Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
ENHANCED TISSUE AND SUBCELLULAR DELIVERY OF
VITAMIN E COMPOUNDS
DESCRIPTION
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention generally relates to an improved method for delivery and
retention
of antitumor and antioxidant Vitamin E compounds to tissues and subcellular
sites. Specifically,
the present invention provides a method to enhance the antioxidant capacity of
normal cells and
subcellular sites such as mitochondria and to inhibit the growth of and kill
tumor cells by
administering an aqueous suspension of tris salts of Vitamin E compounds.
Background Description
It is well established that reactive oxygen intermediates (oxidative stress)
generated from
r
both endogenous and exogenous insults (e.g. drug, chemical, hyperoxia,
hypoxia, radiation,
ischemia/reperfusion, aging, and inflammation) play an important role in the
toxic injury/disease
process (1,2). Recent studies suggest that mitochondria are the most important
endogenous
source of these reactive oxygen species (ROS), generating approximately 85% of
all ROS
produced endogenously in the cell. The overproduction of mitochondria) ROS can
result in the
inhibition of energy production and mitochondria) ROS production is thought to
be the cause of
numerous human diseases, including neurodegenerative diseases, cancer,
cardiovascular disease,
and drug-induced disease, to name a few. To prevent the ROS-induced oxidation
of important
cellular lipids, proteins, and nucleic acids, cells normally contain a battery
of endagenous
protective systems (antioxidants) to insure the maintenance of viability as
well as metabolic and
functional performance (2,3). An example of such a protective system is
unesterified d-a-
-1-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
tocopherol (a-T), also known as vitamin E. Though a-T appears to function as
the predominant
chain-breaking antioxidant in cellular membranes (4-6), this lipophilic
compound is not
synthesized by mammalian cells but rather is derived solely from exogenous
sources. The
antioxidant properties of a-T result from its ability to trap reactive peroxyl
radicals by donating a
hydrogen atom, becoming a tocopherol radical in the process. In order to
preserve cellular oc-T
and its membrane antioxidant activity, other cellular hydrophilic reductants
such as ascorbate,
glutathione and possibly NADPH can regenerate active a-T by donating a
hydrogen atom to the
tocopheroxyl radical (7-9). However, if the tocopherol radical is attacked by
another peroxyl
radical resulting in a 2-electron oxidation of oc-T (e.g. tocopherylquinone
formation), the cell's
ability to regenerate active a-T is apparently lost (7,8). The continual need
in cellular membranes
for the replacement of consumed oxidized a-T with dietary active oc-T suggests
that the cellular
uptake and subcellular distribution of this important antioxidant is crucial
to its ability to protect
membrane constituents and cellular integrity (especially during an oxidative
challenge), thus
limiting cell injury and disease.
Previous studies have demonstrated that oc-T is an extremely lipophilic
molecule and as
such is absorbed from the intestine in chylomicrons through the lymphatic
system and is
transported in plasma by a tocopherol-binding protein incorporated in
lipoproteins (10). The
cellular uptake of a-T has been reported to be mediated by both lipoprotein
receptor-dependent
and -independent pathways (11). It is generally accepted that in lipid
bilayers and biomembranes,
oc-T intercalates between phospholipids with the chroman head group (phenolic
hydrogen) toward
the surface (in close proximity to water-soluble reducing agents for
regeneration) and with the
hydrophobic phytyl chain buried in the hydrocarbon region ( 12).
Interestingly, in biological
membranes only one tocopherol molecule is present for every 500-1000
polyunsaturated fatty
acids (PLJFA) (13). This concentration of membrane-bound a-T is thought to be
close to the
threshold of a-T required to effectively protect phospholipid bilayers [0.2
mol %, ( 14)J and
biomembranes [0.4 mol % (15)J against oxidative damage, thus again emphasizing
the importance
of replenishing oxidized a-T by regenerating a-T with hydrophilic reductants
or by incorporating
new active a-T into the membrane.
Biomembranes or lipid bilayers are not limited to this ratio of oc-T to PUFA.
In fact, Lai et
-2-
Printed by VisualPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
al. (16) have shown that lecithin liposomes can be prepared with up to 40 mol
% oG-T, while
numerous reports indicate that increasing the oc-T content of biomembranes
decreases the
susceptibility of these membranes to lipid peroxidation (13, 17). At present,
it is not understood
v~rhy the concentra~on of a-T in biomembranes is kept at such a low mol %
(close to the
threshold). The amount of oc-T embedded in intracellular membranes appears to
result from the
concentration of a-T available from the diet and its intracellular transport
as well as from the rate
of consumption by oxidation and by transport out of the cell.
During an oxidative challenge when membrane-bound a-T is being rapidly
consumed, a
rate limiting factor in providing intracellular membranes with additional
active a-T may be the
requirement for a tocopherol transporting protein. Niki et al. ( 18) have
demonstrated using
artificial phospholipid membranes that the extreme water insolubility
conferred on oc-T by the
phytyl tail greatly inhibits its ability to exchange between membranes in the
absence of any
transporting factors. Other investigators ( 19-21 ) have also suggested that
the intracellular
transport of a-T requires a tocopherol transport protein that can cony oG-T to
subcellular
locations. Such a protein has been identified in rat and rabbit liver ( 19,
20) and heart (21 ) for the
transfer of oc-T to the nucleus (22), mitochondria (23) and microsomes (24)
and the binding of
t~-T to this protein is saturable. However, the ability of this tocopherol
transfer pmtein to rapidly
supplement intracellular membranes with active a-T remains unclear and seems
doubtful based on
our limited knowledge of oc-T transport. In fact, the observed inability of a-
T to freely exchange
between intracellular membranes may limit the ability of acute a-T
administration, given in vitro
or in vivo, to pmtect cells, organs and organisms from the toxic effects of
oxidative stress.
In light of the enormous cost of health care in the U. S. and the considerable
role of
oxidative stress in causing and exacerbating human disease, it is clear that
therapeutic strategies to
combat oxidative injury are required. To develop pharmaceuticals that protect
us from the adverse
effects of oxidative stress, one strategy is to ra i augment the intracellular
content of a-T at
the appropriate concentration, time and subcellular site (particularly
mitochondria) to diminish or
eliminate oxidative stress-mediated injury and disease.
The in vivo administration of'vitamin E (and related compounds) as
antioxidants and
fmtitumor agents is hampered by insolubility in the aqueous solutions which
are required for
-3-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
w0 00/59488 PCT/US00/08524
parenteral injection. Thus, these agents must be solubilized with additional
additives that may
also cause undesirable and even toxic effects in the patient. It would be
highly advantageous to
have a means of increasing the solubilization of these compounds in a non-
toxic vehicle thus
makvig them amenable to parenteral injection.
Numerous studies have demonstrated that the succinate derivative of vitamin E
(vitamin E
succvnate, d-oc-tocopheryl succinate, TS) administration protects experimental
animals, tissues,
cells and subcellular organelles from toxic cell death (25, 27, 31 ). These
cytoprotective effects of
TS da not appear to be selective for a particular toxic insult, cell type, or
species. Interestingly,
the mechanism for TS cytoprotection appears to be indirect; cellular esterases
cleave TS,
releasing antioxidant d-ot-tocopherol (a-T). It is the released ot-T which
confers cytoprotection.
In a curious contrast to the results that are obtained with normal cells, the
incubation of
tumor cells with TS results not in protection but rather in growth inhibition
and cell death (26).
The antitumor activity of TS has been reported for a wide variety of tumor
cells regardless of
species or cell type. It appears that the antitumor activity of TS is the
result of the intact
compound, TS, and not the release of aT. A plausible explanation is suggested
by the lack of
esteraise activity in tumor cells. Tumor cells often exhibit little or no
esterase activity; therefore,
the intact TS compound is taken up by and ern sists in tumor cells, leading to
tumor cell death. It
is also of interest that antitumor agents may trigger apoptosis (cell death)
in tumor cells through
their :interactions with mitochondria. It would be beneficial to have
available a method that takes
advantage of both the cytoprotective and the tumor killing properties of TS
and related vitamin E
compounds for the prevention and treatment of cancer.
SUMMARY
The present invention provides a method for the delivery of vitamin E
compounds to
tissues, cells and subcellular sites (including mitochondria) in order to 1 )
increase the antioxidant
capacity and protect normal (non-tumor) tissue, cells, mitochondria, and other
subcellular
organelles or substances 2) inhibit the growth of and kill tumor cells, and 3)
both protect normal
tissue while killing tumor cells by administering vitamin E compounds.
According to the
invention, the delivery of vitamin E compounds (either a single vitamin E
compound or a plurality
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
of vitamin E compounds) is achieved by making an aqueous suspension of the
tris salts of those
compounds by sonication. The aqueous suspension may be administered
intravenously,
transdermally, parenterally, by inhalation of an aerosol, orally, or by other
delivery routes. In
addition, the cells to which the vitamin E compound is delivered may be either
in vivo or in vitro.
1~urther, the subcellular sites to which the vitamin E compounds are delivered
are, in particular,
the outer and inner mitochondria) membranes.
ABBREVIATIONS
ALT:Alanine aminotransferase; LDH: lactate dehydrogenase; CCl4: carbon
tetrachloride;
CYP2E1: cytochrome P450, 2E1 form; G6Pase: glucose-6-phosphatase; ip:
intraperitoneal; iv-
:intavenous; PNP: p-nitrophenol; a-T: d a-tocopherol; TA: d a-tocopheryl
acetate; TS: d a-
~tocopheryl hemisuccinate; TS-FA: d a-tocopheryl hemisuccinate free acid; TS-
tris: d a-
tocopheryl hemisuccinate tris salt; TSE: d a-tocopheryloxybutyrate; TSE-tris:
d a-
tocopheryloxybutyrate tris salt; TS-2,2-dimethyl: d-a-tocopheryl 2,2,-
dimethylsuccinate; TG-2,2-
~dimethyl: d-a-tocopheryl 2,2-dimethylglutarate, TS-3-monomethyl d-a-
tocopheryl 3 methyl
~succinate; PUFA: polyunsaturated fatty acids; OCM-1: cell line derived from
ocular melanoma;
'TRF: tocotrienol rich traction; TRF-succinate-tris salt: tris salt of the
hemisuccinate ester of TRF;
NADPH: nicotinamide adenine dinucleotide phosphate (reduced form); NADP+ :
nicotinamide
;adenine dinucleotide phosphate (oxidized form); NaOH: sodium hydroxide; KCI:
potassium
chloride.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Graph showing the effect of oxidative stress on the induction of
lipid peroxidation in
mitochondria isolated from the livers of rats treated with TS-tris and TSE-
Iris. A d-oc-tocopheryl
hemisuccinate tris salt (TS-tris) and a d-oc-tocopheryloxybutyrate tris salt
(TSE-tris) were
administered intraperitoneally to rats at a dose of 0.19 mmol/kg 18 h prior to
isolation. An
increase in fluorescence indicates an increase in lipid peroxidation.
-5-
Printed by VisuaIPatenf
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
Figure 2A and 2B. 2A. Graph showing the effect of TS-tris suspension in water
(after
s~~nication) (~)on the growth and viability of OCM-1 cells. 2B. Graph showing
the effect of taxol
dissolved in ethanol (~), and taxol combined with TS-tris and sonicated in
water (O) on the
viability of OCM-1 cells.
Figure 3. Graph showing the effect of an 18 hour pretreatment of rats (n=3)
with a single ip
uijection of ocT or TS-tris (TS-T) (0.19 mmol/kg) on tocopherol and tocopherol
succinate levels
in liver homogenates. Data points represent the mean ~ SE.
