WO2004047732A2 - Tocopherol and tocotrienol medicaments - Google Patents

Tocopherol and tocotrienol medicaments Download PDF

Info

Publication number
WO2004047732A2
WO2004047732A2 PCT/US2003/037135 US0337135W WO2004047732A2 WO 2004047732 A2 WO2004047732 A2 WO 2004047732A2 US 0337135 W US0337135 W US 0337135W WO 2004047732 A2 WO2004047732 A2 WO 2004047732A2
Authority
WO
Grant status
Application
Patent type
Prior art keywords
gamma
medicament
tocopherol
tocotrienol
delta
Prior art date
Application number
PCT/US2003/037135
Other languages
French (fr)
Other versions
WO2004047732A3 (en )
Inventor
Bruce N. Ames
Qing Jiang
Original Assignee
Children's Hospital & Research Center At Oakland
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/13Amines
    • A61K31/155Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/185Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic, hydroximic acids
    • A61K31/19Carboxylic acids, e.g. valproic acid
    • A61K31/20Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. cannabinols, methantheline
    • A61K31/3533,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355Tocopherols, e.g. vitamin E
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/41Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425Thiazoles
    • A61K31/4261,3-Thiazoles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/56Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives, e.g. steroids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof

Abstract

Anti-inflammatory compositions include medicaments comprising predetermined amounts of a phytyl substituted chromanol and a prostaglandin E2 inhibitor, wherein: said medicament is in unit dosage form suitable for pharmaceutical administration; said phytyl substituted chromanol is a gamma-tocopherol, delta-tocopherol, gamma-tocotrienol or delta-tocotrienol; said PGE2 inhibitor is a non-steroidal anti-inflammatory drug or an omega-3 fatty acid, such as docosahexaenoic acid and eicosapentaenoic acid. Anti-obesity compositions include medicaments comprising predetermined amounts of a phytyl substituted chromanol and an obesity-promoting drug, wherein: said medicament is in unit dosage form suitable for pharmaceutical administration; said phytyl substituted chromanol is a gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, gamma-tocotrienol or delta-tocotrienol; said obesity-promoting drug is a corticosteroid or an anti-diabetes drug such as a hypoglycemic drug, starch blocker, glucose production blocker or insulin sensitizer.

Description

Tocopherol and Tocotrienol Medicaments

INTRODUCTION Field of the Invention The field of the invention is the use of tocopherols and tocotrienols in medicaments.

Background of the Invention

Inflammatory diseases such as rheumatoid arthritis, asthma and hepatitis are among the leading causes of death and disability in the world. Chronic inflammation contributes to the development of degenerative diseases including cancer (1), cardiovascular diseases (2) and neuro-degenerative disorders (3). During inflammation, various eicosanoids derived from arachidonic acid (AA) play a key role in mediating inflammatory response (4). For instance, prostaglandin E2 (PGE2), which is synthesized from cyclooxygenase (COX)- catalyzed oxidation of AA, is believed to cause pain and fever (4, 5), as well as activate cytokine formation (6). PGE2 can be produced by either the constitutive form (COX-1) or the inducible form (COX-2) of cyclooxygenase (7). In most inflammatory conditions, COX-2 is up-regulated and is the primary enzyme responsible for the formation of pro-inflammatory PGE2 (7). Leukotriene B4 (LTB4), another oxidized product derived from AA through the 5- lipoxygenase-catalyzed pathway, is one of the most potent chemotactic agents (8). Because of the central roles of PGE2 and LTB4, COX-2 and 5-lipoxygenase have been recognized as key targets for the drug therapy in inflammation-associated diseases. In particular, COX-2 inhibitors, which are classified as non-steriod anti-inflammatory drugs (NSAIDs), have been proven to be effective in attenuating inflammatory response and beneficial for certain inflammation-associated diseases (9). Vitamin E consists of eight compounds; four tocopherols (alpha-, beta-, gamma-, and delta-) and four tocotrienols (alpha-, beta-, gamma-, and delta-). Among them, only alpha- tocopherol has been extensively studied. Gamma- tocopherol (gamma-T) is the major form of vitamin E in the US diet. However, it has drawn little attention compared with alpha- tocopherol, the primary form of vitamin E found in most supplements. Delta-tocopherol (delta-T) is another form of vitamin E that is rich in some food sources (often found with

" gamma-tocopherol, e.g. in soybeans and soybean oil). Tocotrienols are mainly abundant in palm oil.

We recently found that gamma-tocopherol (gamma-T), and its physiological metabolite, 2, 7, 8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman (gamma-CEHC), inhibits COX-2-catalyzed formation of PGE2, as assayed in lipopolysaccharide-stimulated macrophage and interlukin-lb activated epithelial cells (10; see also our review, 18). This indicates that gamma-T and its metabolite may have anti-inflammatory properties that are similar to those of NSAIDs. In contrast, alpha-tocopherol (alpha-T), the predominant form of vitamin E in the tissues and most supplements, is much less effective in this regard (10). It was not clear, however, whether gamma-tocopherol would reach significant levels in tissues to exert significant effects in vivo.

