions are as follows: cx-TS, PEG 1000. choles-h-p and CS-tris were
purchased from Sigma Chem. Co. and T-P-Na,_ was purchased from U.S.
Biochemical Co.; CG-
T, 'y-T and 8-T were gifts from Henkel Corp.; TS-PEG, Rice-B-oil fraction,
Palm oil fraction,
'y-T3, and 'y-T3 acetate were gifts from Dr. John Hyatt, Eastman Chemical Co.;
TRF was a
gift from Dr. Paul Sylvester (Washington State Univ.) and was originally
obtained from the
Palm Oil Research Institute of Malaysia; betulinic acid was a gift from Dr.
Joun Pezzuto,
University of Illinois. Chicago. Other antitumor compounds were synthesized by
Dr. J. Doyle
Smith, Virginia Commonwealth University, Richmond, VA. The succinate esters,
PEG esters,
butyrate ethers and the tris salts were prepared by procedures previously
described (US Patent
No. ).
All test compounds (except tris salts and betulinic acid) were dissolved in
100%
ethanol and administered to cells such that the final concentration of ethanol
in medium was
less the 0.2%. Tris salt derivatives were administered in distilled water,
following two 15
second sonications. Betulinic acid was administered in dimethylsulfoxide
(DMSO).
Method for anti-tumor compound screening
Alamar Blue Assay:
Alamar blue dye was used to evaluate cell survival and proliferation. Living
cells
metabolize the non-fluorescent dye to a fluorescent metabolite which can be
detected by a
fluorescence plate reader. There is a positive correlation between the level
of fluorescence and
the number of living cells. The fluorescence intensity of the cells treated
with a test compound
was compared to that of a control group which has no added test compound
(vehicle only).
The result was expressed as "Cell number (% control)". A reduction in the cell
number
indicates inhibition of cell growth. or an increase in cell death.
Procedure:
1 ) On day one, cells were plated at a density of 2.5 or 5 x 103 cells/well,
depending on the cell
type, in a 96-well flat-bottomed plate in RPMI 1640 with 10% fetal bovine
serum for the
following cell lines: HL-60, NIH-H69 and HT29; and in DMEM with 10% fetal
bovine serum
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serum for all other cell lines. On the second day, the medium was replaced
with 200~,L RPMI
1640 ( 10% fetal bovine serum) medium containing the desired concentration of
test
compound. The concentration of test compound used ranged from 0 to SO~,M.
200~,L of
medium without cells was plated as a blank.
2) Cells were maintained in a humidified atmosphere in 5% CO, at 37°C
for 42-70 hours,
depending on the cell type. 2-6 hours prior to the end of exposure to the test
compound, 10-
20~,L of Alamar blue stock solution was added to each well. After incubation
at 37°C for an
additional 2-6 hours, the plate was maintained at room temperature for 30
minutes. The exact
time of addition of Alamar blue, the precise amount of Alamar blue added, and
the length of
incubation with Alamar blue varied depending on the cell type and level of
activity of the
compound being tested. After 30 minutes at room temperature, the fluorescence
intensity of
each well was measured using a CytoFluor series 4000 multiplate reader
(excitation
wavelength, 530run; emission, 590nm; gain. 40). Fluroescent values from blank
cells were
subtracted from fluorescent values of cells treated with test compounds. The
resulting values
were then divided by the corresponding values obtained from the control
samples to give the
number of viable cells (% control). The ICS° values (concentration
required to inhibit cell
growth by ~0%) were extrapolated from the plot of cell number (%control)
versus compound
concentration. The cell number (% control) plotted was the mean t SD (n=6).
Animals
Male Sprague-Dawley rats from Simonsen Labs (Gilroy, CA) weighing 175-225 g
were used throughout the course of this study. Animals received water and food
(Purina Rat
Chow 5001, Ralston Purina, St. Louis, MO) ad libitum for at least three days
prior to the
onset of the experiment. Powdered TS-tris, TSE-tris and TS-PEG were suspended
in saline
with brief sonication (30 sec) and were given intraperitoneally at a dose of
0.19 mmol/kg body
weight. Saline was given to rats at a dose of 4 ml/kg. received CC14 by oral
lavage 6 or 18 h
after tocopherol administration.
After 24 hours, rats were anesthetized with diethyl ether, and blood samples
(4-5 ml)
were withdrawn from the inferior aorta. Blood samples were immediately mixed
with 1 ~ mg
tripotassium EDTA, and aliquots were centrifuged at low speed to prepare
plasma samples.
All procedures were approved by the Washington State University Animal Care
and Use
Committee.