Figure 4. Graph showing the effect of an 18 hour pretreatment of rats (n=3) a
single ip injection
of aT or TS-tris (TS-T) (0.19 mmol/kg) on tocopherol and tocopherol succinate
levels in liver
nutochondria. Data points represent the mean t SE.
Figure 5. Graph showing the effect of an 18 hour pretreatment of rats (n=3) a
single ip injection
of aT or TS-tris (TS-T) (0.19 mmol/kg) on tocopherol and tocopherol succinate
levels in liver
nutochondrial outer membranes. Data points represent the mean ~ SE.
Figure 6. Graph showing the effect of an 18 hour pretreatment of rats (n=3)
with a single ip
vijection of ocT or TS-tris (TS-T) (0.19 mmol/kg) on tocopherol and tocopherol
succinate levels
in liver mitochondrial inner membranes. Data points represent the mean ~ SE.
Figure 7. Graph showing the effect of an 18 how pretreatment of rats (n=3)
with a single ip
vzjection of aT or TS-tris (TS-T) (0.19 mmol/kg) on the antioxidant capacity
of inner and outer
nutochondrial membranes. Antioxidant capacity was determined by examining the
susceptibility of
these membranes to lipid peroxidation using the plate reader assay of
Tirmenstein et al. (49). Lag
time indicates the amount of time prior to the onset of lipid peroxidation. A
lag time of 300
vidicates complete protection against lipid peroxidation (or maximal
antioxidant capacity). Data
points represent the mean f SE.
Figure 8. Graph showing the effect of an 18 hour pretreatment of rats (n=3)
with a single ip
W jection of aT (~) or TS-tris (TS-T) (~) (0.19 mmol/kg) on the susceptibility
of hepatocytes
(iisolated from these rats) to iron (ferrous ammonium sulfate) induced cell
death. Hepatocytes
v~rere exposed to a variety of iron concentrations for one hour and cell death
was measured as
L,DH leakage. Data points represent the mean t SE.
-6-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
DETAILED DESCRIPTION OF THE INVENTION
The cytoprotective effects of vitamin E compounds such as TS appear to be the
result of
uptake of TS by cells, followed by cleavage of the compound by endogenous
esterases. This
results in the release of tissue, cellular, and subcellular T from TS,
providing an increased
antioxidant capacity. Tn contrast, the antitumor activity of TS appears to be
the result of the
intact, uncleaved compound. Thus, the administration of TS or related vitamin
E compounds has
the; potential to provide a two-fold, coordinate result: 1 ) the killing of
tumor cells via
accumulation of the intact compound (e.g. TS); and 2) cytoprotective,
antioxidant activity via
hydrolysis of TS to release the antioxidant aT.
The present invention provides a method for the administration of TS (or
related vitamin E
compounds) in order to both individually and in coordination 1 ) treat or
prevent tumor cell
growth and/or formation, and 2) provide increased cytoprotective, antioxidant
capacity to normal
cellls and mitochondria. By "coordinate" or "coordinately" we mean that the
administration of a
single form of the vitamin E compound has more that one beneficial effect on
the tissue or cells to
Wuch it is administered. The beneficial effects occurs in concert via two
related but distinct
mc;chanisms. For example, traditional anticancer agents often cause extensive
oxidative damage to
normal cells even as they are killing cancer cells. According to the method of
the present
invention, the TS or related vitamin E compound enhances the antioxidant
capability of normal
tissue to prevent or attenuate such damage. Further, intact TS compound will
be taken up by and
selectively persist in tumor cells, augmenting tumor killing by the anticancer
agent.
Thus, this dual antioxidant and cytotoxic activity for TS (and related vitamin
E
compounds) in normal and tumor cells, respectively, should prove useful in the
development of
more effective therapies for the prevention and treatment of oxidative stress-
related diseases
including cancer.
In this application, we present data in support of the use of an aqueous
formulation of
vitamin E (the tris salts of anionic vitamin E esters and ethers) which
provides enhanced tissue,
cellular and subcellular delivery and retention of these vitamin E esters and
unesterified vitamin E.
Ot~r data suggest that once this formulation of vitamin E (the tris salts of
anionic vitamin E esters)
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
has accumulated in normal tissue, cells or subcellular sites (mitochondria),
endogenous cellular
esterases can cleave these vitamin E esters, thereby releasing the potent
antioxidant a-7.', thus
providing enhanced antioxidant protection at this site. in addition, a single
administration of
Vitamin E ester-tris scats results in elevated blood, tissue, and
mitochondriaa levels of a-T and TS
for a longer period of time (i.e. oc-T and TS have a longer half life) when
administered in this
manner, compared to the administration of a-T alone. TS-tris salt provides a
tissue, cellulau and
s,ubcellular reservoir of T (in the form of TS) that can be slowly released
over time.
In addition, we demonstrate that the tris salts of anionic vitamin E esters
and ethers are a
fbrmulation that provides an aqueous solution for parenteral injection. In
fact, the administration
of such a preparation results in an enhanced concentration of the antitumor
agent, vitamin E
s,uccinate (TS), in tissue, cells and subcellular fractions (e.g.
mitochondria) for an extended length
of time.
The present invention provides a method for enhancing the delivery of
amtitumor amd
antioxidant Vitamin E compounds to tissues and subcellular organelles
(mitochondria). The
invention is based on the discovery that, upon sonication in saline (0.9% NaCI
in water) or in
water, the Tris salts of vitamin E compounds, (especially succinate
derivatives of Vitamin E),
form a suspension that can be administered parenterally. Further, ass will be
seen in the Examples,
parenteral administration of the suspension results in high levels of the
Vitamin E compound being
present and sustained in the normal tissues and in subcellular organelles
(mitochondria), and
affords protection against oxidative stress-induced lipid peroxidation and
cell death.
The method described herein can be useful for treating cancer in mammals (for
example
liver cancer, prostate cancer, ocular melanoma, cutaneous melanoma, colon
cancer, lung cancer
au~d the like) by delivering TS and related vitamin E derivatives. The method
of the present
invention can also be used to protect tissue, cells and subcellular organelles
(e.g. mitochondria)
against oxidative stress-induced injury or disease in mammals. Such injuries
or diseases may
include but acre not limited to: neurodegenerative diseases such as
Alzheimer's and Parkinson's
disease; vascular disease; heart disease (atherosclerosis and ischemic
damage); carcinogenesis;
aging; cigarette smoking-induced diseases; smog-induced pathologies; diabetes-
induced tissue
damage; and many other diseases for which the inception and progression of the
disease is
_g_
Printed by VisualPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
thought to be, at least in part, due to oxidative stress. It will be
understood by those of skill in the
art that the present invention can be practiced in the treatment of any
condition for which
protection of the tissue, cells, and subcellular organelles (e.g.
mitochondria) against oxidative
stress is desirable. Those of skill in the art will also recognize that the
present invention can be
practiced in both human and veterinary applications.
It will be recognized by those of skill in the relevant arts that the
suspensions and methods
of the present invention will also be useful for the protection of tissue and
cells in vitro, in
addition to the in vivo uses outlined above. For example, the methods of the
present invention
may be used to increase the antioxidant capacity of tissue and cells during
storage, such as tissue,
cells and organs to be used for transplants or for other uses. This may
include, for example, liver
cells or tissue, kidney cells and tissue, whole organs, sperm cells, cells in
blood, etc. In addition,
the methods of the present invention may also be used in such procedures as
bone marrow
transplants, wherein the patient's bane marrow is removed and could be treated
with TS to
selectively kill tumor cells, and then replaced in the patient. Those skilled
in the art will recognize
that the treatment of cells and tissue according to the methods of the present
invention can be
used in any procedure in vivo, in vitro, or both combined, in which it is
desirable to confer
protection from oxidative stress, or to kill tumor cells. The methods may also
be useful for
research purposes in tissue culture procedures, for example, for the passage,
maintenance, or
storage of immortal or primary tissue culture cells, or subcellular fractions.
In a preferred embodiment, the suspension of a Vitamin E compound is made by
sonicating the salt of the compound in saline or water. However, those skilled
in the art will
recognize that other suitable aqueous suspensions may also be used in the
practice of the present
invention. In addition, any dispersal technique that results in a suitable
suspension of the
compounds) may be utilized in the practice of the invention (fox example,
vigorous vortexing).
In a preferred embodiment of the invention, the Vitamin E compounds which are
used in
the practice of the present invention include the tris salts of the anionic
esters or ethers of Vitamin
E (tocopherol and its various forms) prepared individually or in combination,
for example: the tris
salts of TS, TSE, and TS-2,2-dimethyl and T1RF-succinate, including the d and
dl isomers of a, (3,
y and 8 forms of tocopherol related compounds. However, it will be readily
understood by those
-9-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
of skill in the art that other derivatives of other Vitamin E compounds ( e.g.
TG-2,2-dimethyl and
T'S-3-monomethyl) can also be used in the practice of the present invention.
In a preferred embodiment of the invention, the tris salts of the Vitamin E
compounds are
utilized. However, it will be readily understood by those of skill in the art
that other
pharmaceutically acceptable salts of a Vitamin E compound which is capable of
forming an
aqueous suspension suitable for administration may be used in the practice of
the present
vlvention.
In a preferred embodiment of the invention, the suspension of the salt of a
Vitamin E
compound 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 suspension of the salt of a Vitamin E compound may be
administered by
vnhalation of an aerosol. This method has the advantage of delivering the
antitumor or antioxidant
compound directly to the lungs where it could, for example, provide protection
against
carcinogens and oxidants 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
close 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.
Administration may also be achieved transdermally using a patch impregnated
with the
aqueous solution of the salt of the Vitamin E compound, by ocular
administration (eye drops),
sublingual administration, nasal spray administration and rectal
administration (suppository).
Administration may also be oral. As demonstrated in Example 4, the oral
administration of
7.'SE-tris resulted in high (9.2 nmol/ml) plasma TSE levels ( 18 hours
following administration)
that were similar to the levels observed for the same dose of TSE-tris given
intraperitoneally ( 11.3
-10-
Printed by VisualPafent
CA 02366884 2001-10-O1
b'VO 00/59488 PCT/US00/085Z4
nmol/ml) Thus, these data indicate that vitamin E ethers and vitamin E ester
compounds that are
not hydrolyzable (eg. TS-2,2 dimethyl, data not shown) can be absorbed
following ingestion or
oral administration. In the case of oral administration of hydrolyzable
vitamin E esters, absorption
may be accomplished by coating the tris salt vitamin E compounds (liposomes)
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
polyl,ysine could be used. Impermeable polymer films would be degraded by
microflora found in
the colon. Thus, the vitamin E ester 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 esters and promoting
vitamin E ester or ether
absorption following oral administration can also be used in the practice of
the present invention.
For oral administration, the Vitamin E 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 Vitamin E compound may be administered as a compbsition which also
includes a
pharmaceutically acceptable carrier. The 'Vitamin E compound 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 Vitamin E
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 fbrnnulations can also include lubricating agents, wetting agents,
emulsifying agents,
preservatives, and sweetening or flavoring agents.
The dose of the Vitamin E compound to be delivered will vary depending on a
variety of
factors including the route of administration, the particular condition being
treated, the condition
of the individual patient, the patient's age, gender, weight, etc., and other
various factors that will
vary From situation to situation. 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
-11-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
wU OOI59488 PCT/US00/08524
'to 100 mg/kg of body weight. For administration via inhalation, the dose rnay
be less and will vary
;according to the exact delivery technology that is employed.