Here we show that this bioactivity of gamma-T is demonstrable in vivo, and more importantly, establish that there are three pathways by which gamma-T inhibits inflammation, and indicates that gamma-T has superior and supplemental pharmaceutical potential over the commonly used cyclooxygenase inhibitors. We disclose the use of gamma-T, delta-T, and their corresponding metabolites (gamma-CEHC, Delta-CEHC), as well as combinations of gamma-T and delta-T, and these tocopherols with gamma-tocotrienol or delta-tocotrienol, in the form of supplements or drugs, particularly in combination with existing drugs such as NSAIDs to treat inflammation, particularly inflammatory diseases such as arthritis, bowel diseases, and asthma. These subject medicaments can also be used to treat and prevent chronic diseases associated with inflammation such as cancer and cardiovascular disorders.

US Patent Nos. 6,204,290; 6,242,479; 6,410,589; and 6,239,171 appear relevant to these aspects of the disclosure.

Vitamin E consists of eight compounds; four tocopherols (alpha-, beta-, gamma-, and delta-) and four tocotrienols (alpha-, beta-, gamma-, and delta-). Among them, only alpha- tocopherol has been extensively studied. Gamma- tocopherol (gamma-T) is the major form of vitamin E in the US diet. However, it has drawn little attention compared with alpha- tocopherol, the primary form of vitamin E found in most supplements. Delta-tocopherol (delta-T) is another form of vitamin E that is rich in some food sources (often found with gamma-T, e.g. in soybeans and soybean oil). Tocotrienols are mainly abundant in palm oil.

Chronic use of a number of medications is known to contribute to obesity. For example, the treatment of diabetes using anti-diabetes drugs, including the thiazolidinedione class (troglitazone, rosiglitazone and proglitazone), commonly leads to weight gain and obesity (Malinowski JM et al, 2000, Clinical Therapeutics, 22, 1151-68). UK Prospective Diabetes Study has clearly demonstrated that weight gain associated with diabetes treatment partially cancels the beneficial effects of tight blood glucose control on cardiovascular events and mortality (UK Prospective Diabetes Study, Group, 1998, Lancet, 352, 854-65). Here we show that tocopherol and tocotrienol compositions can be used to reduce triglyceride accumulation in adipocytes, particularly accumulation resulting from obesity-promoting drug use. Furthermore, the use of combinations of tocopherols or tocotrienols with anti-diabetes drugs provides a superior therapy.

Ismermann et al. 1999, Diabetes Care 22, 1227-1228 report that alpha-tocopherol induces leptin expression in healthy individuals; Ohrvall et al., J Intern Med 1993 Jul;234(l):53-60 report lower tocopherol serum levels in subjects with abdominal adiposity; Sjoholm et al, Biochem Biophys Res Commun 2000 Oct 22;277(2):334-40, report that gamma-tocopherol partially protects insulin-secreting cells against functional inhibition by nitric oxide. US Patent Nos. 6,239,171 and 5,821,264 appear relevant to these aspects of the disclosure.

SUMMARY OF THE INVENTION The invention provides methods and compositions for inhibiting inflammation. The compositions include medicaments comprising predetermined amounts of a phytyl substituted chromanol and an inhibitor of prostaglandin E2 (PGE2) formation, wherein: said medicament is in unit dosage form suitable for pharmaceutical administration; said phytyl substituted chromanol is selected from the group consisting of gamma-tocopherol, delta- tocopherol, gamma-tocotrienol and delta-tocotrienol; and said PGE2 inhibitor is selected from the group consisting of an omega-3 fatty acid cyclooxygenase substrate and a non-steroidal anti-inflammatory drug (NSAID) cyclooxygenase inhibitor.

The phytyl-substituted chromanol is typically isolated or purified to homogeneity or near homogeneity. In particular embodiments, the medicament comprises less alpha- tocopherol than is present in natural Vitamin E compositions, preferably less than 5% alpha- tocopherol, more preferably less than 0.5%, more preferably less than 0.05%. The medicament may comprise various mixtures of gamma- and delta-tocopherol and gamma and delta-tocotrienol.

Exploiting the synergy of the components of the medicaments, the PGE2 inhibitor may be provided at a dosage that is suboptimally therapeutic, or to various degrees, subtherapeutic, when administered alone. In particular embodiments, the PGE2 inhibitor is anNSAID cyclooxygenase inhibitor of Table 1; and/or a cyclooxygenase-2 (COX) selective inhibitor. In other embodiments, the PGE2 inhibitor is an omega fatty acid cyclooxygenase substrate, such as docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA).

The invention also provides methods of inhibiting inflammation by administering to a patient a subj ect medicament.

The invention provides methods and compositions for reducing triglyceride accumulation in adipocytes, particularly accumulation resulting from obesity-promoting drug use. The compositions include medicaments comprising predetermined amounts of a phytyl substituted chromanol and an obesity-promoting drug, wherein: said medicament is in unit dosage form suitable for pharmaceutical administration; said phytyl substituted chromanol is selected from the group consisting of gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, gamma-tocotrienol and delta-tocotrienol.

The phytyl-substituted chromanol is typically isolated or purified to homogeneity or near homogeneity. In particular embodiments, the medicament comprises less alpha- tocopherol than is present in natural Vitamin E compositions, preferably less than 5% alpha- tocopherol, more preferably less than 0.5%, more preferably less than 0.05%. The medicament may comprise various mixtures of gamma- and delta-tocopherol and alpha-, gamma- and delta-tocotrienol.

In particular embodiments, the obesity-promoting drug is a cortico steroid; in other embodiments, the obesity-promoting drug is an anti-diabetes drug such as a hypoglycemic drug, a starch blocker, a glucose production blocker, or an insulin sensitizer.