Tocopherol determinations
Tocopherol and tocopherol ester levels were measured according to the methods
described by Fariss et al. ( 11 ). TSE levels were measured according to the
procedures of
Tirmenstein et al. (12). Samples were analyzed by reversed-phase high-
performance liquid
chromatography equipped with fluorimetric detection. Retention times for d-S-
tocopherol
(internal standard), a-T and TSE were 8.0, 11.5 and 13.4 min respectively.
Preparation of Taxol and TS-tris Suspensions and Liposomes:
TS-tris suspensions were prepared by adding 1 ml of water or saline to 30 mg
of
TS-tris in a microfuge tube and sonicating for 1 ~ sec., twice. For taxol-TS-
tris suspensions, 1
mg of taxol or taxol monosuccinate tris salt or taxol disuccinate tris salt)
was added to 30 mg
of TS-tris prior to the addition of water or saline and sonication. (Therefore
on a molar basis
there is 50 times more TS-tris than taxol). To prepare washed liposomal
suspensions, the
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suspensions mentioned above were spun at 10.000rpm for 15 min and the
resulting pellet
(liposomes) was washed with saline or water. This procedure (spinning and
washing) was
conducted 3 times and the final washed liposomal pellet was resuspended by
sonication in the
original volume of water or saline. The presence of liposomes in each
suspension was
confirmed by light microscopy.
EXAMPLE 1.
Testing of compounds in human liver tumor (Hep3B) cells
2.5 x 103 cells/well were plated in a 96-well flat bottom plate. On the next
day, the
medium was replaced with 200~.L RPMI 1640 ( 10% FBS) containing from 0 to ~O~M
of test
compound. Cells were maintained as described in Methods. After 70 hours, 10~L
of Alamar
blue stock solution was added to each well and the plates were incubated at
37°C for an
additional 2 hours. Plates were then kept at room temperature for 30 min.,
following which
fluorescence intensity was measured and the values obtained were processed as
described in
Methods. The results are given in FigurelA-F. Figure 2A-F, Figure 3A-F, Figure
4A-F, Figure
SA-F and figure 6, and Table 1 gives the corresponding ICS° values. As
can be seen, the
compounds that appear to be most effective in inhibiting the growth of human
Hep 3B cells
are T-DMAB-T, T-DMAB-Q, T-DMAE-ether, TS-PEG, TSE-PEG, TRF-S, TRF-S-T, 'y-T3,
Choles-h-p, and Btl-acid. Antitumor activity was also observed for all of the
compounds
tested except cc-T, y-T. T-P-Na,, PEG 1000, and 'y-T3 acetate.
Table 1. ICSO (~M) of Test Compounds in Human Liver Cancer (Hep 3B) Cells
Compound ICSO (~1M)
aTS 27
cxTS-T 22
aT-MS 28
TSE 50
TSE-T 35
8TS 37
yTS-T 47
2,2-Dim-TS 26
T-DMAB-T 6
T-DMAB-Q 19, 12
8T 45
~yT >50
T-P-Na, >50
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PEG 1000 >50
TS-PEG 8
TSE-PEG 16
TRF 26
TRF-S 17
TRF-S-T 16
Rice B-oil 76
Palm oil 87
yT3 25
yT3-S 45
yT3-acetate >50
C S-tri s 24
CS-PEG 31
CSE-tris 32
Choles-h-p 13
Btl-acid 15
Btl acid-S ~50
T-DMAE ether 14
EXAMPLE 2.
Testing of Compounds in Human Prostate Cancer (Dul ~t~) Cells
2.5 x 10' cells/well were plated in a 96-well flat bottom plate. On the next
day, the
medium was replaced with 200~.L RPMI 1640 (10% FBS) containing from 0 to SO~,M
of test
compound. Cells were maintained as described in Methods. After 70 hours, 10~,L
of Alamar
blue stock solution was added to each well and the plates were incubated at
37°C for an
additional 2 hours. Plates were then kept at room temperature for 30 min.,
following which
fluorescence intensity was measured and the values obtained were processed as
described in
Methods. The results are given in Figure 7A-F, Figure 8A-F, and Figure 9A-D,
and Table 2
gives the corresponding ICSO values. As can be seen, the compounds that appear
to be most
effective in inhibiting the growth of human prostate cancer (Du 145) cells are
T-DMAB-T, T-
DMAB-Q, Cc-TS-T, TS-PEG, TSE-PEG, y-T3 and Choles-h-p. Antitumor activity was
also
observed for all of the compounds tested except PEG1000.