The Vitamin E compounds may be used alone (i.e. one Vitamin E compound per
;suspension) or as mixtures of Vitamin E compounds (i.e. more than one Vitamin
E compound per
auspension). For example, TRF-succinate tris salt forms liposomes, indicating
that the five
different tocopherol and tocotrienol isomers that are contained in TRF are
contained together in
the liposome. It may be advantageous to deliver to tissue more than one isomer
or form of vitamin
:E compound, especially if different forms of ot-T are able to distribute to
different subcellular
;sites, or protect against different types of oxidative stress, or are
hydrolyzed at different rates.
The Vitamin E compounds may be used by themselves or in combination with other
drugs.
:For example, the Vitamin E compounds may be used with other antitumor drugs
such as taxol, or
with other antitumor agents such as doxorubicin, and with other tocopherol
derivatives. When
used in combination with other drugs, the Vitamin E compounds may be
administered prior to,
oifter, concomitant with, or in the same preparation as the other drugs.
MATERIALS AND METHODS
f'hemicals
Absolute ethanol was obtained from J. T. Baker Inc. (Phillipsburg, NJ).
Ascorbic acid, HPLC-
grade methanol, chloroform and hexane were obtained from Fisher Scientific
(Pittsburgh, PA).
'Che tocopherol compounds a-T (96%) and d &-tocopherol (96%) (internal
standard) were a
generous gift from Henkel (La Grange, IL). TS-tris (99%) was prepared as
described by Fariss et
f~l. (7), The compound TSE-tris (>95%) was synthesized according to the
procedures reported by
1~ariss et al. (9). CC14 (99.9+%) was of the highest purity available and was
obtained from Aldrich
(TVtilwaukee, WI). Heroin, bilirubin, TS-FA (99%), p-nitrophenol and all other
chemicals used for
this study were obtained from Sigma (St. Louis, MO). Taxol was a gift from Dr.
David Bailey at
Hauser, Inc. ~-TS-tris and TRF-S-tris were synthesized by Dr. Doyle Smith.
-12-
Printed by VisuaIPatenf
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
Cell lines
The: cell line OCM1, derived from human ocular melanoma , was used for the
taxol studies and
wa:~ a gift from Dr. June Kan-Mitchell at the University of California, San
Diego, CA.
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 libiturr~ for at least three days prior to
the onset of the
experiment. a-T was dissolved in olive oil (approx. 100 mg/ml) and
administered by ip injection
at a dose of 0.19 mmol/kg body weight. The vehicle, olive oil, was
administered at a dose of 1
ml/!kg body weight. Powdered TS-tris and TSE-tris were suspended in saline
with brief sonication
(30 sec) and were given intraperitoneally at a dose of 0.19 mmol/kg body
weight (approximately
100 mglkg). Saline was given to rats at a dose of 4 ml/kg. In most cases,
after the administration
of the tocopherol compounds animals were sacrificed 18, 72 or 120 hours later
for tissue
procurement (frozen in liquid nitrogen), liver homogenization and subcellular
fractionation. In the
tissue distribution studies, animals were fasted 1.8 hours prior to sacrifice.
In experiments in which
rats were sacrificed 6 h after receiving TS-tris, rats were fasted for a 12 h
period prior to
receiving TS-tris and for an additional & h until sacrifice.
Rats received CC14 by oral lavage 6 or 18 h after tocopherol administration.
CCl4 was
dissolved in peanut oil (0.5 g lml) and given at a dose of 1.0 g/kg. Food was
restored 1 h after
receiving CCl4. In some experiments, rats were sacrificed 4 h after receiving
CC14 and plasma and
liver samples were collected for hepatotoxicity determinations. Rats were
anesthetized with
diethyl ether, and blood samples (4-5 ml) were withdrawn from the inferior
aorta. Blood samples
were immediately mixed with 15 mg tripotassium EDTA, and aliquots were
centrifuged at low
speed to prepare plasma samples. Liver microsomes were also isolated after 4 h
for the
determination of lipid peroxides, tocopherol, G6Pase activity and p-
nitrophenol (PNP)
hydroxylase activity levels. All procedures were approved by the Washington
State University
Animal Care and Use Committee.
-13-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/USOO108524
Tumor cell methods and viability assay
Preparation of Tagol 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
micibfuge tube and sonicating fox 15 sec., twice. For taxol-TS-tris
suspensions, 1 mg of taxol was
added to 30 mg of TS-tris prior to the addition of water or saline, and the
mixture was sonicated
for l 5 sec., twice.
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 gmup 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.
PRaICEDURE:
1) On day one, OCM-1 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 DMEM (10% fetal bovine serum)
medium. 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
rangy from 0 to 50~tM. 200~L of medium without cells was plated as a blank.
2) Ct;lls were maintained in a humidified atmosphere in 5% COZ 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, 530nm;
emirsion, 590nm; gain, 40). Fluroescent values from blank cells were
subtracted from fluorescent
-14-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/U500/08524
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 ICso values (concentration required to inhibit cell growth by
50%) were extrapolated
from the plot of cell number (%control) versus compound concentration. The
cell number (%
control) plotted was the mean t SD (n=6).
Rat liver homogenization and subcellular fractionation
Livers were excised from rats and minced in ice cold homogenization buffer
(250 mM sucrose, 10
mM tris and 1 mM EDTA, pH 7.4). T'he minced liver was subsequently rinsed
several times with
ice cold homogenization buffer and finally suspended in 2.5 volumes of
homogenization buffer.
The liver mince was then homogenised with five strokes of a Potter-Elvehjem
tissue grinder.
Aliquots of the homogenate were retained for tocopherol and protein
determinations. The
remainder of the liver homogenate was used for subcellular fractionation
according to previously
described procedures (27, 28). In all cases, the subcellular markers
corresponding to the
appropriate fractions were substantially enriched and were similar to
previously reported values
(28). Following isolation, subcellular fractions were resuspended in phosphate
buffered saline and
,aliquots were withdrawn for tocopheral and protein determinations. Proteins
were measured
;according to the procedures of Lowry et al.(29) as modified by Peterson (30).
.Preparation of respiring mitochondria
:Mitochondria were prepared from either naive rats, or following pretreatment
with T or TS as
outlined in the Animals section above. Rats were anaesthetized with 0.6 mg/kg
pentobarbitone,
and livers rapidly excised, weighed and placed in ice-cold 0.25M sucrose, 1mM
EDTA in IOmM
'iris Hcl, pH 7.4 containing 0.5% BSA. The liver was lightly minced, then
homogenized by 3
ipasses of a drill-assisted Dounce homogenizer. The homogenate was centrifuged
at 3,000 xg, 4°C
;for 10 minutes with the pellet containing debris and nuclei being discarded.
The supernatant was
centrifuged at 10,000 xg for 10 minutes, supernatant aspirated and
mitochondria) pellet collected.
'The pellet was resuspended in ice cold 0.25M sucrose in l OmM Tris HCI, pH
7.4 containing
0.5% BSA, and centrifuged as before. The mitochondria) pellet was washed one
further time
-15-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00159488 PCT/US00/08524
before being resuspended to a volume being 1 gram wet weight liver per ml.
Mitochondria were
stored on ice and used immediately for swelling experiments.
Measurement ofMitochondrial Swelling
The swelling of freshly isolated mitochondria, ( 1 g wet weight liver/ml)
during exposure to ferrous
ammonium sulfate was measured spectrophotometrically as a decrease in
absorbance at 540nm. In
these experiments, analysis buffer comprised of 0.25M sucrose, 1 rnM potassium
dihydrogen
orthophosphate and 2 ~1M rotenone in lOmM MOPS buffer, pH 7.4. At 1 minute
prior to the start
of the experiment, a 0.1 ml aliquot of 60mM succinate, prepared in analysis
buffer was added to
0.9 ml of analysis buffer in a 1.5 ml cuvette. A 0.1 ml aliquot of
mitochondria was added,
inverted to mix and left at room temperature for a further minute. The
swelling agent was added
and the monitoring of change in absorbance commenced immediately and continued
for up to 10
minutes. Prior to investigating mitochondria) swelling to ferrous ammonium
sulfate (0-2500~1M),
the ability to swell in the presence of calcium (20~.M) was used as a positive
control.
Preparation of inner and outer mitochondria) membranes.
Mitochondria were prepared as outlined above with the absence of BSA in the
homogenization
.and washing solutions. The two washings remove the majority of residual
microsomal
contamination. After the final wash, mitochondria were resuspended in 5 ml of
0.25M sucrose and
1 mM EDTA in l OmM TrisHCI buffer, pH 7.4. Mitochondria were snap frozen in
liquid nitrogen
and stored at 80°C until use. After thawing, a 1 ml aliquot of
mitochondria were diluted with 1 ml
of phosphate buffered saline, pH 7.4 and sonicated on ice for 3 seconds. The
mitochondria were
then placed on a sucrose gradient of three 1 ml layers of 51%, 37.7% and 25.3%
and centrifuged
at 140,000 xg for 3 hours 15 minutes. 'The outer membranes of the mitochondria
form a band at
the 25.3:37.7 interface. The inner mitochondria) membranes form a band at the
37.7:51 interface.
l3oth bands are individually collected, resuspended in 40m1 of PBS and
centrifuged at 12,000 xg
iPor 20 minutes. The inner mitochondria) membranes form a smoky red pellet,
which is
resuspended in 1 ml of PBS and snap fi;ozen in liquid nitrogen until use. The
pellet from the outer
membrane sample is discarded, and supernatant centrifuged at 120,000 xg for 90
minutes. The
-16-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PGT/US00/085Z4
clear red pellet is resuspended in 1 ml of PBS and snap frozen until use.
Protein content of the
cellular and subcellular fractions were determined by the BCA protein kit
(Pierce, Rockford, U,).
Susceptibility of Mitochondria to Lipid Peroxidation:
The mitochondria (as well as the inner and outer mitochondrial membranes)
isolated as described
above from TS-tris and TSE-tris animals (18 hour treatment) were examined for
their
susceptibility to lipid peroxidation by the fluorescence plate reader assay of
Tirmenstein et al.
(49).
Preparation of Rat Hepatocyte Suspensions and Cytotoxicity Studies:
Adult male Sprague-Dawley rats (200-300g) were obtained from Simonsen
Laboratories Inc.
(Gilroy, CA). Rats were housed in small groups with food and water ab lib. in
a standard 12 hour
light/dark cycle for at least 1 week prior to use. Rats were administered
either 0.19 mmol/kg
a-tocopherol dissolved in olive oil or oc-tocopherol succinate tris by
intraperitoneal injection 18
hours prior to cell isolation. Tocopherol succinate (Tris salt) was prepared
in saline with two 15
second sonications on ice to form a fme aqueous suspension.
Hepatocytes were isolated using the 2 step collagenase perfusion method
described in
Fariss et al. (50). A yield of 5-7 x 1 Og cells was routinely obtained with
viability of >94%, as
determined via trypan blue exclusion. Hepatocyte suspensions (2 x 106
cells/mL, 12 mL, total)
were prepared in modified Waymouth's medium. After a 15 minute equilibration
time, an aliquot
of cells was taken as the 0 time point. After collection of the 0 time point,
the cytotoxicant under
investigation was immediately added. Ferrous ammonium sulphate (0.1 to 5 mM)
was prepared in
water immediately before addition. A 0.5 mL aliquot of cell suspension was
collected at
half hourly or hourly intervals for up to 6 hours. The aliquot was centrifuged
at 12,000 rpm for 5
seconds, supernatant collected and stored at 4°C until lactate
dehydrogenase (LDH) activity
analysis.