The invention also provides methods of reducing obesity-promotion, by administering to a patient a subject medicament, as well as methods for reducing triglyceride accumulation in adipocytes by contacting a patient predetermined to have or be predisposed to undesirably high triglyceride accumulation in adipocytes with an effective amount of a phytyl substituted chromanol selected from the group consisting of gamma-tocopherol, delta-tocopherol, alpha- tocotrienol, gamma-tocotrienol and delta-tocotrienol.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION We disclose that gamma-T, unlike alpha-T which has no activity in our studies, can be used as a drug to treat or prevent inflammatory diseases. We have shown in an animal model of inflammation, which mimics joint diseases such as arthritis and other inflammatory diseases, that gamma-T inhibits inflammation by blocking three pathways: pro-inflammatory prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and TNF-alpha. PGE2 is known to mediate inflammatory responses, including pain and fever, as well as cytokine formation; LTB4 is one of the most potent chemotactic agents; and TNF-alpha is a key cytokine that regulates inflammatory response. Because most commonly used non-steroid anti-inflammatory drugs (NSAIDs), such as cyclooxygenase inhibitors, only inhibit cyclooxygenase-catalyzed PGE2, the inhibition of PGE2, LTB4 and TNF-alpha indicates that tocopherols (including gamma-T) have superior and supplemental pharmaceutical values over commonly used anti- inflammatory drugs .

Combinations of gamma-T, delta-T or gamma-tocotrienol and delta-tocotrienol are superior to the use of just one of these compounds because each compound exhibits distinct activities against PGE2, LTB4, and TNF-alpha and distinct bio-distribution. Although tocopherols in the vitamin E family differ only in one or two methyl groups, their relative bioactivities are not predictable simply based on the number of methyl groups. For instance, while alpha-T, beta-tocopherol (beta-T), and alpha-tocotrienol showed weak to no inhibitory effect on PGE2 (e.g. ref. 10); gamma-T, delta-T, and gamma-tocotrienol effectively inhibit PGE2, with the apparent IC50s of 5-10 μM (gamma-T), 2.5-5 μM (delta-T), and 1-2.5 μM (gamma-tocotrienol). Our data indicate that delta-T and gamma-tocotrienol are better cyclooxygenase inhibitors than gamma-T in some systems. In addition, the relative distribution of gamma-T, delta-T and gamma-tocotrienol in the body is different due to the differential transport and metabolism of these compounds. For instance, upon supplementation with vitamin E, tocotrienols are more likely to be found in the skin and adipose tissue, whereas gamma-T and delta-T levels are elevated in the skin, muscle, adipose, heart and other tissues. Delta-T also appears to be retained in the body for a shorter time than gamma-T. Hence, a combination of delta-T and gamma-T provides better therapeutic effects, as delta-T is more potent against PGE2 while the effects of gamma-T can last longer. Similarly, a combination of gamma-tocotrienol and gamma-T is superior for treating certain inflammatory conditions, such as inflammatory skin (dermal) diseases.

Combinations of tocopherols (e.g. gamma-T and delta-T), and tocotrienols (e.g. gamma-tocotrienol and delta-tocotrienol) with other anti-inflammatory drags, such as cyclooxygenase inhibitors, provide enhanced therapeutic effects, compared with the use of these drugs alone. Some of the NSAIDs are much more potent inhibitors of PGE2 than gamma-T. Though gamma-T is considered to be a moderate inhibitor of PGE2, it has additional inhibitory activity on LTB4, lipid peroxidation, and TNF-alpha. A combination of gamma-T with other NSAIDs improves therapeutic effects and lowers the required dose of

NSAIDs, thereby reducing such side effects as ulcerogenesis.

Combinations of tocopherols and tocotrienols with dietary supplementation of omega- 3 fatty acid also provide additive or synergistic anti-inflammatory effects. Omega-3 fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have mild anti-inflammatory activity by way of lowering arachidonic acid, the substrate of cyclooxygenase and lipoxygenase for the generation of PGE2and LTB4. Because tocopherols and tocotrienols inhibit cyclooxygenase- and lipooxygenase-catalyzed synthesis of PGE2 and LTB4, combinations of tocopherols (or tocotrienols) with dietary supplementation of DHA or EPA results in a more potent decrease in the pro-inflammatory PGE2 and LTB4, and thus provide stronger anti-inflammatory effects than either of them alone.