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Table 2. ICSO (~,M) of Test Compounds in Human Prostate Cancer (Du 145) Cells
Compound ICSO (~1M)
aTS 34
aTS-T 23
txT-MS 31
TSE >50
2,2-Dim-TG 37
2,2-Dim-TS 30
T-DMAB-Q 21
T-DMAB-T 10
PEG 1000 >50
TS-PEG 15
TSE-PEG 22
TRF 42
TRF-S 32
yT3 24
yT3-S 36
CS-tris ~JO
CS-PEG 32
Choles-h-p 11
EXAMPLE 3.
Testing of compounds in human small cell lung cancer (NIH H69) cells
x 103 cells/well were plated in a 96-well flat bottom plate. On the next day,
the
medium was replaced with 200~L RPMI 1640 (10% FBS) containing from 0 to SO~,M
of test
compound. Cells were maintained as described in Methods. After 66 hours, 20~.L
of Alamar
blue stock solution was added to each well and the plates were incubated at
37°C for an
additional 6 hours. Plates were then kept at room temperature for 30 min.,
following which
fluorescence intensity was measured and the values obtained were processed as
described in
Methods. The results are given in Figure l0A-F, Figure 1 lA-F. Figure 12A-E,
Figure 13A-F,
and Figure 14A-E, and Table 3 gives the corresponding ICSO values. As can be
seen, the
compounds that appear to be most effective in inhibiting the growth of human
small cell lung
cancer (NIH-H69) cells are cc-T-MS, T-DMAB-Q, T-DMAB-T, TS-PEG, TSE-PEG, TRF-
S,
y-T3, choles-h-p, 2,2-Dim-TS, and CS-tris. Antitumor activity was observed for
all
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compounds tested except y-T, 8-T. PEG 1000,
y-T3-acetate and T-P-Na,.
Table 3. ICSO (~.M) of Test Compounds in Human Lung Cancer (NIH-H69) Cells
Compound ICso (~,M)
aTS 33
aTS-T 40
aT-MS 18
TSE 24. 46
8TS ~SO
yTS-T ~50
2,2-Dim-TS 11
T-DMAB-Q 13
T-DMAB-T 8
8T >50
yT >50
T-P-Na, >50
PEG 1000 >50
TS-PEG 8
TSE-PEG 15
TRF 37
TRF-S 25
TRF-S-T 3 0
Rice B-oil >SO
Palm oil 99
yT3 21
yT3-S 37
yT3 acetate >50
CS-tris 20
CS-PEG 25
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CSE-tris 33
Choles-h-p 8
EXAMPLE 4.
Testing of compounds in human colon cancer (HT29) cells
2.5 x 10' cells/well were plated in a 96-well flat bottom plate. On the next
day, the
medium was replaced with 200~L RPMI 1640 (10% FBS) containing from 0 to ~O~M
of test
compound. Cells were maintained as described in Methods. After 70 hours, 10~.L
of Alamar
blue stock solution was added to each well and the plates were incubated at
37°C for an
additional 2 hours. Plates were then kept at room temperature for 30 min.,
following which
fluorescence intensity was measured and the values obtained were processed as
described in
Methods. The results are given in Figure 15A-F, Figures 16A-E, Figure 17A-F,
Figure 18A-B,
and Table 4 gives the corresponding IC5° values. As can be seen, the
compounds that appear
to be most effective in inhibiting the growth of human colon cancer (HT29)
cells are oG-TS-T,
T-DMAB-Q, T-DMAB-T. TS-PEG, TRF-S. and choles-h-p. Antitumor activity was also
observed for all of the compounds tested except TSE and PEG1000.
Table 4. ICS° (~1M) of Test Compounds in Human Colon Cancer (HT29)
Cells
Compound IC5 (~1M)
a-TS 22
a-TS-T 18
aT-MS 24
TSE >50
TSE-T 25
2,2-Dim-TG 28
2.2-Dim-TS 29
T-DMAB-Q 19
T-DMAB-T 18
PEG 1000 >5
TS-PEG 14
TSE-PEG 24
TRF 32
TRF-S 14
yT3 30
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yT3-S 26
CS-tris 24
CS-PEG 31
CSE-tris 24, >50
Choles-h-p 11
EXAMPLE 5.
Testing of compounds in human breast cancer (MCF 7 and MD-231) cells
x 103 cells/well were plated in a 96-well flat bottom plate. On the next day,
the
medium was replaced with 200~L RPMI 1640 (10% FBS) containing from 0 to 50~,M
of test
compound. Cells were maintained as described in Methods. After 68 hours, 10~,L
of Alamar
blue stock solution was added to each well and the plates were incubated at
37°C for an
additional 4 hours. Plates were then kept at room temperature for 30 min.,
following which
fluorescence intensity was measured and the values obtained were processed as
described in
Methods. The results for MCF-7 cells are given in Figure 19A-E and Figure 20A-
C: the
results for MD-231 cells are given in Figure 21 A-D and Figure 22A-D; and
Table 5 gives the
corresponding ICso values.