Lactate dehydrogenase activity:
LDH activity was determined by monitoring the enzymatic formation of NADH for
NAD+ in the
-17-
Printed by Visua/Patent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
presence of L-lactic acid. Post-centrifugation supernatants were diluted 1:80
with PBS. A 100 ~tL
aliquot was added to a well of a 96 well plate and mixed with 100 ~L of
reagent to give a final
concentration of 3.75 mM NAD+ and 25 mM L-lactic acid in 125mM Tris-HCI
buffer, pH 8.9.
The increase in fluorescence was immediately monitored at room temperature
(gain 70) using a
CytoFluor 4000, Perceptive Biosystems (Framingham, MA). The percent LDH
leakage was
calculated by comparing values to total LDH activity. Total LDH was measured
from a sample of
hepatocytes collected at 0 time and lysed with a final concentration of 0.2%
Triton X-100.
Tocopherol determinations
Tocopherol, tocotrienol, and tocopherol ester levels were measured according
to the methods
described by Fariss et al. (31 ). TSE levels were measured according to the
procedures of
Tirmenstein et al. (32). Samples were analyzed by reversed-phase high-
performance liquid
chromatography equipped with fluorimetric detection. Retention times for d b-
tocopherol
(internal standard), a-T and TSE were 8.0, 11.5 and 13.4 min respectively.
Microsomal enzyme assays
Microsomes isolated from vehicle and tocopherol-treated animals were washed
and resuspended
in 0.154 M KCI, 50 mM tris, pH 7.4 buffer and were stored frozen at -
80° C prior to assays. PNP
hydroxylase activity was measured according to the procedures of Koop (33) as
modified by
Speerschneider and Dekant (34). Aliquots of microsomes were resuspended in 0.1
M potassium
phosphate buffer, pH 7.4 at a concentration of 0.75 mg protein/ml with a final
volume of 1 ml.
The NADPH-generating system consisted of 1 mM NADP+, 9 mM glucose-6-phosphate
and 0.2
units/ml glucose-6-phosphate dehydrogenase. Microsomes were preincubated with
the NADPH
generating system at 37° C for 5 min. Incubations were started by
adding PNP to a final
concentration of 450 ~1M. After 20 min at 37° C with shaking,
incubations were stopped by
adding 0.5 ml 0.6 N perchloric acid to 1 ml of samples. Samples were
centrifuged at 16,OOOg for
2 min, and 1 ml of the supernatant was mixed with 0.1 ml of 10 N NaOH. The
amount of the
reaction product, 4-nitrocatechol, formed was determined by measuring the
absorbance at 546 nm
(extinction coefficient of 9.5 3 mM- i cm-1 ).
-18-
Printed by VisualPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
Microsomal lipid peroxide and G6Pase activity determinations
In the studies described in Table 3, rats were sacrificed 4 h after CC14
administration and rat liver
microsomes were prepared as previously described (27,28). Microsomes were
washed and
resuspended in 0.154 M KCI, 50 mM tris HCI, pH 7.4 buffer. After isolation,
microsomes were
stored frozen at -80° C. Lipid peroxide levels in microsomes were
determined according to the
procedures of Ohkawa et al. (35). Aliquots of the washed microsomes were also
assayed for
G6Pase activity as described by Aronson and Touster (36).
Statistics
Results are presented as means t SD. Analysis of variance was performed with
the InStat 2.03
(GraphPad Software, Inc., San Diego, CA) statistical package. Differences
between groups were
determined using the Dunnett multiple comparison post test.
EXAMPLES
The foregoing are Examples which represent preferred embodiments of the
present
invention, but should not be construed so as to limit the invention in any
way.
EXAMPLE 1.
Subcellular distribution of tocopherol analogs
The subcellular and liver homogenate levels of a-T, TS and TSE measured at
either 6 h
or 18 h after the ip administration of tocopherol analogs (0.19 mmol/kg) to
rats are reported in
Table 1, and the values are normalized per mg protein. The administration of
the tris salts of TS
and TSE yielded much greater incorporation of TS and TSE, respectively, into
the liver than an
equimolar dose of T. TS-tris administration increased the total tocopherol (TS
+ a-T) levels
found in liver homogenates and subcellular fractions by a factor of 8-36 fold
over those seen in
vehicle controls. The highest total tocopherol values expressed per mg of
protein were observed
in mitochondria and in plasma membranes. If tocopherol values were not
normalized per mg
protein, the majority of the tocopherol found in hepatocytes was associated
with mitochondria
-19-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
w0 00/59488 PCT/US00/08524
and ~rucrosomes. Our data indicate that these fractions consistently contained
the majority of the
cell's tocopherol levels, regardless of the tocopherol treatment.
Supplementation of rats with
tx-T was much less effective in increasing total tocopherol levels in rat
liver homogenates and
subcellular fractions were increased only about 2 fold in rats receiving a-T.
a-T is insoluble in water~and was dissolved in olive oil prior to
administration. In
conbrast, powdered TS-tris and TSE-tris can be placed in saline and suspended
by sonication.
Light microscopic analysis of these TS-tris and TSE-tris suspensions suggested
that they
consisted of lipsomes of variable size. The presence of TS-tris and TSE-tris
liposomes was
confirmed by negative-stain electron microscopy (data not shown). Previous
findings by Lai et al.
(51 ) have shown that TS is capable of forming liposomes, and Janoff et al.
(52) have
characterized a-tocopherol hemisuccinate vesicles. In agreement with our
findings, 3uzmoto et
al. (4.5) have previously shown that powdered TS shaken in tris buffer forms
liposomes at pH
7.4.
TS-tris administration to rats also proved to be the most effective means of
increasing
a-T :levels in the liver and exceeded the ability of TS administration to
increase hepatic oc-T
levels (Table 1 and reference 27). The administration of a-T produced a modest
but significant
increase in a-T levels in all of the fractions except microsomes. In the
microsomal fraction, a-
T administration produced a nonsignificant 1.? fold increase in a-T levels. In
contrast, TS-tris
administration increased microsomal oc-T levels 7.6 fold. These findings
suggest that TS-tris
administration is the most effective means for increasing the antioxidant
capacity of tissue,
cells and subcellular fractions. This was confirmed in studies demonstrating
that hepatic
mitochondria isolated from rats treated with TS-tris were less susceptible to
lipid peroxidation
than mitochondria isolated from control or TSE-tris treated rats (Fig. 1 ).
Large quantities of the intact TS molecule were also present in each of the
subcellular
fractions (except cytosol) and liver homogenates from animals supplemented
with TS-tris. In
these animals, high TS levels (expressed as nmol/mg protein) were found in
mitochondria,
plasma membranes, microsomes and nuclei. As compared with TS administration,
TS-tris
treatrnents resulted in an up to 10-fold increase in homogenate and
subcellular TS levels (Table 1
and reference 27). This superior ability of'CS-tris administration to increase
cellular and
-20-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
subcellular TS levels was especially high in mitochondria. These findings
suggest that this
large accumulation of TS could serve as a a-T reservoir for the release of
site-specific a-T
over time (thus increasing the antiaxidant capacity of the cell or cellular
organelle) or could
serve to enhance the antitumor abilities of TS administration.
The administration of TSE-Iris to rats led to the uptake and incorporation of
TSE into
hepatocyte membranes that exceeded by up to 10-fold that observed following
TSE
administration, again with mitochondria showing the largest increase (Table l
and reference
27). However, TSE-tris did not introduce as much TSE into liver homogenates
and
subcellular fractions as an equimolar dose of TS-tris. Supplementation of rats
with TSE-tris
did not significantly increase homogenate or subcellular levels of a-T
supporting the
observation that TSE is a non-hydraylzable form of TS that will not produce an
increase in
the antioxidant capacity of a tissue or subcellular fraction (as noted in
Example 3, Fig. 1).
However, the administration of TSE-tris may prove useful in enhancing the
antitumor activity
of this compound (26).
TABLE 1. Tocopherol analog levels in liver homogenates and subcellular
fractions from rats
treated with d a-tocopherol (a-T), d-a-tocopheryl hemisuccinate tris salt (TS-
tris) and
d a-tocopheryloxybutyrate tris salt (TSE-tris).
Homogenate Mitochondria
Treatment' a-T TS TSE a-T TS TSE
(nmoUmg (nmoUmg
protein) protein)
Vehicle-18h O.lSt ND' ND 0.341 ND ND
0.02b 0.03
a-T-18 h 0.301 ND ND 0.91 ND ND
0.034 0.334
TS-tris-6 0.291 2.52 ND 1.041 10.511 ND
h
O.OId 0.65 0.164 1.98
-21-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
'CS-tris-18 0.401 1.611 ND 1.181 6.491 ND
h
0.084 0.41 0.224 1.78
'rSE-tris-18h0.131 ND 0.651 0.391 ND 3.321
0.01 0.19 0.04 1.11
Microsomes Plasma
Membranes
'Treatment' a-T TS TSE a-T TS TSE
(nmoUmg (nmoUmg
protein) protein)
'Vehicle-18h0.221 ND ND 0.471 ND ND
0.01 0.07
.a-T-18 h 0.371 ND ND 0.96 ND ND
0.08 0.13
'TS-tris-6 1.41 2.16 ND
h
0.174 0.30
'TS-tris-18 1.68 2.041 ND 1.241 6.15 ND
h
0.544 0.68 0.17 2.34
'TSE-tris-18h0.27 ND 1.04 0.681 ND 5.661
0.01 0.33 0.06 2.94
Nuclei Cytosol
'Treatment" a-T TS TSE a-T TS TSE
(nmoUmg (nmoUmg
protein) protein)
Vehicle-18h 0.11 ND ND 0.04 ND ND
0.02 0.01
a-T-18 h 0.251 ND ND 0.081 ND ND
O.OSd 0.024
-22-
Printed by VisualPatent
CA 02366884 2001-10-O1
WO OOI59488 PCTlUS00/08524
TS-tris-6 0.11 0.38 ND
h
0.1 Od 0.02
TS-tris-18 0.421 3.55a ND 0.121 0.021 ND
h
0.074 Ø93 0.044 0.09
TSE-tris-18hO.lSt ND 2.83 0.041 ND O.llt
0.02 0.58 0.00 0.03
a Rats received a 0.19 mmol/kg ip injection of the tocopherol analog, 18 h
prior to liver
homogenization and subcellular fractionation. Each rat was fasted during this
18 h period
and the vehicle was saline.
b Values expressed as the mean t SD (n = 3 to 6).
c ND, not detected.
d Values are significantly different (p < 0.05) from vehicle treated rats.
EXAMPLE 2.
Tocopherol protection against CCIa induced microsomal lipid peroxide formation
and
enzyme inactivation: increase in hepatic c~ T in TS-treated rats following
toxic insult.
The effects of CCl4 on microsomal a-T, lipid peroxide and enzyme activity
levels are
reported in Table 2. The administration of CC14 decreased microsomal a-T
levels by about
20% after 4 h. This decrease could not be explained by CCl4 induced liver cell
death. Four
hours following CCI4 treatment, animals had plasma ALT values (89 t 30
units/L) near
controls (53 f 11 units/L) and no liver injury as judged by histopathology
(data not shown).
Microsomal a-T levels in rats supplemented with TS-tris at 6 and 18 h prior to
CCI4
administration were 7.5 and 16 times higher than controls, respectively. In
contrast, the
micmsomal a-T levels in rats pretreated with a-T and then administered CCl4
were only
about 1.7 times higher than the microsomal a-T levels measured in control
animals.
Interestingly, during a toxic insult (CCI4) it appears that the hepatic
microsomal a-T levels in
TS-tris treated animals increases significantly from 1.68 (no toxic insult) to
3.83 (toxic insult)
nmol/mg protein. These findings suggest that during a toxic insult,
subcellular stores of TS
can be converted to a-T at an accelerated rate, possibly as a means for
increasing the
antioxidant capacity of the organelle. Microsomal lipid peroxide levels were
significantly
-23-
Printed by VisualPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
increased only in CC14 treated animals. Supplementation of rats with a-T or TS-
tris at either
6 or 18 h prevented CC14-induced increases in microsomal lipid peroxides.