Gamma-T, delta-T, gamma-tocotrienol, or their combinations are also useful in cancer prevention or therapy. COX-2 and PGE2 are elevated in inflammation-associated diseases, including cancer, and frequent intake of non-steroid anti-inflammation drugs, such as aspirin, reduce the risk of certain cancers. In addition to the cyclooxygenase-related mechanism, the lipoxygenase-relative pathways (one of the products of 5-lipoxygenase is leukotriene B4), also contributes to the development of certain cancers. We confirmed that gamma-T showed anti-proliferative effects on human prostate epithelial cancer cells and lung epithelial cancer cells by mechanisms associated with the inhibition of both cyclooxygenase and lipoxygenase catalyzed reactions. The anti-inflammatory effects of gamma-T are also beneficial in preventing or treating cardiovascular disease, another inflammation-associated disorder. Accordingly, the invention provides methods and compositions for inhibiting or reducing inflammation or any manifestation thereof, and/or for reducing the likelihood of developing or for promoting a resistance to inflammation or any manifestation thereof. The compositions include medicaments comprising predetermined amounts of a phytyl- substituted chromanol and an inhibitor of prostaglandin E2 (PGE2) formation, wherein said PGE2 inhibitor is selected from the group consisting of an omega-3 fatty acid cyclooxygenase substrate and a non-steroidal anti-inflammatory drug (NSAID) cyclooxygenase inhibitor. In preferred embodiments, said medicament is in unit dosage form suitable for pharmaceutical administration; and/or said phytyl-substituted chromanol is selected from the group consisting of gamma-tocopherol, delta-tocopherol, gamma-tocotrienol and delta-tocotrienol. The phytyl-substituted chromanol is typically isolated or purified to homogeneity or near homogeneity. In various embodiments, the chromanol is, prior to admix, purified to at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 99%o homogeneity. In particular embodiments, the medicament comprises less alpha-T than is present in natural Vitamin E source compositions. In various embodiments, the alpha-T is reduced to less than 50%, preferably less than 20%, more preferably less than 5% of its natural source concentration. In particular embodiments, the pre-admix chromanol/tocopherol is less than 5% alpha-T, more preferably less than 0.5%), more preferably less than 0.05%. The medicament may comprise various mixtures of gamma- and delta-tocopherol and gamma and delta-tocotrienol, including gamma-T + gamma and/or delta tocotrienol, delta-T + gamma and/or delta tocotrienol, and gamma-T + delta-T + gamma and/or delta tocotrienol.

Exploiting the synergy of the components of the medicaments, the PGE2 inhibitor may be provided at a dosage that is suboptimally therapeutic, or to various degrees, subtherapeutic, when administered alone. In various embodiments, the PGE2 inhibitor is provided at or less than 50%> of conventional dosages or conventional dosage ranges, preferably at or less than 20% of conventional dosages or ranges. In particular embodiments, the PGE2 inhibitor is an NSAID cyclooxygenase inhibitor, such as an NSAID of Table 1. In other embodiments, the PGE2 inhibitor is a cyclooxygenase-2 (COX) selective inhibitor. In other embodiments, the PGE2 inhibitor is an omega-3 fatty acid cyclooxygenase substrate such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

Figure imgf000009_0001

The subject medicament components can be purchased commercially and/or prepared from readily available starting materials using conventional methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

The subject medicament compositions may be administered in conjunction with a earner, vehicle or excipient suitable for use in pharmaceutical compositions. Without being limited thereto, such materials include diluents, binders and adhesives, lubricants, plasticizers, disintegrants, colorants, bulking substances, flavorings, sweeteners and miscellaneous materials such as buffers and adsorbents in order to prepare a particular medicated composition. Such carriers are well known in the pharmaceutical art as are procedures for preparing pharmaceutical compositions.

Depending on the intended route of delivery, the compositions may be administered in one or more dosage form(s) including, without limitation, liquid, solution, suspension, emulsion, tablet, multi-layer tablet, bi-layer tablet, capsule, gelatin capsule, caplet, lozenge, chewable lozenge, bead, powder, granules, dispersible granules, cachets, douche, suppository, cream, topical, inhalant, aerosol inhalant, patch, particle inhalant, implant, depot implant, ingestible, injectable, or infusion. The dosage forms may include a variety of other ingredients, including binders, solvents, bulking agents, plasticizers, etc.

A wide variety of orally administrable compositions may be used. In a particular embodiment, the oral compositions are provided in solid discrete, self-contained dosage units, such as tablets, caplets, lozenges, capsules, gums, etc., which may comprise or be filled with liquid or solid dosages of the recited medicament constituents. A wide variety of dosages may be used, depending on the application and empirical determination; typical dosages range from 1 mg to 1 g, preferably at least 10 mg, more preferably at least 100 mg.

The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.

Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

The above described components are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pa., which is incorporated herein by reference.

The dosage forms of the present invention involve the administration of an active therapeutic substance or multiple active therapeutic substances in a single dose during a 24 hour period of time or multiple doses during a 24 hour period of time. The doses may be uneven in that each dose is different from at least one other dose. The subject compositions may be administered to effect various forms of release, which include, without limitation, immediate release, extended release, controlled release, timed release, sustained release, delayed release, long acting, pulsatile delivery, etc., using well known procedures and techniques available to the ordinary skilled artisan. A description of representative sustained release materials can be found in the incorporated materials in Remington's Pharmaceutical Sciences.

The subject compositions may be formulated for administration by any route, including without limitation, oral, buccal, sublingual, rectal, parenteral, topical, inhalational, including itnranasal, injectable, including subcutaneous, intravenous, intramuscular, etc., topical, including transdermal, etc. The subject compositions are administered in a pharmaceutically (including therapeutically, prophylactically and diagnostically) effective amount. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. The following formulation examples illustrate representative pharmaceutical compositions of this invention. The present invention, however, is not limited to the following exemplified pharmaceutical formulations:

Formulation 1— Capsules. Acetaminophen and gamma-T are blended with a starch diluent in an approximate 1:3:1 weight ratio. The mixture is filled into 250 mg capsules (approx. 50 mg each of Acetaminophen and 150 mg gamma-T per capsule). Formulation 2~Capsules. Acetaminophen and delta-T are blended with a starch diluent in an approximate 1:3:1 weight ratio. The mixture is filled into 250 mg capsules (approx. 50 mg each of Acetaminophen and 150 mg delta-T per capsule).

Formulation 3~Capsules. Acetaminophen, gamma-T and gamma-tocotrienol are blended with a starch diluent in an approximate 1:3:3:1 weight ratio. The mixture is filled into 400 mg capsules (approx. 50 mg each of Acetaminophen, 150 mg gamma-T and 150 mg gamma-tocotrienol per capsule).

