Note: Descriptions are shown in the official language in which they were submitted.
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WO 99/34794 1 PCT/US99/00596
METHODS FOR TREATING DIABETIC
DYSLIPIDEMIA USING TOCOTRIENOLS
TECHNICAL FIELD OF THE INVENTION
This invention relates to the treatment of diabetic dyslipidemia using
tocotrienols. Specifically, this invention relates to the use of individual
tocotrienols
(such as P25 tocotrienol), mixtures of tocotrienols and mixtures of one or
more
tocotrienols with other substances (such as the TRF25 mixture). The methods of
this
invention are particularly well suited for treating diabetic dyslipidemia in
Type 2
diabetic patients.
BACKGROUND OF THE INVENTION
Inadequately controlled hyperglycemia has been cited as a primary cause of
diabetic complications. Increased serum levels of lipids in diabetics (as
observed by
high levels of triglycerides, total cholesterol and LDL-cholesterol) are
partially
responsible for and further exacerbate the damaging effects of diabetic
hyperglycemia.
Because there has been no effective and inexpensive therapeutic option to
control
glycemic or lipidemic levels in diabetic patients, these patients often suffer
severe
complications, including nephropathy, retinopathy, neuropathy and
atherosclerosis.
Although improved glycemic control can reduce the incidence and progression of
diabetic complications, implementation and monitoring of glycemic control is
arduous
and expensive. In addition, results from the recently completed Diabetes
Control and
Complications Trial (DCCT) have shown that even with intensive insulin
treatment,
there is still a significant occurrence of complications in the diabetic
population.
Many diabetic complications are believed to occur through the oxidative
action of glucose. In particular, the high oxidant activity in diabetics
coupled with
dyslipidemia can lead to the formation of advanced glycatian endproducts
(AGEs).
The presence of AGES is associated with the formation of arterial atheromas
and
ultimately, to the development of atherosclerosis. These complications can be
mitigated in part by certain antioxidants, including superoxide dismutase,
catalase and
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WO 99/34794 2 PCT/US99/00596
gluathione. Blocking the oxidative action of glucose responsible for diabetic
vascular
dysfunction has been validated as one approach to reduce the occurrence of
diabetic
complications. For example, aldose reductase inhibitors have been shown to
prevent
or reduce different components of vascular dysfunction, cataract formation,
S neuropathy and nephropathy in animal model. Encouraging results have also
been
observed in diabetic patients in the prevention of neuropathy and retinopathy
using
aldose reductase inhibitors. In addition, glycation inhibitors (such as
aminoguanidine)
have been shown to prevent or reduce vascular dysfunction and microvascular
complications in animal models. Furthermore, antioxidants (such as vitamin E,
vitamin C and alpha Iipoic acid) and antiplatelet agents (such as aspirin and
ticlopidine) are being tested to determine their efficacy against the
progression of
certain diabetic complications, such as non-proliferative diabetic
retinopathy.
Although some strides have been made to control glycemic levels and the
associated oxidant activity in diabetics, little progress has been made to
address the
underlying problem of diabetic dyslipidemia. By lowering levels of
triglycerides, total
cholesterol and LDL-cholesterol in diabetics, development or progression of
atherosclerosis and other diabetic complications could be slowed or
eradicated.
Advantageously, such therapy could be combined with hypoglycemic medications
to
synergistically treat diabetic patients. Therefore, it is of great importance
for the long-
term quality of life for diabetic patients that therapeutic options be made
available to
treat diabetic dyslipidemia.
SUMMARY OF THE INVENTION
The present invention satisfies the need for therapeutic agents effective in
the
treatment of diabetic dyslipidemia. Specifically this invention provides
methods for
treating diabetic dyslipidemia comprising the step of administering to a
diabetic
patient an effective amount of a tocotrienol, a mixture of tocotrienois or a
mixture of
one or more tocotrienols with other substances.
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WO 99/34794 3 PCT/US99/00596
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 A and 1 B show the effects of y-tocotrienol (GT301 ) on superoxide
production in human peripheral blood neutrophils.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the following definitions apply (unless expressly noted to the
contrary):
"Composition" as used herein refers to a preparation for administration via
any
acceptable route known to those of ordinary skill in the art. Such routes
include, but
are not limited to oral, parenteral, transdermal, intravenous or topical
administration.
"Composition" encompasses pharmaceutical compositions as well as dietary
supplements, foodstuffs, food additives and the like.