The administration of CCI4 to rats is known to inactivate microsomal enzymes
such as
CYP2E1 (48) and G6Pase (37). These enzymes were assayed in our study to
determine if a-
T or TS-tris supplementation could protect against CC14-dependent enzyme
inactivation. As
expected, CCl4 administration signiftcantly decreased both G6Pase and PNP
hydroxylase
activities (Table 2). PNP hydroxylase activity was reduced by 86% 4 h after
CC14
administration. Supplementation of rats with either a-T or TS-tris provided no
protection
against CCl4-induced inactivation of PNP hydroxylase activity.
CC14 administration also decreased G6Pase activity levels by about 60% as
compared
to controls after 4 h. Pretreatment of rats with a-T did not significantly
protect against
G6Pase inactivation by CCl4. In contrast, supplementation of rats with TS-tris
provided
partial but significant protection against G6Pase inactivation by CC14 G6Pase
activity levels
were decreased by only 23% 4 h after CC14 administration in animals pretreated
with TS-tris
18 h prior to CC14 and by 36% in animals pretreated with TS-tris 6 h prior to
CC14.
CCl4 is known to be metabolized to the trichloromethyl free radical (CC13) by
CYP2E1. Once formed, CC13 can initiate the peroxidation of polyunsaturated
fatty acids or
covalently bind to cellular proteins. In addition, CCl3 can react with
molecular oxygen and
generate secondary radicals such as the trichloromethylperoxyl free radical
(CC13 Oz) which
can also initiate free radical reactions. Since a-T effectively inhibits lipid
peroxidation, we
examined the relative capacity of tocopherol compounds to protect against CCl4-
induced lipid
peroxide formation (Table 2). As expected, 4 hours after CC14 administration
there was a
significant decrease in microsomal a-T levels and an increase in lipid
peroxide levels. Rats
pretreated with either a-T or TS-tris (6 or 18 h pretreatment) , and then
administered CCl4
had microsomal a-T levels above those found in control animals and were
protected against
microsomal lipid peroxidation. However, despite this attenuation of microsomal
lipid
peroxidation, rats pretreated with a-'T were not protected against CC14-
induced hepatic
necrosis. These results suggest that early microsomal lipid peroxidation can
be dissociated
from CC14-induced hepatic necrosis. This dissociation between CCl4-induced
lipid
peroxidation has also been reported by other researchers (38-41 ). It is
important to note,
however, that in our study microsomal lipid peroxides were only measured 4
hours after CC14
-24-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
administrafion. Lipid peroxidation may continue at later time points in
response to oxidative
stress induced by CC14 . Morrow et al. (42) concluded that lipid peroxidation
continued for at
least 24 h following CC14 administration.
Lipid peroxidation at these later time points may not be due to CCl4 radical
formation,
but instead may involve activation of Kupffer cells and infiltration of
neutrophils. Activated
Kupffer cells produce reactive oxygen species and blocking Kupffer cell
function has been
shown to be protective against CCl4 -induced hepatotoxicity (43). The amount
of a-T
introduced into hepatocytes following a-T administration may be sufficient to
protect against
the initial lipid peroxidation generated by CC14 radicals but may eventually
be depleted by the
generation of reactive oxygen species at later time points. TS-tris in
contrast may continue to
be hydrolyzed to free a-T by cellular esterases and inhibit lipid peroxidation
over a longer
period of time. Studies by Kagan et al. (44) demonstrated that esterases
located in
microsomes can hydrolyze tocopherol esters. These results may explain why we
found TS-
tris especially effective in increasing microsomal a-T levels 18 h after
administration.
TABLE 2. Effects of carbon tetrachloride (CC14) administration on rat liver
microsomal
d a-tocopherol (a-T), lipid peroxide and enzyme activity levels: protective
effects of oG-T
and the tris salt of d-oc tocopheryl hemisuccinate (TS-tris).
Treatment' Ot-T Glucose-6- p-NitrophenolMicrosomal
nmoUmg Phosphatase Hydrogylase Lipid
protein Activity Activity Peroxides
~t"/mg protein~16/mg proteinnmoUmg
protein
Vehicle-18 0.24 ~ 0.05 31.2 + 4.1 1.26 t 0.06 2.96 + 0.43
h f
Vehicle-18 0.19 ~ 0.05 12.8 ~ 2.1 0.18 t 0.03 4.83 t 0.50
h + '
CCl4
ac-T-18 h 0.40 + 0.13 13.6 + 1.5' 0.17 t 0.02 2.72 t 0.51
+
CCh
TS-tris-6 1.81 t 0.40' 19.9 t 0.8''f0.28 t 0.03' 3.07 f 0.26
h +
CC14
-25-
Printed by VisualPatent
CA 02366884 2001-10-O1
WO 00159488 PCT/US00/08524
TS-tris-18 3.83 + 0.55' 23.9 t 4.8'~f0.15 t 0.03' 2.85 t 0.56
h +
CCl4
a Rats received a 0.19 mmol/kg ip injection of the tocopherol analog, 6 or 18
h prior to
receiving CC14 ( 1 g/kg). Each. rat was fasted for an 18 h period prior to
sacrifice, and the
vehicle was saline. Rats were sacrificed and microsomes were isolated 4 h
after CCl4
administration. Following isolation, rnicrosomes were washed and resuspended
in 0.154 M
KCI, 50 mM tris, pH 7.4 buffer.
b Units are expressed as ~,mol phosphate formed per h at 37° C.
c Units are expressed as nmolp-nitrocatechol formed per min at 37° C.
d Values expressed as the mean =~ SD (n = 3 or 4).
a Values are significantly different (p < 0.05) from vehicle treated rats.
f Values are significantly different (p < 0.05) from CCl4 treated rats.
EXAMPLE 3.
E, f~''ect of the Administration of TS-tris and TSE-tris on Mitochondria)
Lipid Peroxidation
Using an ADP/Fe system to generate an oxidative stress, Fig. 1 shows that
hepatic
mitochondria isolated from TS-tris treated rats are protected against
oxidative stress-lipid
peroxidation. (Lipid peroxidation is measured by an increase in fluorescence.)
By contrast,
hepatic mitochondria isolated from TSE-tris treated or vehicle (saline)
treated rats (data not
shown) are susceptible to oxidative stress-induced lipid peroxidation. From
Table 1 we know
that TS-tris administration results in an elevation of mitochondria) oc-T and
TS levels as
compared to the levels measured in TSE-tris and control mitochondria). Thus
these findings
support the hypothesis that an elevation in tissue, cellular or subcellular cx-
T and TS
concentrations result in an enhanced antioxidant capacity and prevention of
oxidative-stress
mediated damage.
EXAMPLE 4.
The E,f,~''ect of d a Tocopherol ('G~=T), d a Tocopherol Hemisuccinate Tris
Salt (TS Tris), and
d l~-Tocopheryloxybutyrate Tris Salt (TSE-tris) Administration on Tocopherol
Analog
Levels in Tissue from Rats, 18 Hours after a Single Dose.
In rats receiving a single ip dose of a-T, the tissue concentrations of oc-T
were
-26-
Printed by VisualPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
significantly elevated (as compared with control rats) in all tissues measured
(liver, kidney,
heart, lung, plasma, and blood), except for the brain. In TS-Iris (ip) treated
rats, the amount
of T-equivalents (a-T + TS) measured in each tissue (except brain) exceeded
that observed
for oc-T treated animals. The greatest concentration of TS and T-equivalents
was found in
liver. However, the tissue a-T concentration in TS-tris (ip) treated rats
rarely exceeded that of
a-T treated animals, 18 h following vitamin E administration. These data
suggest that TS-tris
administration can deliver more vitamin E to tissues (than a-T) but 18 hr
following
administration may not be sufficient time for adequate release of a-T from the
TS tissue
reservoirs. The observation that TS-Iris treated animals do not have
significantly greater
tissue a-T levels, as compared with ~c-T administration, may result from
tissue esterase
activities that limit the rate of a-T release from TS or the rate of
elimination of tissue TS or
TS-released oc-T from each tissue may be accelerated (transport out of the
tissue or
utilization). These possibilities will be examined in future studies (Tables 4
and S) by
measuring tissue levels of vitamin E analogs in tissue, 72 and 120 h following
a single
administration of these vitamin E compounds. The iv administration of TS-tris
resulted in a
tissue distribution of TS and a-T that was nearly identical to that achieved
with TS-tris
administered intraperitoneally. The only exception was in the lung, where TS
levels of
intravenously treated animals were almost 5-fold the levels observed in the
lungs of rats which
had received TS-tris intraperitoneally. These results are most likely due to
larger liposomes
being trapped by the first capillary bed which with iv administration would be
the lung
(whereas with ip injections the large liposomes may not leave the
intraperitoneal space).
The administration of TSE-tris (intraperitoneally) resulted in TSE
accumulation in all
tissues measured except the brain. Tissue TSE accumulation was considerably
lower than that
observed for TS-tris (ip) and the liver contained the highest concentration of
TSE.
Interestingly, the oral administration of TSE-tris by oral administration
resulted in plasma
levels that were nearly identical to that observed with ip administration.
These findings
indicate that the oral absorption of TS-tris is excellent and that oral
administration is a viable
route for this anti-tumor agent. These data also suggest that other non-
hydrolyzable vitamin E
derivatives (such as TS-2,2 dimethyl) can also be administered orally.
-27-
Printed by VisuatPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
TABLE 3. Tocopherol Analog Levels in Tissue from Rats, 18 Hours after single
Treatment
with d-a-Tocopherol (a-T), d-a-Tocopherol Hemisuccinate Tris Salt (TS-Tris),
and d-a-
Tocopheryloxybutyrate Tris Salt ('rSE-tris).
Analog Tissue
Concentration
(nmoUg
or nmol/ml)
Administered'Detected Liver Brain Kidney Heart
Control a-T 25.7 t 21_7 ~ 0.9 19.1 ~ 32.1 t 1.1
1.36 I I 1.5 I
T (ip) a-T 101.3 ~ 25.712.9 29.0 t 40.915.6
21.0 I 4.5 ~
TS-tris (ip)a-T 85.0 t 25.3 t 3.9 32.0 ~ 34.9 t 2.6
2.5 2.2
TS 313.4141.1ND 21.0 ~ 22.5 ~ 3.9
2.8
T-equiv. 398.4 ~ 25.7 t 3.9 53.0 t 57.4 t 1.8
40,0 1.1
TS-tris (iv)a-T l 13.3 17.7 ~ 0.3 31.8 ~ 38.7 t 2.2
t 6.4 2.4
TS 342.237.5 1.1 X0.6 31.02.7 33.811.9
T-equiv. 455.6 t 18.8 t 0.9 62.8 t 72.5 t 3.4
34.4 5.0
TSE-tris a-T 23.3 ~ 33.6 ~ 7.2 33.6 ~ 33.2 f 6.2
(ip) 2.9 9.3
TSE 137.918.2 ~ 0.50.9 ~ 8.211.7 ~ 3.60.4
Analog Tissue Concentration
(nmoUg or
ml)
Administered Detected Lung Plasma Blood
Control a-T 37.7 t 7.6 12.9 t 0.8 12.6 t 1.0
T (ip) a-T 85.3 ~ 23.5 20.715.8 17.0 ~ 3.9
TS-tris (ip) a-T 52.4 t 6.8 24.4 t 5.6 19.6 t 2.5
TS 49.017.5 29.87.2 2.5.713.4
T-equiv.d 101.3 t 11.7 39.3 t 6.7 47.6 t 6.8
-28-
Punted by VisuaIPatent
CA 02366884 2001-10-O1
WU 00/59488 PCT/US00/08524
TS-tris (iv) a-T 68.2 t 1.9 25.9 t 3.4 24.1 t 3.1
TS 229.0 t 28.9 23.0 t 2.6 18.4 ~ 1.8
T-equiv. 297.2 t 29.7 48.9 f 5.6 42.4 ~ 4.6
TSE-tris (ip)a-T 48.9 t 1.6 18.7 t 1.5 13.7 t 0.7
TSE 18.04.7 11.32.5 10.1 t 1.5
TSE-tris (oral)a-T 12.5 t 2.2
TSE 9.2 ~ 1.7
' Rats received a 0.19 mmol/kg dose (ip, iv, or oral) of the tocopherol
analog, 18 hours prior
to sacrifice. Immediately following sacrifice, tissues were obtained by freeze-
clamp method.
b Values expressed as the mean t SD (n=3-6).