Formulation 4-Capsules. Aspirin and gamma-T are blended with a starch diluent in an approximate 1:3:1 weight ratio. The mixture is filled into 250 mg capsules (approx. 50 mg each of aspirin and 150 mg gamma-T per capsule). Formulation 5~Capsules. Aspirin and delta-T are blended with a starch diluent in an approximate 1:3: 1 weight ratio. The mixture is filled into 250 mg capsules (approx. 50 mg each of aspirin and 150 mg delta-T per capsule).

Formulation 6~Capsules. Aspirin, gamma-T and gamma-tocotrienol are blended with a starch diluent in an approximate 1:3:3:1 weight ratio. The mixture is filled into 400 mg capsules (approx. 50 mg each of aspirin, 150 mg gamma-T and 150 mg gamma-tocotrienol per capsule).

Formulation 7— Capsules. Naproxen and gamma-T are blended with a starch diluent in an approximate 1:3:1 weight ratio. The mixture is filled into 250 mg capsules (approx. 50 mg each of naproxen and 150 mg gamma-T per capsule). Formulation 8~Capsules. Ibuprofen and gamma-T are blended with a starch diluent in an approximate 1:3:1 weight ratio. The mixture is filled into 250 mg capsules (approx. 50 mg each of ibuprofen and 150 mg gamma-T per capsule).

Formulation 9~Capsules. Docosahexaenoic acid (DHA) and gamma-T are blended with a starch diluent in an approximate 1:3:1 weight ratio. The mixture is filled into 250 mg capsules (approx. 50 mg each of DHA and 150 mg gamma-T per capsule). Formulation 10— Liquid. Acetaminophen (100 mg) and gamma-T are blended (300 mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through aNo. 10 mesh

U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (1189, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water is then added to produce a total volume of 5 mL.

Formulation 11 —Liquid. Aspirin (100 mg) and gamma-T are blended (300 mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (1189, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water is then added to produce a total volume of 5 mL.

Formulation 12~Liquid. Docosahexaenoic acid (DHA) (100 mg) and gamma-T are blended (300 mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through a

No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (1189, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water is then added to produce a total volume of 5 mL.

Formulation 13-Ointment. Acetaminophen and gamma-T are blended with isopropyl myristate 81 g, fluid paraffin oil 9 g and silica (Aerosil 200, 9 g, Degussa AG, Frankfurt). Formulation 14— Ointment. Aspirin and gamma-T are blended with isopropyl myristate 81 g, fluid paraffin oil 9 g and silica (Aerosil 200, 9 g, Degussa AG, Frankfurt).

Formulation 15-Ointment. Docosahexaenoic acid (DHA) and gamma-T are blended with isopropyl myristate 81 g, fluid paraffin oil 9 g and silica (Aerosil 200, 9 g, Degussa AG,

Frankfurt). Formulation 16-Non-ionic ater-m-oil cream. Acetaminophen and gamma-T are blended with a mixture of emulsified lanolin 39 g alcohols, of waxes and of oils (Anhydrous eucerin, BDF), methyl para-hydroxybenzoate 0.075 g, propyl para-hydroxybenzoate 0.075 g and sterile demineralized 100 g water.

Formulation 17-Non-ionic water-in-oil cream. Aspirin and gamma-T are blended with a mixture of emulsified lanolin 39 g alcohols, of waxes and of oils (Anhydrous eucerin, BDF), methyl para-hydroxybenzoate 0.075 g, propyl para-hydroxybenzoate 0.075 g and sterile demineralized 100 g water.

Formulation 18-Non-ionic water-in-oil cream. Docosahexaenoic acid (DHA) and gamma-T are blended with a mixture of emulsified lanolin 39 g alcohols, of waxes and of oils (Anhydrous eucerin, BDF), methyl para-hydroxybenzoate 0.075 g, propyl para- hydroxybenzoate 0.075 g and sterile demineralized 100 g water.

Formulation 19-Lotion. Acetaminophen and gamma-T are blended with polyethylene glycol (PEG 400) 69 g and 95% Ethanol 30 g.

Formulation 20-Lotion. Aspirin and gamma-T are blended with polyethylene glycol (PEG 400) 69 g and 95% Ethanol 30 g. Formulation 21-Lotion. Docosahexaenoic acid (DHA) and gamma-T are blended with polyethylene glycol (PEG 400) 69 g and 95% Ethanol 30 g.

Formulation 22-Hydrophobic ointment. Acetaminophen and gamma-T are blended with isopropyl myristate 36 g, silicone oil (Rhodorsil 36.400 g 47 V 300, Rhone-Poulenc), beeswax 13 g and silicone oil (Abil 300 100 g cst, Goldschmidt). Formulation 23-Hydrophobic ointment. Aspirin and gamma-T are blended with isopropyl myristate 36 g, silicone oil (Rhodorsil 36.400 g 47 V 300, Rhone-Poulenc), beeswax 13 g and silicone oil (Abil 300 100 g cst, Goldschmidt).

Formulation 24-Hydrophobic ointment. Docosahexaenoic acid (DHA) and gamma-T are blended with isopropyl myristate 36 g, silicone oil (Rhodorsil 36.400 g 47 V 300, Rhone- Poulenc), beeswax 13 g and silicone oil (Abil 300 100 g cst, Goldschmidt).