"Patient' refers to a warm-blooded mammal and preferably, a human.
"P18 tocotrienol" refers to a tocotrienol having the formula
P~8 tocotrienol and P,8 are trademarks of Bionutrics, Inc. (Phoenix, Arizona).
"P2, tocotrienol" refers to the compound 3,4-dihydro-2-methyl-2-(4,8,12-
trimethyltrideca-3'(E), 7'(E), 11'-trienyl)-2H-benzopyran-6-ol. This specific
tocotrienol has been referred to as "tocotrienol" in some of the published
literature
cited herein.
"P25 tocotrienol" refers to the tocotrienol 3,4-dihydro-2-(4,8,12-
trimethyltrideca-
3'(E),7'(E), 11"-trienyl)-2H-1-benzopyran-6-ol) which has the formula:
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WO 99/34794 4 PCT/US99/00596
P25 tocotrienol and P25 are trademarks of Bionutrics, Inc. (Phoenix, Arizona).
"Tocotrienol" refers to compounds possessing the following three structural
characteristics: (1) a hydrogen donor group (or a group that can be hydrolyzed
to a
hydrogen donor group) attached to an aromatic ring system; (2) a side chain
attached to the
aromatic ring system comprising one or more isoprenoid or isoprenoid-like
units and (3) a
methylene unit or a functional group having at least one lone pair of
electrons positioned
adjacent to the atom to which the side chain is attached to the aromatic ring,
said electrons
being conjugated to the aromatic ring system (preferably CHZ, C=O, CHOH, O, S
or NH).
Preferred tocotrienols for use in the methods of this invention are those
which are naturally
occurring. These naturally occurring tocotrienols may be conveniently isolated
from
biological materials or synthesized from commercially available starting
material.
Preferably, the tocotrienols for use in the methods of this invention are
obtained from
biological materials that have been stabilized and extracted, such as by the
processes
described in PCT publication WO 91/17985 (the entire disclosure of which is
hereby
incorporated by reference). Examples of preferred biological materials,
tocotrienols and
methods for obtaining tocotrienols synthetically and from biological materials
are referred
to in co-owned US patent 5,591,772 and PCT publication WO 91/17985 (the entire
disclosures of which are hereby incorporated by reference). Preferred
biological materials
from which the tocotrienols of this invention may be obtained include
stabilized brans and
especially, stabilized rice bran.
Specific preferred tocotrienols of this invention include those of formula
(I):
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WO 99/34794 5 PCT/US99/00596
R~
R2 R4
~(CH2)n
R
R3 / R
Rs
Z
wherein
5 R~ and R3 are each independently selected from the group consisting of H,
halogen,
OH, OCH3 and C~-C6 branched or unbranched alkyl (preferably, H, halogen and C,-
C3 branched or unbranched alkyl and more preferably, H and methyl);
RZ is a hydrogen donor group selected from the group consisting of OH, NHRB,
COZY,
C(R$)zC02H and C,-C8 branched or unbranched alkyl substituted with OH, NHRg,
CHZY
or C(R8)ZCOzH (preferably, OH and C,-C3 branched or unbranched alkyl
substituted with
OH and more preferably, OH);
R4 is selected from the group consisting of O, NH, CH-Rg, C=O and CH-OH
(preferably, O, CHZ and C=O);
RS is selected from the group consisting of CH2, C=O, CHOH, O, S and NH
(preferably, O, CH2 and C=O and more preferably, O and C=O);
R6 is selected from the group consisting of H and C1-C6 branched or unbranched
alkyl
(preferably, H and C,-C3 branched or unbranched alkyl and more preferably, H
and
methyl);
R, is selected from the group consisting of isoprenoid and isoprenoid-like
side chains,
and more preferably from the group consisting of side chains of formulas (a)-
(c):
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WO 99/34794 6 PCT/U599/00596
R1o
R11 (a)
m
R1o
~ O /mRl1
11
(C)
wherein each Rio is independently selected from the group consisting of H, NHZ
and C,-C6
branched or unbranched alkyl and R~~ is selected from the group consisting of
H, C~-C6
branched or unbranched alkyl, CHZOH, COZH and OH (preferably, R, is a side
chain of
formula (a), wherein Rio and Rl ~ are each independently is selected from the
group
consisting of H and CI-C3 branched or unbranched alkyl and more preferably, H
and
methyl);
each Rg and R9 is independently selected from the group consisting of H and C~-
C6
branched or unbranched alkyl (preferably, H and C,-C3 branched or unbranched
alkyl
and more preferably, H and methyl);
Y is H or and C,-C,$ branched or unbranched alkyl (preferably H and C1-C6
branched
or unbranched alkyl and more preferably, H and C1-C4 branched or unbranched
alkyl);
Z is selected from the group consisting of H, halogen, OH, CHZOH, CH3, OCH3
and
COCH3 (preferably H and CH3);
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WO 99/34794 7 PCT/US99/00596
n is an integer selected from the group consisting of 0, 1, 2, 3 and 4
(preferably 0 and
1 ); and
m is an integer selected from the group consisting of'1-30 (preferably 1-20,
more
preferably 3-10 and most preferably, 3-7).