ND, not detected.
d T-equiv. : total T and/or T derivatives detected
EXAMPLE 5.
The E,~''ect of d a-Tocopherol (a T), and d a-Tocopherol Hemisuccinate Tris
Salt (TS
Tris) Administration on Tocopherol Analog Levels in Tissue from Rats, 72 Hours
after a
.Single Dose.
Seventy two hours following a single injection of a-T, tissue a-T levels
continued to
be elevated (as observed after 18 hrs) except for brain, plasma and blood
levels which were
now back to control levels. By contrast, TS-tris treated animals continued to
show plasma
aad blood a-T levels that were markedly elevated and similar to the levels
observed at 18h (2x
contml). Furthermore substantial levels of TS were detected in all tissues
except brain.
(Plasma, blood and liver TS levels at 72 h had declined by approx. 50% from
the 18 h time
point. Interestingly, though approximately 200 nmol/g of TS was lost between
the 18 and 72
hr measurements, liver a-T levels did not change appreciably. These results
suggest that the
liver eliminates hepatic stores of TS and a-T. Our findings also suggest that
TS-tris
administration results in the dramatic maintenance of plasma and blood a-T
levels, thus
providing a continual source of a-T for other tissues. In the kidney, heart
and lung, T-
_29_
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
equivalents (TS + a-T) are maintained at levels similar to that observed at 18
hrs and the a-T
level in each of these tissues is, if anything, on the rise.
TABLE 4. Tocopherol Analog Levels in Tissue from Rats, 72 Hows Following
Single
Treatment with d-a-Tocopherol (a-T) and d-a-Tocopherol Hemisuccinate Tris Salt
(TS-tris)
Tocopherol Tissue
Analog Concentration
(nmoUg
or ml)
Administered'Detected Liver Brain Kidney Heart
Control a-T 25.7 t 21.7 t 0.9 19.1 ~ 1.5 32.1 t
1.3b 1.1
a-T a-T 77.8 + 22.910.8 69.918.7 52.212.8
13.9
TS-Tris (ip)a-T 78.4 t 25.4 t 0.0838.4 t 1.7 62.0 ~
1.3 9.5
TS 151.4173.80 15.45.3 29.816.2
T-equiv. 229.8 + 25.4 t 0.0853.8 t 7.0 91.8 t
75.1 25.7
Tocopherol Tissue
Analog Concentration
(nmol/g
or ml)
Administered'DetectedLung Plasma Blood Spleen Muscle
Control a-T 37.7 ~: 12.9 + 12.6 t 32.6 ~ 5.3
7.6 I 0.8 I 1.0 I
a-T (ip) a-T 74.4 + 9.9 + 12.0 t 996.91150.025.3 t
13.9 1.0 0.3 9.4
TS-Tris (ip)a-T 78.4 ~: 19.1 t 24.3 t 100.0 X6.8 15.3 t
2.6 2.9 1.6 1.5
TS 36.2 ~: 13.812.5 12.1 ~ 232.41114.36.S t
5.2 1.7 2.2
T-equiv.d114.6 32.915.4 36.413.3 332.41121.1I 21.8
a7.8 I I f4.0
~
' Rats received a 0.19 mmol/kg ip injf;ction of the tocopherol analog, 72 hows
prior to
sacrifice. Immediately following sacrifice, tissues were obtained by freeze-
clamp method.
b Values expressed as the mean t SD (n=3-5).
ND, not detected.
d T-equiv. : total T and/or T derivatives detected
-30-
Printed by VisuaIPatant
CA 02366884 2001-10-O1
WO 00/59488 PCTNS00/08524
EXAMPLE 6.
Tihe E, ffect of d !x -Tocopherol (c~ -T), and d a -Tocopherol Hemisuccinate
Tris Salt (TS
Tris) Administration on Tocopherol Analog Levels in Tissue from Rats, 120
Hours after a
Single Dose.
One hundred and twenty hours following a single dose of a-T, the plasma and
blood
a-T levels are at control levels and the a-T level in the remaining tissues
are declining as
compared with the 72 hour time point. In contrast, the total a-T equivalents
(a-T + TS)
found in kidney, heart, lung, plasma and blood obtained from TS-tris (ip)
treated animals is
similar to that observed at 72 h. In these animals, the plasma and blood a-T
and TS levels at
72 and 120 h have not changed. The kidney, heart and lung TS levels [from TS-
tris (ip)
treated rats] are declining at 120 h (as compared with 72 h) but the loss in
TS appears to
result in an concomitant increase in a-T levels in these tissues. These
results suggest that TS-
triis administration offers the advantage of maintaining tissue levels of a-T
and TS for an
e~,;tended length of time and at an enhanced level. Importantly, the loss of
TS in many tissues
appears to be related to its hydrolysis to a-T which is retained by the
tissue. These findings
suggest that this enhanced maintenance of tissue a-T levels will provide an
increased
antioxidant capacity which may protect these tissues from oxidative stress-
induced damage
and disease. In the liver, the a-T concentration is maintained at approx. 70-
80 nmol/g, a level
observed at 18, 72 and 120 h., even though hepatic stores of TS are continuing
to be lost
dtu~ing this time period.
These results suggest that the liver has a limited capacity to store high
levels of a-T
an.d/or TS. In one rat administered TS-tris (intravenously), the tissue
distribution of a-T and
TS was nearly identical to that described above for TS-tris (ip). This again
suggests that TS-
tris administered intraperitoneally and intravenously result in similar tissue
distributions of TS
anal a-T.
TABLE 5. Tocopheml Analog Levels in Tissue from Rats, 120 Hours after Single
Treatment
with d-a-Tocopherol (a-T), d-a-Tocopherol Hemisuccinate Tris Salt (TS-tris) or
d-a-
Tocopheryloxybutyrate (TSE-tris).
-31-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
'WO 00/59488 PCT/US00/08524
Tocop6erol Tissue Concentration
Analog (nmoUg
or ml)
Administered'Detected Liver Brain Kidney Heart
Control f a-T f 25.7b ~ 21.78 t 19.1 ~ 32.1 ~ 1.1
1.3 0.9 1.5
a-T (ip) a-T ~ 50.8 ~ 18.525.4 t 3.4 31.5 ~ 44.8 t 6.7
, ~ ~ 11.8
TS-tris (ip)a-T 74.0 ~ 25.028.2 t 3.0 42.0 ~ 68.7 t 13.8
i 1.2
TS 63.5 ~ 40.8ND' 10.2 ~ 15.4 t 6.2
7.3
T-equiv. 137.5 ~ 28.2 ~ 3.0 52.2 t 82.5 ~ 20.0
65.8 18.5
TS-tris (iv)a-T 47 28 39 59
TS 103 1 2 17
T-equiv. 150 29 41 76
Tocopherol Tissue Concentration
Analog (nmoUg
or ml)
Administered'Detected Lung Plasma Blood Spleen
Control a-T 37.7 ~ 7.6 12.9 ~ 0.8 12.6 t 32.6 ~ 6.3
1.0
a-T (ip) a-T 46.6 ~ 8.0 11.5 t 1.6 13.9 t 477 t 312
1.2
TS-tris (ip)a-T 92.7 ~ 10.125.6 t 4.8 28.1 t 101.1 t
5.3 14.8
TS 17.2 ~ 7.2 13.0 t 5.9 8.215.3 70.4142.7
T-equiv ' 109.9 I 38.6 t ~ 36.3 171.5 ~
a t 17.3 10.7 t 10.6 57.5
TS-tris (iv)a-T 85 19 23 92
TS 16 10 11 128
T-equiv. ~ 101 , 29 ~ 34 ~ 220
Rats received a 0.19 mmol/kg ip or iv injection of the tocopherol analog, 102
hovers prior to
sacrifice. Immediately following sacrifice, tissues were obtained by a freeze-
clamp method.
-32-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
'WO 00/59488 PCT/L1S001085Z4
b Values expressed as the mean t SD (n=3-S), except iv treatment (n=1).
ND, not detected.
d T-eduiv. : total T and/or T derivatives detected
EXAI.VIPLE 7.
E, fect of TS TRIS Suspension in the Presence and Absence of Taxol on the
Viability of
Ocularr Melanoma Tumor (OCM 1) Cells .
These experiments were conducted to examine the antitumor properties of TS-
tris and
the ability of TS-tris treatment to enhance the ability of other traditional
antitumor agents such
as taxol to induce tumor cell death. As can be seen in Figure 2A, suspensions
of TS-tris
(sonicated in water) added to a human ocular melanoma tumor cell line resulted
in significant
tumor cell killing at a medium concentration of 25 micromolar. The addition of
T at similar
concentrations is not cytotoxic to these tumor cells (data not shown). The
effect of taxol and
taxol in combination with TS-tris on tumor cell viability was also examined.
As can be seen in
Figure 2B, taxol alone is a potent antitumor agent with approximately 95% cell
kill observed
with a 10 nanomolar concentration in the medium. However, even at 10
micromolar
concentrations of taxol in the medium, 100°ro cell kill was not
observed. Interestingly, the
addition of TS-tris and taxol allows for I OOai° kill of tumor cells
that normally is not observed
with t~axol alone (see Fig. 2B). 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
combination confers a distinct advantage over administration of taxol alone.
EXAPvIPLE 8.
The E,;~''ect of d y Tocopherol Hemisuccinate Tris Salt (y TS-tris) and
Tocotrienol-rich
Fraction Hemisuccinate Tris Salt (TRP S-tris) Administration on Tacopherol and
Tocoh~ienol Analog Levels in Tissues from hats, 18 and 120 Hours after a
Single Dose
In previous examples in this application we have clearly demonstrated that a-
TS-tris
has a distinct and significant advantage (as compared to unesterified a-
tocopherol) in terms of
delivering to tissue, cells and subcellular fractions large amaunts of TS that
can serve as a a-T
reservoir for the release of a-T over time. In addition, we have shown that oc-
TS-tris also has
an advantage in terms of providing a dramatic maintenance of plasma and blood
oG-T levels (in
-33-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT1US00/08524
concentration and over time), thus providing a continual source of a-T for
blood (lipoproteins
and formed elements) and other tissues. In the present example, we
investigated whether a
change in the structure of the tocopherol molecule would have an effect on the
tissue delivery
properties of a-TS-tris mentioned above. Vitamin E is a generic term that
includes, in nature,
eight substances, d-oc-, d-(3-, d-y-, d-S-tocopherol and d-ot-, d-(3-, d-y-, d-
8-tocotrienol.