The invention also provides methods of inhibiting inflammation by administering to a patient a subject medicament. In a particular embodiment of this aspect, the invention provides a method for treating a patient with an inflammatory disease which method comprises administering to said patient a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective inflammatory disease-treating amount of a subject medicament. Additionally, this invention is directed to a method for preventing the onset of an inflammatory disease in a patient at risk for developing the inflammatory disease which method comprises administering to said patient a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective inflammatory disease- preventing amount of a subject medicament. In preferred embodiments of this invention, the inflammatory disease treated and/or prevented in the above methods is rheumatoid arthritis, septic shock, erythema nodosum leprosy, septicemia, uveitis and the like.

This aspect of the invention may be implemented by a first diagnostic step, e.g. determining that the patient is suffering from, subject to, or predisposed to a target disease or condition followed by prescribing and/or administering to the patient a subject medicament, optionally followed by an evaluation/confirmation/prognosis step, e.g. determining an effect of the treatment, such as an amelioration of symptoms of a targeted disease or condition or an indicator thereof.

We disclose that gamma- and delta-T, as well as alpha-, gamma- and delta- tocotrienol, can be used as drugs to reduce triglyceride accumulation in adipocytes. In contrast, alpha-T was much less effective as an anti-obesity agent in our studies.

Accordingly, we disclose the use of gamma-T, delta-T, and alpha-tocotrienol, gamma- tocotrienol, and delta-tocotrienol, as well as combinations of these compounds to treat and prevent obesity and/or undesirable weight gain, and their associated diseases including diabetes, cardiovascular diseases and cancers. In addition, these tocopherols and tocotrienols may be given in the form of supplements or neutraceutical drugs or in combination with existing drugs to treat and prevent obesity associated with anti-diabetes and steroid drug therapy.

We have found that tocopherols, particularly gamma-T and delta-T, and tocotrienols, particularly, alpha-, gamma-, and delta-, show dose-dependent anti-adipogenesis activity. The compounds also ameliorate high fat diet-induced obesity and type 2 diabetes in mice.

Our data demonstrate that tocopherols and tocotrienols and their combinations can reduce the development of obesity and its associated disorders such as diabetes. Accordingly, our invention provides methods and compositions for inhibiting or reducing triglyceride accumulation in adipocytes, or any manifestation thereof, and/or for reducing the likelihood of developing, and/or for promoting a resistance to undesirable or excess triglyceride accumulation in adipocytes, or any manifestation thereof. The compositions include medicaments comprising predetermined amounts of a phytyl-substituted chromanol and an obesity-promoting drug.

As our methods target triglyceride accumulation in adipocytes and adipogenesis, our methods are applicable to addressing obesity and/or undesirable weight-gain from a wide variety causes, including environmental, pharmaceutical, psychological and behavioral. For example, the invention is generally applicable to the wide variety of drags known to promote obesity. Well-known examples of obesity-promoting drugs are corticosteroids and anti- diabetes drugs like hypoglycemic drugs, starch blockers, glucose production blockers, and insulin sensitizers. In preferred embodiments, said medicament is in unit dosage form suitable for pharmaceutical administration; and/or said phytyl-substituted chromanol is selected from the group consisting of gamma-T, delta-T, alpha-tocotrienol, gamma- tocotrienol and delta-tocotrienol.

The phytyl-substituted chromanol is typically isolated or purified to homogeneity or near homogeneity. In various embodiments, the chromanol is, prior to admix, purified to at least 80%), preferably at least 90%), more preferably at least 95%, more preferably at least

99% homogeneity. In particular embodiments, the medicament comprises less alpha-T than is present in natural Vitamin E source compositions. In various embodiments, the alpha-T is reduced to less than 50%, preferably less than 20%), more preferably less than 5% of its natural source concentration. In particular embodiments, the pre-admix chromanol/tocopherol is less than 5% alpha-T, more preferably less than 0.5%, more preferably less than 0.05%. The subject medicaments may advantageously include various mixtures of gamma- and delta-T and alpha-, gamma- and delta-tocotrienol, including gamma- T + alpha, gamma and/or delta tocotrienol, delta-T + alpha, gamma and/or delta tocotrienol, and gamma-T + delta-T + alpha, gamma and/or delta tocotrienol. Exemplary obesity-promoting steroids include prednisone (Deltasone®, Orasone®), methylprednisolone (Medrol®), prednisolone (Prelone®, Pediapred®), dexamethasone (Decadron®), and triamcinolone (Aristocort®). Exemplary obesity-promoting anti-diabetes drugs include hypoglycemic drugs such as glyburide (DiaBeta®, Micronase®), Amaryl®, Glucotrol®, repaglinide (Prandin®), or nateglinide (starlix®); starch blockers such as acarbose (Precose®), and miglitol (Glyset®); glucose production blockers such as metformin

(Glucophage®); and insulin sensitizers such as a thiazolindinedion drag such as rosiglitazone (Avandia®) and pioglitazone (Actos®).

The subject medicament components can be purchased commercially and/or prepared from readily available starting materials using conventional methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

The subject medicament compositions may be administered in conjunction with a carrier, vehicle or excipient suitable for use in pharmaceutical compositions. Without being limited thereto, such materials include diluents, binders and adhesives, lubricants, plasticizers, disintegrants, colorants, bulking substances, flavorings, sweeteners and miscellaneous materials such as buffers and adsorbents in order to prepare a particular medicated composition. Such carriers are well known in the pharmaceutical art as are procedures for preparing pharmaceutical compositions.