As can be seen, the compounds that appear to be most effective in inhibiting
the
growth of hormone- dependent human breast cancer (MCF7) cells is TS-PEG.
Antitumor
activity was, however, also observed for all of the compounds tested except
PEG 1000.
The most effective compounds for inhibiting the growth of hormone-independent
MD-
231 human breast cells were TS-PEG, TRF-S, and DMAB-T.
Table 5. ICSO (~iM) of Test Compounds in Hormone-Dependent (MCF-7) and Hormone-
Independent (MD-231) Human Breast Cancer Cells
Compound MCF-7 Cells MD-231 Cells
ICso (N~M) ICSO (N~M)
aTS 41 25
aT-MS 42
TSE ~50 50
2,2-Dim-TG 42
T-DMAB-T 18
PEG 1000 >50
TS-PEG 15 10
TSE-PEG 36
TRF 42 3 8
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TRF-S 3 7 22
yT3 27
~yT3-S 41
CS-tris ~50
CS-PEG 45
a-TS-tris 28
2,2-Dim-TS 28
EXAMPLE 6.
Testing oJcompounds in human leukemia cancer (HL60) cells
~ x 103 cells/well were plated in a 96-well flat bottom plate. On the next
day, the
medium was replaced with 200~L RPMI 1640 (10% FBS) containing from 0 to 100~M
of
test compound. Cells were maintained as described in Methods. After 42 hours.
20~,L of
Alamar blue stock solution was added to each well and the plates were
incubated at 37°C for
an additional 6 hours. Plates were then kept at room temperature for 30 min.,
following which
fluorescence intensity was measured and the values obtained were processed as
described in
Methods. The results are given in Figure 23A-D, and Table 6 gives the
corresponding ICSo
values. As can be seen, the compounds that appear to be most effective in
inhibiting the
growth of human leukemia (HL-60) cells are TS-PEG, Choles-h-p and Y-T3.
Antitumor
activity was also observed for all of the compounds tested except 'y-T, PEG
1000, and CS-
PEG.
Table 6. ICSO (~1M) of Test Compounds in Human Leukemia (HL-60) Cells
Compound ICS (~M)
aTS 27
aT-MS 24
TSE >50
8TS 27
~yTS-T 34
8T 46
yT >50
T-DMAB-T 39
2,2-Dim-TS 24
PEG 1000 >50
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TS-PEG 12
TSE-PEG 24
TRF 39
TRF-S 40
TRF-S-T 40
yT3 15
yT3-S 36
yT3-S-tris 36
yT3-acetate >50
CS-tris 27
CS-PEG >50
Choles-h-p 13
EXAMPLE 7.
Testing of compounds in human cartaneous melanoma cancer (A375) cells
2.5 x 103 cells/well were plated in a 96-well flat bottom plate. On the next
day, the
medium was replaced with 200~,L RPMI 1640 (10% FBS) containing from 0 to SO~M
of test
compound. Cells were maintained as described in Methods. After 70 hours, 10~,L
of Alamar
blue stock solution was added to each well and the plates were incubated at
37°C for an
additional 2 hours. Plates were then kept at room temperature for 30 min.,
following which
fluorescence intensity was measured and the values obtained were processed as
described in
Methods. The results are given in Figure 24A-D, Figure 25A-C, and Figure 26A-
D, and Table
7 gives the corresponding ICS° values. As can be seen. the compounds
that appear to be most
effective in inhibiting the growth of human cutaneous melanoma (A375) cells
are TS, TS-T,
(xT-MS, TS-PEG. TSE-PEG, TRF-S. Btl-acid, Btl-acid-S. T-DMAB-T, and y-T3.
Antitumor
activity was also observed for all of the compounds tested.
Table 7. IC5" (p,M) of Test Compounds in Human Cutaneous Melanoma (A375) Cells
Compound ICS (~tM)
aTS 25
aT-MS 27
TSE 43
TS-PEG 15
TSE-PEG 29
TRF 34
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TRF-S 26
yT3 17
yT3-S 32
CS-tris 32
CS-PEG 29
Btl-acid 14
Btl-acid-S 25
2,2-Dim-TS 33
T-DMAB-T 23
TS-tris 26
TSE-tris >50
EXAMPLE 8.
