CA2318218A1 - Methods for treating diabetic dyslipidemia using tocotrienols - Google Patents

Abstract

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.

Description

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 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.

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:

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):

R~

~(CH2)n 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):

R1o R11 (a) m R1o ~ O /mRl1 (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~-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);

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).

WO 99/34794 g PCT/US99/00596 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 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 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;

WO 99/34794 11 PC'T/US99/00596 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:

Protocol I: Dry Heat Stabilization Extruder: Wenger Model X-25 Standard ScrewBarrel Setup Barrel No. Standard Port Screw No. Standard Port 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 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.

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 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 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 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) 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.

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) 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%), 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.

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 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.

Claims (25)

WHAT IS CLAIMED IS:

1. A method for treating dyslipidemia in a diabetic patient comprising the step of administering to the patient a therapeutically effective amount of a composition comprising a tocotrienol.

2. The method according to claim 1, wherein the composition comprises a mixture of tocotrienols.

3. The method according to claim 1, wherein the composition comprises one or more tocotrienols and one or more additional agents selected from the group consisting of antioxidants, aldose reductase inhibitors, glycation inhibitors and anti-platelet agents.

4. The method according to claim 3, wherein the composition comprises TRF 25.

5. The method according to claim 1, wherein the composition comprises one or more additional agents selected from the group consisting of biguanide, glucosidase inhibitors, sulfonylureas, and insulin.

6. The method according to claim 5, wherein the composition comprises TRF25.

7. The method according to claim 1, wherein the composition comprises one or more tocotrienols selected from the group consisting of P21 tocotrienol, .alpha.- tocotrienol, .beta.-tocotrienol, .gamma.- tocotrienol, .delta.- tocotrienol, P18 tocotrienol and P25 tocotrienol.

8. The method according to claim 1, wherein the tocotrienol is P25 tocotrienol or P18 tocotrienol.

9. The method according to claim 1, wherein the composition is administered orally.

10. The method according to claim 9, wherein the composition is in the form of a tablet, capsule, liposome, caplet, emulsion, suspension or powder.

11. The method according to claim 10, wherein the composition comprises between about 0.1 and about 5000 mg of tocotrienol/dose.

12. The method according to claim 1, further comprising the step of administering to the patient one or more additional agents selected from the group consisting of antioxidants, aldose reductase inhibitors, glycation inhibitors and anti-platelet agents.

13. The method according to claim 1, further comprising the step of administering to the patient one or more additional agents selected from the group consisting of biguanide, glucosidase inhibitors, sulfonylureas, and insulin.

14. A method for treating dyslipidemia in a diabetic patient, comprising:
(a) diagnosing a diabetic patient with dyslipidemia; and (b) administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising a tocotrienol.

15. The method according to claim 14, wherein the composition comprises a mixture of tocotrienols.

16. The method according to claim 14, wherein the composition comprises one or more tocotrienols and one or more additional agents selected from the group consisting of antioxidants, aldose reductase inhibitors, glycation inhibitors and anti-platelet agents.

17. The method according to claim 16, wherein the composition comprises TRF25.

18. The method according to claim 14, wherein the composition comprises one or more additional agents selected from the group consisting of biguanide, glucosidase inhibitors, sulfonylureas, and insulin.

19. The method according to claim 18, wherein the composition comprises TRF25.

20. The method according to claim 14, wherein the composition comprises one or more tocotrienols selected from the group consisting of P2, tocotrienol, .alpha.-tocotrienol, .beta.-tocotrienol, .gamma.-tocotrienol, .delta.-tocotrienol, P18 tocotrienol and P25 tocotrienol.

21. The method according to claim 14, wherein the tocotrienol is P25 tocotrienol or P18 tocotrienol.

22. The method according to claim 14, wherein the composition is administered orally.

23. The method according to claim 22, wherein the composition is in the form of a tablet, capsule, liposome, caplet, emulsion, suspension or powder.

24. The method according to claim 23, wherein the composition comprises between about 0.1 and about 5000 mg of tocotrienol/dose.

25. The method according to claim 14, further comprising the step of administering to the patient one or more additional agents selected from the group consisting of antioxidants, aldose reductase inhibitors, glycation inhibitors and anti-platelet agents.