More preferred tocotrienols of this invention include P2, tocotrienol, a-
tocotrienol, ~i-tocotrienol, 'y-tocotrienol, 8-tocotrienol, P~8 tocotrienol
and Pas tocotrienol.
This invention expressly encompasses the prodrug form of tocotrienols. Upon
administration to a patient, such a prodrug undergoes biotransformation to
their active
form. Prodrugs include the esterified form of the tocotrienols used in this
invention
which comprise a carboxylic acid functionality.
The tocotrienols for use in the methods of this invention may be in their
isomerically pure form or be present as mixtures of isomers. For example, the
tocotrienols of this invention may exist as the d- or 1-isomer or the d,l-
racemic
mixture. The naturally occurring isomer (usually the d-isomer) and the d,l-
racemic
mixture are preferred.
"TRF" refers to a tocotrienol rich fraction obtained by the stabilization and
extraction of a biological source. TRF typically contains varying amounts of
P2,
tocotrienol, a-tocotrienol, (3-tocotrienol, y-tocotrienol and 8-tocotrienol
and may also
contain quantities of the newly discovered tocotrienols, P~g tocotrienol and
Pzs
tocotrienol. Most commonly, TRF will be comprise at least about 50% to about
90%
tocotrienols w/w (preferably, at least about 60% to about 90% and more
preferably, at
least about 70% to about 90%).
"TRFZS" refers to a TRF comprising a significant weight percentage of Pzs
tocotrienol. Preferably, TRFZS comprises at least about 5% PZS, more
preferably, at
least about 10% PZS, and even more preferably, at least about 15% PZS w/w. An
example of the preparation of a specific TRFZS is set forth in A.A. Qureshi et
al., Nutr.
Biochem., 8, pp. 290-98 (1997). TRFzs is a preferred component of the
compositions
and methods described herein. TRFZS is a trademark of Bionutrics, Inc.
(Phoenix,
Arizona).
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This invention provides a method for treating diabetic dyslipidemia in a
diabetic patient comprising the step of administering to the patient a
therapeutically
effective amount of a composition comprising a tocotrienol, a mixture of
tocotrienols
or a combination of one or more tocotrienols with one or more additional
substances.
Preferably, the "diabetic patient" is a patient with the symptoms of Type 2
diabetes
mellitus (non-insulin dependent diabetes). "Therapeutically effective amount"
refers
to an amount sufficient to reduce the levels of triglycerides, total
cholesterol and/or
LDL-cholesterol in a diabetic patient. Typically, a therapeutically effective
amount of
a composition according to this invention will reduce the levels of one or
more of
these serum factors by at least about the following percentages: total
cholesterol
reduced by at least about 10%, preferably, by at least about 15% and more
preferably,
by at least 20%; triglycerides reduced by at least about 3%, more preferably,
by at
least about 5% and more preferably, by at least about 10%; and LDL-cholesterol
levels reduced by at least about 10%, preferably, by at least about 15% and
more
preferably, by at least 20%. "Therapeutically acceptable means" refer to means
effective to impart a therapeutic effect.
The methods described herein may be used alone or in conjunction with
conventional therapeutic methods for treating diabetic complications (such as
those
described above). Advantageously, the methods of this invention may be used
together with methods for treating hyperglycemia. Such therapies attack the
underlying causes of diabetic complications on at least two levels by reducing
high
glucose levels (with their attendant oxidant activity) and simultaneously
reducing high
levels of lipids (the oxidative target).
Without wishing to be bound by theory, tocotrienols are useful in treating
diabetic dyslipidemia due to their unique lipid lowering properties.