Testing of compounds in human ocular melanoma cancer (OCMI) cells
2.5 x 103 cells/well were plated in a 96-well flat bottom plate. On the next
day, the
medium was replaced with 200~,L RPMI 1640 (10% FBS) containing from 0 to 50~,M
of test
compound. Cells were maintained as described in Methods. After 70 hours, 10~,L
of Alamar
blue stock solution was added to each well and the plates were incubated at
37°C for an
additional 2 hours. Plates were then kept at room temperature for 30 min.,
following which
fluorescence intensity was measured and the values obtained were processed as
described in
Methods. The results are given in Figure 27A-E. Figure 28A-F, Figure 29A-F,
Figure 30A-D,
and Figure 31A-C, and Table 8 gives the corresponding IC5° values. As
can be seen, the
compounds that appear to be most effective in inhibiting the growth of human
ocular
melanoma (OCM-I ) cells are choles-h-p, Btl-acid, TS-PEG, T-DMAB-Q, and T-DMAB-
T.
Experiments were conducted to determine if taxol, which is insoluble in water:
(a)
could be solubilized by adding this compound to TS-tris followed by the
addition of water and
sonication (which produces liposomes); (b) could retain its antitumor activity
if solubilized in a
suspension of TS-tris; (c) would be sequestered by the TS-liposomes, thus
conferring
excellent antitumor activity on the TS-tris liposomes that are isolated and
washed from the
TS-tris/taxol suspension; and (d) could upon esterification with succinate and
tris salt
formation (to produce the taxol monosuccinate tris salt and the taxol
disuccinate tris salt),
have greater solubility in water, retain its antitumor activity and be
sequestered in TS-tris
liposomes. The data illustrated in Table 8 and Figure 32A-D clearly
demonstrates that the
answer to all of these questions is yes.
In regards to the solubilization of taxol by TS-tris, we observed using light
microscopy
that taxol in water formed a precipitate that had a distinct appearance upon
magification under
the microscope and could be pelleted by centrifugation. However when taxol was
combined
with TS-tris, the resulting suspension formed liposomes without the
precipitate, again
observed using the light microscope. As can be seen in Fig 32B, the suspension
of taxol plus
TS-tris (referred to as taxol, 50X TS-tris) and the washed liposomes obtained
from this
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suspension (referred to as taxo1,50X TS-tris pellet) had excellent antitumor
activity [killing
greater than 85% of the tumor cells at 10 nM taxol (also containing 0.5
micromolar TS, a
nontoxic dose as seen in Fig 32A)]. In fact the antitumor activity of these
two preparations
was nearly identical to that of taxol dissolved methanol (referred to as
Taxol). These results
indicate that taxol can be solubilized by TS-tris in water or saline, that
taxol is contained
within TS-tris liposomes (probably as part of the lipid layer) and that both
the TS-tris/taxol
suspension and liposomal fractions have excellent antitumor activity. In
addition. our data
suggest that the addition of TS-tris and taxol allows for 100% kill of tumor
cells that normally
is not observed with taxol alone (see Fig.32B). These findings suggest that
both taxol and
TS-tris are killing tumor cells by different mechanisms and that the
administration of both of
these compounds in one preparation may improve the antitumor activity of both
compounds.
The data with taxol monosuccinate tris salt treatment in combination with TS-
tris
suspension and liposomes also demonstrates the same increased solubility for
this taxol
derivative in the presence of TS-tris as that observed for taxol. Like taxol,
the monosuccinate
tris of taxol has minimal solubilty in water (as determined by centrifugation
and light
microscopy). In contrast the disuccinate tris salt of taxol displayed complete
solubility in water
at a concentration of 4 mg/ml. These data suggest that the disuccinate taxol
may serve to
provide the means to administer taxol parenterally. Unfortunately, the
antitumor activity of the
disuccinate taxol is not as great as taxol and the monosuccinate derivative.
However, once in
vivo, the hydroylsis of this compound by esterases could liberate taxol and
monosuccinate
taxol which are much more active. The monosuccinate tris salt of taxol
demonstrates excellent
antitumor activity both in ethanol and as a suspension in TS-tris. The loss of
antitumor activity
of the liposomal preparation suggests a greater water solubility for this
derivative. In addition,
our data suggest that the addition of TS-tris and monosuccinate and
disuccinate taxol allows
for 100% kill of tumor cells that normally is not observed with taxol
derivative alone (see
Fig.32 C and 32D). These findings suggest that these taxol derivatives and TS-
tris are killing
tumor cells by different mechanisms and that the administration of both of
these compounds in
one preparation may improve the antitumor activity of both compounds.