Depending on the intended route of delivery, the compositions may be administered in one or more dosage form(s) including, without limitation, liquid, solution, suspension, emulsion, tablet, multi-layer tablet, bi-layer tablet, capsule, gelatin capsule, caplet, lozenge, chewable lozenge, bead, powder, granules, dispersible granules, cachets, douche, suppository, cream, topical, inhalant, aerosol inhalant, patch, particle inhalant, implant, depot implant, ingestible, injectable, or infusion. The dosage forms may include a variety of other ingredients, including binders, solvents, bulking agents, plasticizers, etc.

A wide variety of orally administrable compositions may be used. In a particular embodiment, the oral compositions are provided in solid discrete, self-contained dosage units, such as tablets, caplets, lozenges, capsules, gums, etc., which may comprise or be filled with liquid or solid dosages of the recited medicament constituents. A wide variety of dosages may be used, depending on the application and empirical determination; typical dosages range from 1 mg to 1 g, preferably at least 10 mg, more preferably at least 100 mg. The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.

Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid,

Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

The above described components are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical

Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pa., which is incorporated herein by reference.

The dosage forms of the present invention'involve the administration of an active therapeutic substance or multiple active therapeutic substances in a single dose during a 24 hour period of time or multiple doses during a 24 hour period of time. The doses may be uneven in that each dose is different from at least one other dose.

The subject compositions may be administered to effect various forms of release, which include, without limitation, immediate release, extended release, controlled release, timed release, sustained release, delayed release, long acting, pulsatile delivery, etc., using well known procedures and techniques available to the ordinary skilled artisan. A description of representative sustained release materials can be found in the incorporated materials in Remington's Pharmaceutical Sciences.

The subject compositions may be fonnulated for administration by any route, including without limitation, oral, buccal, sublingual, rectal, parenteral, topical, inhalational, including itnranasal, injectable, including subcutaneous, intravenous, intramuscular, etc., topical, including transdermal, etc. The subject compositions are administered in a pharmaceutically (including therapeutically, prophylactically and diagnostically) effective amount. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

The following formulation examples illustrate representative pharmaceutical compositions of this invention. The present invention, however, is not limited to the following exemplified pharmaceutical formulations:

Formulation 1 —Capsules. Prednisone and gamma-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg prednisone and 150 mg gamma-T per capsule).

Formulation 2— Capsules. Prednisone and delta-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg prednisone and 150 mg delta-T per capsule). Formulation 3— Capsules. Prednisone, gamma-T and gamma-tocotrienol are blended with a starch diluent in an approximate 1:30:30:10 weight ratio. The mixture is filled into 350 mg capsules (approx. 5 mg prednisone, 150 mg gamma-T and 150 mg gamma-tocotrienol per capsule).

Formulation 4— Capsules. Glyburide and gamma-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg glyburide and 150 mg gamma-T per capsule).

Formulation 5— Capsules. Glyburide and delta-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg glyburide and 150 mg delta-T per capsule). Formulation 6— Capsules. Glyburide, gamma-T and gamma-tocotrienol are blended with a starch diluent in an approximate 1:30:30:10 weight ratio. The mixture is filled into 400 mg capsules (approx. 5 mg glyburide, 150 mg gamma-T and 150 mg gamma-tocotrienol per capsule).

Formulation 7— Capsules. Methylprednisolone and gamma-T are blended with a starch diluent in an approximate 1 :30: 10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg methylprednisolone and 150 mg gamma-T per capsule). Formulation 8— Capsules. Prednisolone and gamma-T are blended with a starch diluent in an approximate 1:30: 10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg prednisolone and 150 mg gamma-T per capsule).

Formulation 9— Capsules. Dexamethasone and gamma-T are blended with a starch diluent in an approximate 1 :30: 10 weight ratio. The mixture is filled into 200 mg capsules

(approx. 5 mg dexamethasone and 150 mg gamma-T per capsule).

Formulation 10— Capsules. Triamcinolone and gamma-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg triamcinolone and 150 mg gamma-T per capsule). Formulation 11— Capsules. Amaryl® and gamma-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg Amaryl® and 150 mg gamma-T per capsule).

Formulation 12— Capsules. Glucotrol® and gamma-T are blended with a starch diluent in an approximate 1:30: 10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg Glucotrol® and 150 mg gamma-T per capsule).

Formulation 13— Capsules. Repaglinide and gamma-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg repaglinide and 150 mg gamma-T per capsule).

Formulation 14— Capsules. Nateglinide and gamma-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules

(approx. 5 mg nateglinide and 150 mg gamma-T per capsule).

Formulation 15~Capsules. Acarbose and gamma-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg acarbose and 150 mg gamma-T per capsule). Formulation 16—Capsules. Miglitol and gamma-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg miglitol and 150 mg gamma-T per capsule).

Formulation 17— Capsules. Metformin and gamma-T are blended with a starch diluent in an approximate 1 :30: 10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg metformin and 150 mg gamma-T per capsule).

Formulation 18— Capsules. Rosiglitazone and gamma-T are blended with a starch diluent in an approximate 1 :30: 10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg rosiglitazone and 150 mg gamma-T per capsule).

Formulation 19~Capsules. Pioglitazone and gamma-T are blended with a starch diluent in an approximate 1:30:10 weight ratio. The mixture is filled into 200 mg capsules (approx. 5 mg pioglitazone and 150 mg gamma-T per capsule).

Formulation 20~Liquid. Prednisone (10 mg) and gamma-T are blended (300 mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (1189, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water is then added to produce a total volume of 5 mL.