Tocotrienols
reduce total cholesterol, LDL-cholesterol and triglyceride levels in diabetic
patients.
Furthermore, tocotrienols target several additional mechanisms leading to the
complications caused in part by diabetic dyslipidemia. For example, the
tocotrienols
of this invention inhibit the production of free arachidonic acid (a major
mediator of
inflammatory response). This inhibition is believed to occur by either the
inhibition
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WO 99/34794 9 PCT/US99/00596
of phospholipase AZ or alternatively, through the increase in corticosterone
levels in
the blood. Phospholipase AZ cleaves at C-2 of phosphate head groups, resulting
in the
release of free arachidonic acid. Free arachidonic acid can then be converted
to a
variety of biologically important molecules, such as prostaglandins and
thromboxanes
(via the cyclooxygenase pathway) and the leukotrienes (via the lipoxygenase
pathway). These factors are associated with the increased level of glucose and
triglycerides observed in diabetic dyslipidemic patients.
Furthermore, tocotrienols inhibit the production of a variety of cytokines
(including TNF, IL-1 and growth factors). These cytokines contribute to the
proliferation of smooth muscle and propagation of the inflammatory response
partially
responsible for the development of atherosclerosis and other diabetic
complications
associated with dyslipidemia. Furthermore, tocotrienols reduce the levels of
superoxide production. Superoxide and nitric oxide react to form
peroxynitrite, which
is a causative factor in arterial atherosclerosis. By reducing superoxide and
cytokine
1 S production, tocotrienols reduce the cell proliferation, chemotaxis,
inflammation and
endothelial damage that also play a role in atherosclerosis and other
complications
associated with diabetic dyslipidemia. As a result of their unique combination
of
properties, tocotrienols are capable of combating the root causes and lessen
the
severity of the damaging effects of diabetic dyslipidemia.
Compositions of this invention are prepared by combining one or more
tocotrienols with an acceptable carrier. For pharmaceutical compositions of
this
invention, the carrier must be pharmaceutically acceptable (i.e., a carrier
which is non-
toxic to the patient at the administered level and which does not destroy the
activity of the
active component of the composition). Acceptable carriers, including
pharmaceutically
acceptable carriers, are well known to those of ordinary skill in the art.
The compositions of this invention may be used or administered by any
therapeutically acceptable means to a patient in need of treatment for
diabetic
dyslipidemia. For example, pharmaceutical compositions of this invention may
be
administered orally, topically, transdermally, parenterally, intravenously or
by
inhalation. These compositions may be formulated so as to impart a time-
released
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WO 99/34794 I ~ PCT/US99/00596
benefit. Oral compositions may take the form of tablets, capsules, caplets,
emulsions,
liposomes, suspensions, powders and the like. Topical compositions include,
but are
not limited to, gels, lotions and creams. Parenteral compositions take the
form of
sterile solutions and emulsions and the like. Intravenous compositions
include, but
S are not limited to sterile solutions. The preferred routes of administration
is oral or
transdermal administration.
Dosage levels and requirements are well-recognized in the art and may be
chosen by those of ordinary skill in the art from publicly available sources.
Typically,
dosage levels will range between about 0.1 and about 5000 mg of tocotrienol or
mixture of tocotrienols per dose. Multiple doses may be required over a period
of
time to obtain maximum benefit. For example, a patient may receive oral or
transdermal administration of between about 0. l and about 1000 mg/day for a
period
of several days to several weeks or more. Specific dosage and treatment
regimens
will depend upon factors such as the patient's overall health status, the
severity and
1 S course of the patient's disorder or disposition thereto and the judgment
of the treating
physician. Higher or lower doses may be employed as needed.