Table 8. IC5° (~1M) of Test Compounds in Human Ocular Melanoma
(OCM1) Cells
Compound ICso (~.M)
ecTS 29
aTS-T 20
aT-MS 21
TSE 33
TSE-T 32
2,2-Dim-TG 35
2,2-Dim-TS 25, 22
T-DMAB-T 17, 21
T-DMAB-Q 14, 10
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PEG 1000 >50
TS-PEG 14
TSE-PEG 22
TRF 42
TRF-S 28
yT3 26
yT3-S 34
CS-tris 23
CS-PEG 33
CSE-tris 29
Choles-h-p 14, 14
Btl-acid 15
Btl-acid-S 49
Taxol (in ETOH) <0.01
Taxol-S tris (in ETOH) <0.01
Taxol-DS tris (in ETOH) >1.0
Taxol + TS-tris (sonicated in <0.01
water)
liposomes isolated from Taxol <0.01
+ TS-tris
Taxol-S tris + TS-tris (sonicated<0.01
in water)
liposomes from Taxol-S tris + <0.1
TS-tris
Taxol-DS tris + TS-tris (sonicated>0.1
in water)
liposomes from Taxol-DS tris >1.0
+ TS-tris
EXAMPLE 9.
In vivo plasma concentrations of tocopherol analogs 2=1 hours after
administration
The plasma concentrations of tocopherol analogs were assessed in vivo in rats
24
hours after administration. Rats were given either an intrapeironeal (ip)
injection or an oral
intubation of TS-tris salt, TSE or TS-PEG (0. l9mmol/kg body weight). After 24
hours, rats
were sacrificed and blood samples were obtained and extracted for HPLC
analysis of
concentrations of TS-PEG. TS, TSE and T.
As can be seen in Table 9 below, the in vivo administration of TS-tris and TS-
PEG
result in plasma levels of these compounds that suggest they may be active
anti-tumor agents
in vivo. Furthermore, the near identical TSE plasma concentrations for rats
administered
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TSE-tris by ip and oral administration demonstrates the excellent oral
absorption of this
compound (a similar plasma level was also observed for the oral administration
of TSE, data
not shown). These data indicate that TSE (and other nonhydrolyzable vitamin E
and
cholesteral derivatives, such as the 2.2 dimethyl TS) may be given orally to
treat cancer in
patients.
Table 9. In vivo plasma concentrations of tocopherol analogs 24 hours after
administration.
Analog Plasma concentration
(nmoUml)~
Administered T TS TS-PEG TSE
Vehicle (saline)11.6 ~ 1.1 ndb nd nd
TS-tris (ip) 24.4 ~ 5.6 29.8 ~ 7.2 nd nd
TS-PEG (ip) 22.8 t 0.8 38.4 ~ 7.3 10.6 t 3.0 nd
TSE-tris (ip)18.7 ~ 1.5 nd nd 11.3 ~ 2.5
TSE-tris (oral)12.5 t 2.2 nd nd 9.2 ~ 1.7
a , values are the means ~ SD (n=4)
b , not determined
EXAMPLE 10. Synthesis of d-oc-tocopheryl 2,2-dimethylglutarate (Figure 33A).
In a small reaction vial containing a magnetic stirring bar was placed 1.54 g
(3.58 mM)
of d-oG-tocopherol, 569 mg (4.00 mM) of 2,2 dimethylglutaric anhydride, and 15
mg of
anhydrous potassium carbonate. the vial was swept with nitrogen gas, capped,
and placed in
an oil bath at 140 °C for 8 days. the vial was opened every 2 days and
an additional 120 mg of
the 2,2-dimethylglutaric anhydride was added giving a total of 360 mg (2.5 mM)
of the
anhydride added during the heating. To the reaction mixture was added 15 ml of
chloroform
and 25 ml of water. This mixture was stirred at 38°C for 16 hr to
hydrolyze the unreacted
anhydride. The chloroform layer was removed and dried over anhydrous magnesium
sulfate.
The crude product was run through a silica column using hexane-chloroform.
chloroform, and
chloroform-methanol as the mobile phases. Several fractions were considered to
be pure
product; 768 mg (37%). A number of fractions were mixtures and were run
through a second
silica column to give an additional 252 mg ( 12 %) of pure product. The total
yield was 1.02 g
(49%). The compound is a low-melting solid. IR (thin film) showed that the
compound
absorbs at 1755 cm-' (indicative of an ester) and at 1702 cm'' (indicative of
COOH). Thin layer
chromatography (TLC) in chloroform-methanol 9:1 yielded an Rf value of 0.63.
EXAMPLE 11. Synthesis of d-OC--tocopheryl 2,2 dimethyglutarate TRIS salt
(Figure
33B).