Thus, in the present example we examined the tissue distribution of d-a- and d-
y-tocopherol
and d-a-, d-y- and d-8-tocotrienol in rats administered in a single dose (0.19
mmol/kg ip
injection) of y-TS-tris or TItF-S-tris. For administration, an aqueous
suspension was prepared
for each compound as previously described for a-TS-tris.
The findings from the studies examining the tissue distribution following y-TS-
tris
administration showed that y-TS-tris has tissue distribution properties
similar to those
described in previous examples for a-TS-tris. This is a significant finding
since it is well
known that although y-T is the most abundant form of tocopherol found in
nature (plants) and
has excellent antioxidant properties, it is poorly retained by our tissues. As
a result of these
properties, y-T is found in low concentrations in our cells and tissues
(approx. 1-3 nmollg of
tissue) as compared to OG-T which has a 10-fold higher tissue concentration
(20-40 nmol/g
tissue) (see Tables 5 and 6). Since y-T' has recently been shown to be a more
effective
antioxidant than a-T against some oxidative insults, the ability to enhance y-
T tissue
accumulation may provide important therapeutic benefits. To briefly summarize
the results of
our studies (as shown in Table 6), 18 h following the single administration of
rats with y-
TS-tris, significant y-TS levels were detected in all tissues (except brain)
with the liver
containing 363 nmol/g tissue (a similar concentration as observed following a-
TS-tris
administration (see Table 3). The i>r vavo hydrolysis rate for y-TS appears to
be faster than for
oG-TS as noted by the near absence of tissue y-TS, 120 h following
administration (Table 6).
This finding agrees with that predicted from the structure of y-TS where the
absence of a
methyl group surrounding the ester linkage would be expected to promote an
accelerated
esterase hydrolysis rate. As with oc-TS-tris administration, the injection of
y-TS-tris
suspensions in rats resulted in a dramatic increase and maintenance of plasma
and blood levels
of y-T. These data suggest the enhanced delivery of tocopherols (regardless of
the number of
methyl groups on the chromanol ring) to tissue can be accomplished by
administering an
aqueous suspension of the succinate ester tris salt.
-34-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
The second substance examined, TRF-S-tris, is a mixture of vitamin E compounds
containing the succinate ester tris salt of d-a- tocopherol (28%) and d-a-
(35%), d-'y-(22%),
and d-S-tocatrienol ( 16%). The presence of the succinate ester of each of
these vitamin E
derivatives was confirmed by HPLC analysis using base hydrolysis. In naive rat
tissues, these
tocotrienol compounds were not detectable using our standard analytical
methods. In rats, 18h
following the single injection of TRI~-S-tris (0.19 nmol/kg intraperitoneally)
significant vitamin
E levels in tissues were only observed far oc-TS. In contrast, each
tocotrienol succinate
compound did not appear to accumulate in tissues to any appreciable degree
including the
blood (approximately less than 1 nmol/g tissue was detected). Thus, these
results agree with
our previous experiments using «-TS-tris administration (both had similar
tissue distributions
regardless of administering alone ar in combination with other vitamin E
succinate ester tris
salts). However, the absence of tissue tocotrienol accumulation indicates that
changing the
phytyl tail of the tocopherol molecule to an isoprenoid side chain (as with
tocotrienal
compounds) dramatically alters the transport and/or retention of these
compounds by tissues
in vivo.
TABLE 6. Tocopherol Analog Levels in Tissue from Rats 18 or 120 Hours after
Single
Treatment with d-'y-Tocopherol ('~-T) or d-'y-Tocopherol Hemisuccinate Tris
Salt ('y- TS-
tris).
Tocopherol Tissue Concentration
Analog (nmoUg
or ml)"
Administered'Detected Liver Brain Kidney Heart
Control T 1.910.4 1.6x0.3 2.4x0.6 4.Of0.5
Y-T T 81.6181.8 2.O x 1.5 49.5138.4 12.8 6.9
(18 hrs.)
Y-T T 24.3x9.1 1.5x0.0 23.2x4.5 21.0x5.9
(120 hrs.)
'y-TS-tris T 126.1 x 2.8 x 0.2 15.8 x 24.0 x
(18 6rs.) 16.8 4.2 2.0
-35-
Printed by VisualPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00108524
TS 362.6f 1.011.0 8.35.0 11.2f2.7
105.9
T-equiv. 488.7 f 3.8 f 0.9 24.1 t 35.2 ~
104.6 9.2 4.6
y-TS-tris T 24.4 t 15.62.6 ~ 0.2 10.1 ~ 24.5 ~
(120 hrs.) 2.1 4.7
TS 5.5 t 10.5 1.8 t 3.5 ND' ND
T-equivd 29.9 ~ 25.94.3 ~ 3.5 9.9 f 2.4 23.9 ~
4.5
Tocopherol Tissue Concentration
Analog (nmoUg
or ml)
Administered'Detected Lung Spleen Plasma Blood
Control T 3.9 ~ 0.2 0.9 t 0.1 0.9 ~ 0.1
y-T T 19.2110.3 212.0 1.010.9 1.7~ 1.5
(18 hrs.) 153.8
y-T T 17.913.4 497.4 1.3f0.1 2.20.3
(120 6rs.) 360.6
y-TS-tris T 57.6 ~ 7.6 111.0 ~ 12,2 f 16.8 f
(18 6rs.) 19.6 2.6 1.8
TS 22.3f4.8 318.Sf 12.913.0 10.711.8
219.8
T-equiv. 79.8 t 11.9429.5 t 25.1 t 27.5 t
360.6 5.5 3.6
y-TS-tris T 27.9 t 5.0 42.4 f 9.1 4.0 t 0.9 6.5 t 1.5
(120 hrs.)
TS 0.110.2 10.2 13.2 0.80.1 O.Sf0.1
-36-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
T-equiv 27.9 ~ 4.7 52.6 X20.9 4.7 ~ 0.9 7.0 ~ 1.5
a Rats received a 0.19 mmollkg, intraperitoneally at the indicated number of
hours prior to
sacrifice. Immediately following sacrifice, tissues were obtained by a freeze-
clamp method.
b Values expressed as the mean ~ SD (n=3-6).
ND, not detected.
d T-equiv. : total T and/or T derivatives detected
E?~',AMPLE 9. E,~'-ect of TS TRIS admin:.stration on the delivery of TS and T
to liver,
hepatocytes, and mitochondria and protection against iron-induced lipid
peroxidation and
cell death.
Our previous studies (Exarnples 1 and 3) demonstrate that the administration
of cx-TS-tris,
results in the accumulation of TS and T in mitochondria and that these
mitochondria are
protected against iron-induced lipid peroxidation. To further characterize
this apparent
directed subcellular transport and accumulation of OG-TS, we developed an
improved method
for the purification of isolated mitochondria (respiring mitochondria method)
to reduce the
contamination of lysosomes in the preparation (it is well known that aT
accumulates in
lysosomes). Using this improved isolation method, we measured the amount of aT
and otTS
in liver homogenates, liver mitochondria, liver outer mitochondria) membranes
and liver inner
mitochondria) membranes in rats, 18 h following the administration of a single
dose (0.19
mmol/kg, intraperitoneally) of aT or ot'TS-tris. As shown in Figure 3, oGTS-
tris administration
resulted in a 10 fold increase in ocT equivalents (aT + aTS) and a 2 fold
increase in aT levels
as compared to homogenate from aT-treated rats. In purified hepatic
mitochondria isolated
from aT-treated rats (Figure 4), the ocT concentration did not significantly
differ from that
observed in mitochondria obtained from control (naive-no treatment) rats. In
contrast, TS-tris
administration resulted in dramatic increases in both aT (10-fold higher than
aT-treated) and
aTS levels, indicating that aTS has a unique ability to selectively
concentrate in mitochondria
(as compared to homogenate and ocT-treatment data). Next, respiring
mitochondria were
isolated from the treated rats (as described above) and exposed to high levels
of iron (a well
known oxidative challenge). Using mitochondria) swelling as an indicator of
mitochondria
damage and dysfunction, we found that only mitochondria isolated from the
livers of TS-tris
treated rats were completely protected against iron-induced damage (data not
shown). Since
-37-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
mitochondria are considered the most important cellular site for the
production of reactive
oxyl;en species and thus a potential cause for numerous oxidative-stress
related diseases, the
ability of oGTS-tris administration to load mitochondria with oc'TS and then
release large
amounts of the antioxidant OGT has tremendous implications in terms of a novel
therapeutic
strategy for the prevention and treatment of oxidative stress-related
diseases. To insure that
these mitochondria) stores of aT and OcTS (observed following ocTS-tris
administration) do
indeed accumulate at submitochondrial sites (inner and outer membranes) where
most ROS
are generated, we ~ easured ocT and aTS levels in isolated inner and outer
mitochondria)
membranes from the mitochondria described above, and then measured their
susceptibility to
oxidative damage (lipid peroxidation) following exposure to iron (an
endogenous metal
thought to be responsible for the propagation of ROS in many human diseases).
The results
from these studies are shown in Figures 5-'7 and clearly demonstrate that aTS-
tris
administration results in large accumulations of aTS and aT in the inner and
outer membrane
of hepatic mirochondria, and this dramatic accumulation (as compared with aT-
treatment)
results in complete protection of these membranes against a strong oxidative
challenge (a lag
time of 300 indicates complete protection against membrane lipid peroxidation
in Figure 7).
These findings (Figures 5-7) also demonstrate the inability of T, administered
acutely, to be
rapidly transported and retained by mitochondria and provide antioxidant
protection. Though
it is clear from these studies that otTS-tris administration protects
mitochondria from
iron-mediated damage, the implications of this treatment for cell viability
were unknown. Thus
we investigated whether viable cells isolated from the livers of rats treated
with otTS-tris (as
described above) were also protected against the toxic effects of iron
exposure. As shown in
Figuu~e 8, IxTS-tris treatment does indeed protect hepatocytes from cell death
induced by the
oxidative challenge of iron, unlike control cells or cells isolated from aT-
treated rats. These
findnngs support the conclusion that oxidative stress-mediated toxicity can be
prevented by
enhancing the antioxidant capacity of mitochondria and oc-TS-tris
administration appears to
accomplish this remarkably well.
While the invention has been described in terms of the preferred embodiment of
the
invention, those skilled in the art will recognize that other vitamin E
compounds, other salts,
different concentrations, and different means of administration can be
employed within the
spirit and scope of the appended claims.
-38-
Printed by VisualPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
REFERENCES
1. Ames, B.N., Shigenaga, M.K. and Hagen, T.M. (1993) Oxidants, antioxidants,
and the
degenerative diseases of aging. Proc. Natl. Acad. Sci. USA. 90, 791 S-7922.
2. Kehrer, J.P. (1993) Free radicals as mediators of tissue injury and
disease. Crit. Rev.
Toxicol. 23, 21-48:
3. Reed, D.J. (1985) Cellular defense mechanisms against reactive metabolites.
In
Bioactivation ofForeign Compounds (Anders, M.W., ed.), pp 71-108, Academic
Press,
New York.
4. Tappet, A.L. (1962) Vitamin E as the biological lipid antioxidant. Vitam.
Horm.20, 493-
510.
5. Burton, G.W., Joyce, A. and Ingold, K.U. (1983) Is Vitamin E the only lipid-
soluble, chain
breaking antioxidant in human blood plasma and erythrocyte membranes? Arch.
Biochem.
Biophys.221, 281-290.
6. Cheeseman, K.H., Emery, S., Maddix, S.P., Slater, T.F., Burton, G.W. and
Ingold, K.U.
(1988) Studies on lipid peroxidation in normal and tumor tissues. Biochem. J.
250, 247-
252.