Tocotrienols and mixtures thereof may be used in combination with
conventional therapeutics in the methods described herein. The conventional
therapeutics may be administered separately from the tocotrienols and mixtures
thereof, or they may be formulated together in a single dosage form. Such
combination therapy may advantageously utilize lower dosages of those
conventional
therapies and reduce or avoid possible toxicity incurred when those agents are
used as
monotherapies. For example, the tocotrienols used in the methods of this
invention
may be administered with conventional antioxidants such as those of the
vitamin E,
vitamin C and lipoic acid (preferably alpha lipoic acid) classes, aldose
reductase
inhibitors, glycation inhibitors (such as aminoguanidine), anti-platelet
agents (such as
aspirin and ticlopidine), bile acid sequestrants, such as Cholestyramine and
Colestipol; fabric acid derivatives, such as Clofibrate, Gamfibrozil,
Bezaflbrate,
Fenofibrate and Ciprofibrate; HMGR inhibitors such as statins (including but
not
limited to Lovastatin, Mevastatin, Pravastatin, Simvastatin and SRI-62320;
Probucol;
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Nicotinic Acid and its derivatives and conjugates such as Nicotinamide-N-
oxide, 6-
OH Nicotinamide, NAD, N-methyl-2-pyridine-8-carboxamide, N-Methyl-
Nicotinamide, N-Ribosyl-2-Pyridone-S-Carboxide, N-Methyl-4-pyridone-5-
carboxamide Bradilian, Niceritrol, Sorbinicate and Hexanicit; Neomycin and d-
Thyroxine. Furthermore, the tocotrienols used in the methods of this invention
may
be administered with conventional antidiabetes drugs. Such conventional
antidiabetes
drugs include biguanides (such as Glucophage {metformin hydrochloride)),
glucosidase inhibitors (such as Precose (acarbose)), sulfonylureas (such as
Amaryl
(glimepiride), DiaBeta (glyburide), Diabinese (chlorpropamide), Glucotrol and
Glucotrol XL (glypizide), Glynase (glypizide), and Micronase (glypizide)) and
insulin
(including natural and recombinant forms, insulin zinc, isophane insulin and
human,
bovine or procine forms). Other combination therapies will be obvious to those
of
skill in the art.
EXAMPLES
In order that this invention be more fully understood, the following examples
are set forth. These examples are for the purpose of illustration only and are
not to be
construed as limiting the scope of the invention in any way.
The methods used for obtaining and purifying tocotrienols and mixtures
thereof (including TRF) useful for the methods of this invention are described
in the
Examples section of US patent 5,591,772. Stabilization of rice bran follows
Example
1 of US patent 5,591,772. Purification of TRF, P2~ tocotrienol, PZS
tocotrienol and P~8
tocotrienol follow Examples 2-4 of US patent 5,591,772.
All assays conducted on chicken or swine were done following the protocols
described in A.A. Qureshi et al., Am J. Clin. Nutr., 53, pp. 1021 S-26S (1991
). All
enzymatic assays were conducted following the protocols described in A.A.
Qureshi
et al., Lids, 17, p. 924 (1982). TNF levels were measured using the
radioimmunoassay kit available from Genzyme Corp. (Cambridge, MA).
The following specific protocols are used in the subsequent examples herein:
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WO 99/34794 12 PCT/US99/00596
Protocol I: Dry Heat Stabilization
Extruder: Wenger Model X-25
Standard ScrewBarrel Setup
Barrel No. Standard Port Screw No. Standard Port
28714-9 5 28320-1
4 28318-1 4 8326-9
3 28372-9 3 28326-1
2 28318-1 2 28326-1
1 28350-1 1 28387-9
5 ie Setup
Standard
D
Die/Spacer Measurement Standard Port
Spacer 0.375 28340-11
Back Plate 0.625 28361-51
Intermediate Plate 0.218 28316-723
Front Plate 0.235 28389-507
Operating Conditions
Feed Rate: 1000 lbs/hr
Temperature: 170C at exit die
Pressure: 975-1025 psi
Moisture Feed: 12%
Moisture Discharge: 9.6%
Residence Time: 15 seconds
Run Duration: 8 hours
Sample Size: 50 lbs
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Protocol II: Drv Heat Followed By Wet Heat Stabilization
Dry Heat Stage: Protocol I
Wet Heat Stage:
Extruder: Anderson 4 inch
Screw Barrel Configuration: Standard Cut Flight
Die Setup:
Diameter: 0.1875 inches
Length: 0.75 inches
Operating Conditions:
Feed Rate: 378 lbslhr
Shaft Speed: 279 rpm
Steam Injection: 36 lbs/hr (32 psi
at #8 hole)
Mechanical Pressure: 750 psi (ast.)
Moisture Feed: 11.4%
Discharge Moisture: 15%
Discharge Rate: 450 lbs/hr
Discharge Temp.: 121 C
Protocol III: Dr~ing/Cooling Procedure
The wet heat stabilized product of protocol II (15% moisture) was discharged
onto
aluminum trays and placed in a tray oven at 101.1 °C until the moisture
content was 8-
10% (approximately 1.5 hrs). The trays were then placed on tray racks and
allowed to
cool at ambient temperature (approximately 20°C).