In a small round bottom (R.B.) flask was placed 1.31 g (2.28 mM) of d-cc-
tocopheryl
2,2-dimethylglutarate in 10 ml of chloroform. In a separate container 263 mg
(2.17 mM) of
TRIS (base) was dissolved in 10 ml of methanol. The methanol solution was
poured into the
R.B. flask to give a clear solution. The solvents were removed under reduced
pressure to give
an oil which solidified upon the addition of acetone. The material was
recrystallized using
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acetone to give a white solid, 1.42 g (90 %) with a melting point of 96-99
°C. IR (thin film)
showed that the compound absorbs at 1744 cm-' (ester), and at 1557 cm'
indicative of a -
COO- salt, but no absorption at 1702 cm'', i.e no -COOH group is present.
Elemental analysis was conducted and the results were compared to the
theoretical
results which would be expected from a compound with the formula C~° H"
N Og:
Theoretical: C, 69.22 H, 10.31 N, 2.02
Actual Results: C, 69.19 H, 10.36 N, 2.10
EXAMPLE 12. Synthesis of d-a-tocopheryl ~3-(N,N-dimethylamino)ethyl ether
(free
base) (Figure 33C).
In a small reaction vial with a magnetic stirring bar was placed 800mg ( 1.86
mM) of d-
cc-tocopheroh 445 mg (11.1 mM) of NaOH (crushed), and 2 ml of methyl ethyl
ketone
(MEK). After the mixture had stirred for 15 minutes. 535 mg (3.71 mM) of
2-dimethylaminoethyl chloride hydrochloride was added; the vial was swept with
nitrogen gas;
the vial was capped and the mixture heated for 4 hours at 78 °C. The
liquid phase of the
mixture was transferred to a R.B. flask and the remaining solid was extracted
repeatedly with
4 ml portions of acetone. The acetone extracts were added to the initial
liquid phase. The
solvents were removed under reduced pressure to give a pale yellow oil. This
oil was passed
through a silica column using chloroform as the mobile phase to give 814 mg
(87%) of the d-
cx-tocopheryl ~3-(N,N-dimethylamino) ethyl ether (free base). Thin layer
chromatography
(TLC) in chloroform-methanol 9:1 yielded an Rf value of 0.65.
H NMR (CDC13) b 0.80-1.60(brm, 36H, CHI),
1.77 (m, 2H, CH,),
2.07-2.18 (s, 9H, three CH3),
2.35 (s,6H, N(CH3)Z),
2.56 (t, 2H, CH,),
2.72 (t, 2H, CH,),
3.75 (t, 2H, CH,).
Elemental analysis was conducted and the results were compared to the
theoretical results
which would be expected from a compound with the formula C3;H59NO,
Theoretical: C 78.98 H 11.85 N 2.79
Actual results: C 78.92 H 12.06 N 2.83
EXAMPLE 13. Synthesis of d-cx-tocopheryl ~i-(N,N-dimethylamino)ethyl ether
oxalate
salt (Figure 33D).
In one container was placed 238 mg (0.47 mM) of d-cc-tocopheryl ~3-(N,N-
dimethylamino)ethyl ether (free base) in 4 ml of acetone. In a separate
container 60.0 mg (0.47
mM) of oxalic acid dihydrate was dissolved in 4 ml of acetone. This oxalic
acid solution was
added to the amine (free base) solution to give a large amount of white solid.
This was filtered
off and recrystallized from acetone to give 237 mg (85%) of a white solid with
a melting point
of 170-172 °C. Infrared (KBr) studies showed that the compound absorbs
at 2523 cm'
(indicative of a tertiary amine salt) and at 1650-1580 cm'' (indicative of a -
COO-salt).
Elemental analysis was conducted and the results were compared to the
theoretical
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WO 00/59492 PCT/US00/09141
results which would be expected from a compound with the formula C35H6,NO6.
Theoretical: C 71.02 H 10.39 N 2.37
Actual results: C 70.97 H 10.45 N 2.49
EXAMPLE 14. Synthesis of d-a-tocopheryl ~3-N,N-(dimethylamino)ethyl ether
methiodide (Figure 33E).
In a small reaction vial with a magnetic stirnng bar was placed 76 mg (0. 1 S
1 mM) of
d-octocopheryl (3-(N,N-dimethylamino) ethyl ether (free base). The vial was
cooled and 1.20 g
(8.45 mM) of methyl iodide was added: the vial was capped and the mixture
allowed to stir at
R.T. for 3 days. The vial was opened and warmed to remove the excess methyl
iodide forming
a solid. This solid was recrystallized several times using acetone then dried
over phosphorous
pentoxide (under vacuum) to give 95 mg (97%) of white crystals with a melting
point of 178-
180 °C. Thin layer chromatography (TLC) in chloroform-methanol 9:1
yielded an Rf value of
0.36.