7. Burton, G.W. and Ingold, K.U. (1989) Vitamin E as an in vitro and in vivo
antioxidant Ann.
N. Y. Acad Sci. 570, 7-22..
8. Liebler, D.C. (1993) The role of metabolism in the antioxidant function of
vitaminE. Crit.
Rev. Toxicol. 23, 147-169.
9. Kehrer, J.P. and Lund, L.G. (1994) Cellular reducing equivalents and
oxidative stress. Free
Radic. Biol. Mec~ 17, 65-75.
10. Traber, M.G. (1994) Determinants of plasma vitamin E concentrations. Free
Radic. Biol.
Med. 16, 229-239.
11. Cohn, W., Gross, P., Grun, H., Loechleiter, F. Muller, D.P.R. and Zulauf,
M. (1992)
Tocopherol transport and absorption. Proc. Nutri. Soc. 51, 179-188.
12. Perly, B.L, Smith, C.P., Hughes, L., Burton, G.W., and Ingold, K.U. (1985)
Estimation of
the location of natural alpha tocopherol in lipid bilayers by C-NMR
spectroscopy.
Biochem. Biophys. Acta. 819, 131-135.
13. Kornburst, D.J. and Mavis, R.D. (1980) Relative susceptibility of
microsomes from lung,
heart, liver, kidney, brain and testes to lipid peroxidation: Correlation with
vitamin E
content. Lipids 1 S, 315-322.
-39-
Printed by VisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00I085Z4
14. Liebler, D.C., Kling, D.S., and Reed, D.J. (1986) Antioxidant protection
of phospholipid
biilayers by oc-tocopherol. J. Biol. Chem. 261, 12114-12119.
15. Leedle, R.A. and Aust, S.D. ( 1986) Importance of the polyunsaturated
fatty acid to
vitamin E ratio in the resistance of rat lung microsomes to lipid
peroxidation. J. Free
Radic. Biol. Med. 2. 397-403.
16. Lai, M., Duzgunes, N. and Szoka, F.C. {1985) Cellular reducing equivalents
and oxidative
stress. Biochemistry 24, 1646-1653.
17. Murphy, D.J. and Mavis, R.D. (1981) A comparison of the in vitro binding
of a-
tacopherol to microsomes of lung, liver, heart, and brain of the rat. Biochim.
Biophys.
A~cta. 663, 390-400.
18. Niki, E., Kawakami, A., Saito, M., Yamamoto, Y., Tsuchiya, J., and Kamiya,
Y. (1985)
Effect of phytl side chain of vitamin E an its antioxidant activity. J.
Biachm. 260, 2191-
2196.
19. Catignani, G.L. and Bierei, J.G. (1977) Rat liver ot-tocopherol binding
protein. Biochim.
Bxophys. Acta. 497, 349-357.
20. Murphy, D.J. and Mavis, R.D. (1981) Membrane transfer of oc-tocopherol:
Influence of
soluble a-tocopherol binding factors fmm the liver, lung, heart and brain of
the rat. J.
Biol. Chem. 256, 10464-10468.
21. Dutta.-Roy, A.K., Gordon, M.J., Leishman, D.J., Paterson, B.J., Duthie,
G.G. and James,
W.P\T. (1993) Purification and partial characterization of an a-tocopherol-
binding protein
from rabbit heart cytosol. Mol. Cell. Biochem. 123, 139-144.
22. Gnarnieri, C., Flamigni, F., and Caldareray C.M. (1980) A possible role of
rabbit heart
cy~tosol tocopherol binding in the transfer of tocopherol into nuclei.
Biochem. J. 190, 469-
4i'1.
23. Mowri, H., Nakagawa, Y., Inoue, K. and Nojuma, S. (1981) Enhancement of
the transfer
of a-tocopherol between liposvmes and mitochondria by rat liver protein(s).
Eur. J.
Biochem. 117, 537-542.
24. Bf;hrens, W.A. and Madere, L.T. (1982) Transfer of a-tocopherol to
microsomes
m~liated by a partially purified liver a-tocopherol binding protein. Nutr.
Res. 2, 611-618.
25. Fariss, M. W., Bryson, K. F., Hylton, E. E., Lippman, H. R., Stubin, C.
H., Zhao, X.-G.
(1993) Protection against carbon tetrachloride-induced hepatotoxicity by
pretreating rats
with the hemisuccinate esters of tocopherol and cholesterol. Erwiron. Health
Perspect.
101, 528-536.
-40-
Printed by VisuaIPatenf
CA 02366884 2001-10-O1
WO 00/59488 PCTi'US00108524
26. Fariss, M. W., Fortuna, M. B., Everett, C. K., Smith, J. D., Trent, D. F.,
Djuric, Z. ( 1994)
The selective andproliferative effects of a-tocopheryl hemisuccinate and
cholesteryl
hemisuccinate on marine leukemia cells result fram the action of the intact
compounds.
Cancer Res. 54, 3346-3351.
27. Tirmenstein, M. A., Leraas, T. L., F'ariss, M. W. (1997) a-Tocopheryl
hemisuccinate
administration increases rat liver subcellular a-tocopherol levels and
protects against
carbon tetrachloride-induced hepatatoxicity. Toxicol. Lett. 92, 67-77.
28. Tirmenstein, M. A., Nelson, S. D. ( 1989) Subcellular binding and effects
on calcium
homeostasis produced by acetaminophen and a nonhepatotoxic regioisomer, 3'-
hydroxyacetanilide, in mouse liver. J. Biol. Chem. 264, 9814-9819.
29. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. ( 1951 )
Protein measurement
with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.
3~0. Peterson, G. L. (1977) A simplification of the protein assay method of
Lowry et al. which
is generally more applicable. Anal. Biochem. 83, 346-356.
31, Fariss, M. W., Merson, M. H., O'Hara, T. M. ( 1989) a-Tocopheryl succinate
protects
hepatocytes from chemical-induced toxicity under physiological calcium
conditions.
Toxicol. Lett. 47, 61-75.
32. Tirmenstein, M. A., Watson, B. W., Haar, N. C., Fariss, M. W. (1998)
Sensitive method
for measuring tissue a-tocopherol and a-tocopheryloxybutyric acid by high
performance
liquid chromatography with fluorimetric detection. .J. Chromatogr. B 707, 308-
311.
33. Koop, D. R. (1986) Hydroxylation ofp-nitrophenol by rabbit ethanol-
inducible
cytochrome P-450 isozyme 3a. Mol. Pharmacol. 29, 399-4.04.
34. Speerschneider, P. and Dekant, W . ( 1995) Renal tumorigenicity of 1,1-
dichloroethene in
mice: the role of male-specific expression of cytochrome P450 2E1 in the renal
bioactivation of 1,1-dichloroethene, ToxicoL Appl. Pharmacol. 130, 48-56.
3.'>. Ohkawa, H., Ohishi, N., Yagi, K. (1979) Assay for lipid peroxides in
animal tissue by
thiobarbituric acid reaction. Anal. Biochem. 95, 351-358.
3fi. Aronson, N. N. and Toaster, O. (1974) Isolation of rat liver plasma
membrane fragments
in isotonic sucrose. Methods Enzymol. 31, 90-102.
3 7. Padron, A. G., De Toranzo, E. G. D., Castro, J. A. ( 1996) Depression of
liver
microsomal glucose 6-phosphatase activity in carbon tetrachloride-poisoned
rats.
Potential synergistic effects of lipid peroxidation and of covalent binding of
haloalkane-
derived free radicals to cellular components in the process. Free Radic. Biol.
Med. 21,
81-87.
_41 _
Printed by UisuaIPatent
CA 02366884 2001-10-O1
WO 00/59488 PCT/US00/08524
38. Wolfgang, G. H. L, Donarski, W. J., Petry, T. W. (1990) Effects of novel
antioxidants on
carbon tetrachloride-induced lipid peroxidation and toxicity in precision-cut
rat liver slices.
Toxicol. Appl. Pharmacol. 106, 63-70.
39. Kefalas, V., Stacey, N. H. (1989) Potentiation of carbon tetrachloride-
induced lipid
pemxidation by trichloroethylene in isolated rat hepatocytes: no role in
enhanced toxicity.
Toxicol. Appl. Pharmacol. 101, 158-169.
40. Yamamoto, H. (1990) Relation of Ca++ accumulation and lipid peroxidation
with CCl4
induced toxicity in the rat liver. Pharmacol. Toxicol. 66, 213-216.
4 t . Masuda, Y. and Nakamura, Y. ( 1990) Effects of oxygen deficiency and
calcium omission
on carbon tetrachloride hepatotoxicity in isolated perfused livers from
phenobarbital-
pretreated rats. Biochem. Pharmacol. 40, 1865-1876.
42. Morrow, J. D., Awad, J. A., Kato, T., Takahashi, K., Badr, K. F.;,Roberts,
L. J., Burk, R.
F. (1992) Formation of novel non-cyclooxygenase-derived prostanoids (F2-
isoprostanes)
in carbon tetrachloride hepatotoxicity. J. Clin. Invest. 90, 2502-2507.
43. Laskin, D. L., Pendino, K. J. (1995) Macrophages and inflammatory
mediators in tissue
injury. Annu. Rev. Pharmacol. Toxicol. 35, 655-677.
44. Kagan, V. E., Serbinova, E. A., Bakalova, R. A., Stoytchev, T. S., Erin,
A. N., Prilipko,
L. L., Evstigneeva, R. P. (1990) Mechanisms of stabilization of biomembranes
by alpha-
tocopherol. Biochem. Pharmacol. 40, 2403-2413.
45. Jizomoto, H., Kanaoka, E., Hirano, K. (1994) pH-sensitive liposomes
composed of
tocopherol hemisuccinate and of phosphatidylethanolamine including tocopherol
hemisuccinate. Biochim. Biophys. Acta 1213, 343-348.
46. Wu, J. Zern, M. A. (1996) Modification of liposomes for liver targeting.
J. Hepatol. 24,
757-763.
47. Kao, Y. J., Juliano, R. L. (1981) Interaction of liposomes with the
reticuloendothelial
system effects of reticuloendothelial blockade on the clearance of large
unilamellar
vesicles. Biochim. Biophys. Acta 677, 453-461.
48. Tiemey, D.J., Haas, A.L., and Koop, D.R. (1992) Degradation of cytochrome
P450 2E1:
selective loss after labilization of the enzyme. Arch. Biochem. Biophys. 293,
9-I 6.
49. Tirmenstein, M.A., Pierce, C.A., Leraas, T.L., and Fariss, M.W. (1998) A
fluorescence
plate reader assay for monitoring the susceptibility of biological samples to
lipid
peroxidation. Anal. Biochem. 265, 246-252.
50. Fariss, M.W., Brown, M. K., Schmitz, J. A., and Reed, D. J. (1985)
Mechanism of
chemically induced toxicity. Toxico~ and Appl. Pharmacol. 79, 283-295.
_q.2_
Punted by VisuaIPatent
CA 02366884 2001-10-O1
'WO 00/59488 PCT/US00/08524
51. L;ai, M-Z., Duzgunes, N., and Szoka, F.C. (1985) Effects of replacement of
the hydroxyl
group of cholesterol and tocopherol on the thermodynamic behavior of
phospholipid
membranes. Biochemistry 24:1646-1653.
52. Janoff, A.S., Kurtz, C.L., Jablonski, R.L., Minchey, S.R., Boni, L.T.,
Gruner, S.M., Cullis,
P..R., Mayer, L.D. and Hope, M.J. (1988) Characterization of cholesterol
hemisuccinate
and oG-tocopherol hemisuccinate vesicles. Biochem. Biophys. Acta, 941, 165-
175. .
-43-
Printed by VisuaIPatent