Protocol IV: Oil Extraction
Oil to Hexane Ratio: 1:4
No. of Washings: 3
Extraction Temperature 40C
The hexane was removed from the extract by mild heating (40°C) under a
mild
vacuum.
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Protocol V: Dewaxin~
20 lbs of crude oil were refrigerated for 24 hrs at -15.6°C. The
supernatant
(containing the dewaxed oil) was decanted from the solidified waxes. The waxes
were then centrifuged to removed entrained oil, yielding 0.59 lbs of waxes and
19.41
lbs of dewaxed oil.
Evaluation of Activity of Tocotrienols
Example 1
The effects of tocotrienols (in the form of the TRF mixture and individual
tocotrienols) on plasma levels of thromboxane BZ and platelet factor 4 in
chickens
were determined. These levels are known to correlate with the levels of
inflammatory
cytokines. The serum levels of triglycerides and glucose were also measured.
The
following feeding conditions were used:
Each group of six chickens (6-week old female white leghorn chickens) was
administered a chick mash control diet or a control diet containing one or
more
additives. The amount of feed consumed by all groups was comparable to the
control
group. The feeding period was 4 weeks. The birds were fasted for a period of
14
hours prior to sacrifice (at 0800 hours).
The chicken mash control diet contained the following ingredients:
Ingredient Weight (g)
Corn (8.8% protein) 615.0
Soybean Meal 335.0
Corn Oil 10.0
Calcium Carbonate 10.0
Dicalcium Phosphate 20.0
Iodized Salt 5.0
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WO 99/34794 15 PCTNS99/00596
Mineral Mixture 2.5
Vitamin Mixture 2.5
The mineral mixture contained per kg feed: zinc sulfate ~ H20, 110 mg;
manganese sulfate ~ SH20, 70 mg; ferric citrate ~ H20, 500 mg; copper sulfate
SH20, 16 mg; sodium selenite, 0.2 mg; DL-methionine, 2.5 g; choline chloride
(50%),
1.5 g; ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline), 125 mg; and
thiamine HCI, 1.8 mg. The vitamin mixture contained per kg feed: vitamin A,
1,500
units; vitamin D3, 400 units; vitamin E, 10 units; riboflavin, 3.6 mg; calcium
panthothenate, 10 mg; niacin, 25 mg; pyridoxine HCI, 3 mg; folacin, 0.55 mg;
biotin,
0.15 mg; vitamin B,2, 0.01 mg; and vitamin K" 0.55 mg.
Results are reported as mean t standard deviation. Percentages of control are
reported in parentheses. The following results were obtained
EXPERIMENT 1
Diet ThromboxanePlatelet TriglyceridesGlucose
B2 (mg/100 Factor 4 (mg/100 (mg/100
ml) ml) ml)
(ng/ml)
1) control diet 16.7 ~ 1.697.2 ~ 0.48 90.2 t 1.17124.6 ~
+ S% 2.3
corn oil ( 100.0) ( 100.0) ( 100) ( 100)
2) control diet 15.8 ~ 1.297.5 ~ 0.42 91,5 ~ I 126.7 t
+ 5% .48 2.1
corn oil + waxes (94.6) ( 104.2) ( 1 O 1.4) ( 101.7)
(50 ppm)
3) control diet 12.4 + 1.425.7 t 0.64 73.2 ~ 1.b986.4 t
+ 5% 1.55
corn oil + TRF (74.3) (79.2) (81.2) (69.3)
(50
ppm)
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WO 99/34794 16 PCTNS99/00596
Significant decreases of thromboxane Bz and platelet factor 4 plasma levels
and glucose and triglyceride serum levels were observed in the chickens fed a
control
diet supplemented with TRF.