H NMR (CDC13) b 0.78 - 1.90 (brm, 38H, CH,),
2.08 - 2.26 (s, 9H, three CH3),
2.56 (t, 2H, CHI)
3.68 (s, 9H, -N +(CH3)3),
4.10 (s, 2H, CH,),
4.19 (s, 1 H, CH,).
Elemental analysis was conducted and the results were compared to the
theoretical
results which would be expected from a compound with the formula C34H6~NO,I.
Theoretical: C 63.43 H 9.71 N 2.18
Actual results: C 63.20 H 9.52 N 2.26
While the invention has been described in terms of the preferred embodiments
of the
invention, those skilled in the art will recognize that the invention may be
practiced with
modification within the spirit and scope of the appended claims.
REFERENCES
1. elAttar, T.M. and Lin, H.S. (1995) Vitamin E succinate potentiates the
inhibitory effect of
prostoglandins on oral squamous carcinoma cell proliferation. Prostoglandins
Leukot.
Essential Fatty Acids, 52, 69-73.
2. Schwartz, J. and Shklar, G. (1992) The selective cytotoxic effect of
carotenoids and oc-
tocopherol on human cancer cell lines in vitro. J. Oral Maxillofac. Surg., 50,
367-373.
3. Prasad, K.N. and Edwards-Prasad, J. (1992) Vitamin E and cancer prevention:
recent
advances and future potentials. J. Am. Coll. Nutr., 11, 487-500.
4. Turley, J.M., Sanders, B.G. and Kline, K. (1992) RRR-oc-tocopherol
succinate modulation
-26-
CA 02366807 2001-10-05
WO 00/59492 PCT/US00/09141
of humna premyelocytic leukemia (HL-60) cell proliferation and
differentiation. Nutr. Cancer,
18, 201-213.
5. Prasad, K.N. and Edwards-Prasad, J. (1982) Effects of tocopherol (vitamin
E) acid
succinate on morphological alterations and growth inhibition in melanoma cells
in culture.
Cancer Res., 42, 550-555.
6. Fariss, M.W., Fortuna, M.B., Everett, C.K. Smith, J.D., Trent, D.F. and
Djuric, Z. (1994)
The selective antiproliferative effects of oc-tocopherol hemisuccinate and
cholesterol
hemisuccinate on murine luekemia cells result from the action of intact
compounds. Cancer
Res., 54, 3346-3351.
7. Sharma, S. , Stutzman, J.D., Kelloff, G.J. and Steele, V.E. (1994)
Screening of potential
chemopreventive agents using bioichemical markers of carcinogenesis. Cancer
Res. 54, 5848-
5855.
8. Fariss, M.W., Bryson, K.F., Hylton, E.E. Lippman, H.R., Stubin, C.H. and
Zhoa, X.G.
(1993) Protection against carbon tetrachloride-induced hepatoxicity by
pretreating rats with
the hemisuccinate esters of tocopherol and cholesterol. Environ. Health
Perspect. 101, 528-
536.
9. Fariss, M.W. (1990) Oxygen toxicity: unique cytoprotective properties of
vitamin E
succinate in hepatocytes. Free Rad. Biol. Mea'. 9, 333-343.
10. Djuric, Z., Heibrun, L.K., Lababidi, S., Everett-Bauer, C.K. and Fariss,
M.W. (1997)
Growth inhibition of MCF-7 and MCF-l0A human breast cancer cells by a-
tocopheryl
hemisuccinate, cholesteryl hemisuccinate and their ether analogs. Cancer
Letters 1 I 1, 133-
139.
11. Fariss, M.W., Merson, M.H. and O'Hara, T.M. (1989) cc-tocopheryl succinate
protects
hepatocyted from chemical-induced toxicity finder physiological calcium
conditions. Toxicol
Lett. 47, 61-75.
12. Tirmenstein, M.A. , Watson, B. W. . Haar, N.C., and Fariss, M. W. ( 1998)
Sensitive
method for measuring tissue a-tocopherol and oc-tocopheryloxybutyric acid by
high
performance liqiod chromatography with fluorimetric detection. J. Chromatogr.
B. 707, 308-
311.
13. Fariss, M.W. (1997) Anionic tocopherol esters as antioxidants and
cytoprotectants. In
Handbook of Synthetic Antioxidants, E. Cadenas and L. Packer. eds. Marcel
Dekker Inc.,
New York, Chapter 4, 139-176.
14. Tirmenstein, M. A., Leraas, T. L., Fariss, M. W. (1997) Cc-Tocopheryl
hemisuccinate
administration increases rat liver subcellular cx-tocopherol levels and
protects against carbon
tetrachloride-induced hepatotoxicity. Toxicol. Lett. 92, 67-77.
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