EXPERIMENT 2
Diet Thromboaan Platelet TriglyceridesGlucose
a BZ (pg/ml)Factor 4 (mg/100 mg/ 100
ml) ml)
(ng/ml)
l) control diet 25.84 t 13.9 t 0.4974.3 t 224.30 ~
0.84 1.26 5.30
( 100.00) ( 100.00) ( 100) ( 100)
2) control diet 20.55 t 9.67 ~ 0.4364.73 t 212.50 t
+ TRF 0.95 0.93 0.98
(50 ppm) (79.53) (73.87) (87.44) (94.74)
3) control diet 21.63 f 8.78 t 0.7268.4 t 216.90 t
+ a- 0.89 1.32 1.62
tocotrienol (50 (83,71 ) (67.07) (91.91 (96.70)
ppm) )
4) control diet+Y-19.22 ~ 8.36 ~ 0.7961.7 ~ 208.70 t
0.78 1.37 1.41
tocotrienol (50 (74.38) (63.87) (82.49) (93.05)
ppm)
5) control diet 18.65 t 8.23 t 0.7760.50 t 205.30 t
+ b- 0.99 2.50 1.83
tocotrienol (50 (72.17) (62.87) (81.72) (91.53)
ppm)
6) control diet 16.74 ~ 7.76 t 1.6756.22 t 198.80 t
+ Pz, 1.62 1.11 2.10
tocotrienol (50 (64.78) (59.28) (75.94) (88.63)
ppm)
7) control diet 16.42 ~ 7.27 t 0.7454.65 ~ 196.10 1.74
+ Pzs 1.36 1.42
tocotrienol (50 (63.54) (55.54) (73.82) (87.43)
ppm)
8) control diet 25.46 ~ 12.38 t 71.29 ~ 227.60 ~
+ I .52 1.26 1.19 2.03
Geraniol (100 (98.53) (94.57) (96.30) (101.47)
ppm)
9) control diet+24.89 ~ 12.24 t 64.04 f 220.10 ~
0.88 1.49 1.03 1.32
Lovastatin (100 (96.32) (93.51) (86.SI) (98.13)
ppm)
10) control diet24.95 ~ 12.19 t 66.10 t 24.95 ~
+ 0.97 1.15 0.99 0.97
Geraniol (50 (96.56) (93.12) (89.29) (96.56)
ppm) +
Lovastatin (50
ppm)
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WO 99/34794 17 PCT/US99/00596
Significant decreases of thromboxane BZ and platelet factor 4 plasma levels
and glucose, insulin and glucagon serum levels were observed in the chickens
fed a
control diet supplemented with TRF and individual tocotrienols.
Example 2
The effects of tocotrienols (in the form of the TRF mixture and individual
tocotrienols) on plasma levels of thromboxane Bz and platelet factor 4 in
swine were
determined. These levels are known to correlate with the levels of
inflammatory
cytokines. The serum levels of glucose and triglycerides was also measured.
The
following feeding conditions were used:
Each group of three swine (S-month old swine carrying Lpds and Lpu' mutant
alleles) were administered a control diet or a control diet supplemented with
one or
more additives. After a 12 hour fast, plasma samples were taken at 42 days
from the
start of the feeding period.
The swine control diet contained the following ingredients:
Ingredient Weight Percentage
Corn (9.3% protein) 78.37
Soybean Meal (44.0% protein) 15.42
Lard 3.00
Calcium Carbonate 0.95
Dicalcium Phosphate 0.96
Mineral Mixture 0.30
Vitamin Mixture 1.00
The mineral mixture contained per kg feed: zinc sulfate ~ H20, 110 mg;
manganese sulfate ~ SH20, 70 mg; ferric citrate ~ H20, S00 mg; copper sulfate
SHZO, 16 mg; sodium selenite, 0.2 mg; DL-methionine, 2.5 g; choline chloride
(50%),
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WO 99/34794 18 PCT/US99/00596
l.~ g; ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline), 125 mg; and
thiamine HCI, 1.8 mg. The vitamin mixture contained per kg feed: vitamin A,
1,500
units; vitamin D3, 400 units; vitamin E, 10 units; riboflavin, 3.6 mg; calcium
panthothenate, 10 mg; niacin, 25 mg; pyridoxine HCI, 3 mg; folacin, 0.55 mg;
biotin,
0.15 mg; vitamin Blz, 0.01 mg; and vitamin K,, 0.55 mg.
The gain in body weight in all groups was comparable to the control.
Results are reported as mean t standard deviation. Percentages of control are
reported in parentheses. The following results were obtained:
Diet Thrombox Platelet Glucose Insulin Glucagon
ane B2 Factor (m~ 100 (pg/ml) (pg/100
4 ml) ml)
(pg/100 (ng/ 100
ml) ml)
1) control 75.93 24.12 95.99 t 10.50 t 330.41
diet t 1.45 t 1.75 1.37 0.12 ~ 14.08
(100.00) (100.00) (100) (100) (100)
2) control 64.55 20.32 74.45 t 23.10 t 295.36
diet + t 1.18 t 1.70 1.03 1.08 .t 10.45
TItF (50 (95.23) (84.30) (77.56) (112.68) (89.39)
ppm)
3) control 62.54 20.65 74.46 t 21.21 t 285.41
diet + t 1.39 t 1.15 1.12 0.89 ~ 13.88
r-tocotrienol(92.25) (85.28) (77.51) (103.46) (86.38)
{50 ppm)
4) control 60.48 19.85 72.63 t 21.45 t 278.36
diet + f 1.46 t 1.27 1.58 0.87 t 16.42
PZ, tocotrienol(87.66) (82.59) (75.66) (104.63) (84.25)
(50 ppm)
5) control 57.03 19.15 71.89 t 20.89 f 274.41
diet + t I .95 t 1.45 1.89 1.79 t 1.95
P25 tocotrienol(83.02) (79.36) (74.89) (101.90) (83.02)
(50 ppm)
Significant decreases of thromboxane BZ and platelet factor 4 plasma levels
and glucose and glucagon serum levels were observed in the chickens fed a
control
diet supplemented with TRF and individual tocotrienols. An increase in insulin
levels
was also observed.
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WO 99/34794 I9 PCT/US99/00596
Example 3
The effects of y-tocotrienol on the release of superoxide in human peripheral
blood neutrophils were determined. Neutrophils are an extracellular source of
oxygen
free radicals and, together with nitric oxide, form peroxynitrite (responsible
for
endothelial damage). Activated neutrophils attach to endothelial tissue, where
they
release the potent toxin, superoxide. Superoxide amplifies the inflammatory
response
and impairs local blood circulation.
The neutrophils tested were isolated by density centrifugation on Ficoll-
Hypaque gradients using conventional methods (see E. Serbinova et al., Free
Rad.
Bio. and Med., 10, pp. 263-75 (1991)). The neutrophils were then placed in a
96-well
plate. y-Tocotrienol and phorbol myrstate acetate were added to the wells at
the same
time. The secretion of superoxide was measured as the superoxide dismutase-
inhibitable reduction of ferricytochrome C. The results of this study are
displayed in
I S Figures l A and 1 B.
The amount of released superoxide was reduced from 19.7 nmole (5x105
cells/hour) in the control to 8.0 and 0.0 nmole at y-tocotrienol
concentrations of 10~
and 10-5, respectively.
Efficacy of Tocotrienols in Diabetic Dyslipidemia Animal Models
Several rodent models of diabetes have been developed. Genetically diabetic
mice (C57BL/ks db+/db+) are markedly hyperglycemic, hyperinsulinemic and
insulin
resistant, while demonstrating moderate hyperphagia and obesity. Moreover,
genetically obese mice (C57BL/ks ob+/ob+) are markedly obese and hyperphagic
while demonstrating less hyperglycemia, hyperinsulinism and insulin
resistance.
These genetically diabetic mice are available commercially.
The mice are fed a normal protein control diet (NPCD) or NPCD
supplemented with between about 5 and about 100 ppm of tocotrienols and
tocotrienols mixtures (such as P,B, P25 and TRF25). A typical NPCD consists of
casein
(vitamin-free) 27%, starch 58%, corn oil 10%, mineral mixture 4% and vitamin
mixture 1 % (see Examples 1 and 2 above for composition of mineral and vitamin
CA 02318218 2000-07-11
WO 99/34794 2~ PCT/US99/00596
mixtures). The mice are housed in wire cages under artificial illumination
from 0600
to 1800 hours during each 24 hour period. The mice are fed ad libitum and are
weighed every two weeks. The serum and urine glucose levels are determined at
the
beginning of each experiment and after two weeks during the treatments. The
mice
are fasted on the 52°d day for 36 hours and then refed on the 54'" day
until the end of
the experiment (58 day period). The mice are then sacrificed and the following
measurements are taken: body weight gain, feed consumption, feed efficiency,
serum
and urine glucose levels, plasma glucose levels, serum triglyceride levels,
fatty acid
synthetase and malefic enzyme activities in the cytosolic fraction of the
liver.
While we have described a number of embodiments of this invention, it is
apparent that our basic constructions may be altered to provide other
embodiments
which utilize the compositions and methods of this invention. Therefore, it
will be
appreciated that the scope of this invention is to be defined by the appended
claims,
rather than by the specific embodiments that have been presented hereinabove.