Formulation 21~Liquid. Glyburide (10 mg) and gamma-T are blended (300 mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water is then added to produce a total volume of 5 mL.

Formulation 22-Liquid. Acarbose (10 mg) and gamma-T are blended (300 mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water is then added to produce a total volume of 5 mL.

Formulation 23-Liquid. Metformin (10 mg) and gamma-T are blended (300 mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water is then added to produce a total volume of 5 mL. Formulation 24— Liquid. Rosiglitazone (10 mg) and gamma-T are blended (300 mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water is then added to produce a total volume of 5 mL. The invention also provides methods of reducing weight gain, obesity, and/or triglyceride accumulation in adipocytes by administering to a patient a subject medicament. In a particular embodiment of this aspect, the invention provides a method for treating a patient with an obesity-related disease such as diabetes, and/or seeking to reduced weight gain, which method comprises administering to said patient a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective obesity-related disease / weight-gain -treating amount of a subject medicament. Additionally, this invention is directed to a method for preventing the onset of an obesity-related disease in a patient at risk for developing such disease which method comprises administering to said patient a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective obesity-related disease or weight gain -preventing amount of a subject medicament.

This aspect of the invention may be implemented by a first diagnostic step, e.g. determining that the patient is suffering from, subject to, or predisposed to a target disease or condition followed by prescribing and/or administering to the patient a subject medicament, optionally followed by an evaluation/confirmation/prognosis step, e.g. determining an effect of the treatment, such as an amelioration of symptoms of a targeted disease or condition or an indicator thereof. Hence, in one embodiment, the methods additionally comprise the steps of detecting, confirming, and/or determining the presense of or predisposition to obesity, weight gain and/or undesireably high or excessive triglyceride accumulation in adipocytes and/or the step of detecting, confirming and/or determining a resultant reduction of obesity, weight gain and/or triglyceride accumulation in adipocytes.

The following empirical and experimental examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention.

EMPIRICAL EXAMPLES • I. Oral formulations 1-12 (supra) demonstrate intestinal anti-inflammatory efficacy in vivo For this experimental protocol, we adapted methods originally described by Galvez J, Aliment Pharmacol Ther 2001 Dec;15(12):2027-39, to assess the anti-inflammatory activity of the formulations in the chronic stages of trinitrobenzenesulphonic acid-induced rat colitis. In our methods, rats are rendered colitic by a single colonic instillation of 30 mg of the hapten trinitrobenzenesulphonic acid dissolved in 0.25 mL of 50%o ethanol. A group of colitic animals is given one of formulations 1-24 (supra) orally at doses of 25 mg/kg daily. Animals are sacrificed every week for 4 weeks. Colonic damage is evaluated macroscopically and microscopically. Different biochemical markers of colonic inflammation are also assayed, including myeloperoxidase activity, leukotriene B4 and interleukin-lbeta synthesis, glutathione and malonyldialdehyde levels and nitric oxide synthase activity. The administration of our formulations facilitates tissue recovery during the 4 weeks following colonic insult with trinitrobenzenesulphonic acid, as demonstrated macroscopically and microscopically, as well as biochemically by a reduction in myeloperoxidase activity. The intestinal anti-inflammatory effect is accompanied by a significant reduction in colonic leukotriene B4 and interleukin-lbeta levels, improvement in colonic oxidative stress and inhibition of colonic nitric oxide synthase activity. We conclude that our oral formulations exert a beneficial anti-inflammatory effect in the chronic phase of trinitrobenzenesulphonic acid-induced rat colitis through the down-regulation of some of the mediators involved in the intestinal inflammatory response, including free radicals, cytokines, leukotriene B4 and nitric oxide.

II. Oral formulations 1-12 (supra) reduce carrageenan-induced inflammation in the air- pouch animal model.

For this experimental protocol, we adapted the methods recited in the Experimental Example reported below. As cited below, carrageenan-induced inflammation in the air- pouch model was performed and evaluated substantially as previously described (14).

Formulations were administered as described below, wherein the recited tocopherol was dissolved in tocopherol-stripped corn oil, and continuously administrated by gavage for three days before the induction of inflammation. The PGE2 component of the formulation was empirically varied and adjusted to provide a minimized synergistic concentration. For example, aspirin formulations were adjusted to 50 or lOOmg/Kg with gamma-T at 30mg/Kg.

In controls evaluating the effects of aspirn or gamma-T alone, we increased the aspririn dosage to 150mg/Kg and gamma-T to 30 mg/kg, respectively. These studies demostrate that our formulations provide more potent effects on inhibition of PGE2 and LTB4, TNF-a etc than either alone.

III. Topical formulations 13-24 (supra) demonstrate topical anti-inflammatory efficacy in vivo

For this experimental protocol, we adapted the methods of Parneix-Spake A, et al. (J Dermatolog Treat 2001 Dec; 12(4): 191-7) to compare the healing properties of our topical formulations 13-24 (0.05% cream) with their emollient base, hydrocortisone l°/0 cream and with no treatment, in a single-center, double-blind, intra-individual, comparative study involving 18 volunteers with nickel-induced contact dermatitis. Following a positive patch test to nickel, sub-therapeutic amounts (10 micro 1 *= 3 mg cm(-2)) of each of the treatments are applied twice daily for seven days to each of the four test sites.

In terms of the primary endpoint, a physician's global assessment after 7 days of treatment, each of formulations 13-24 (0.05% cream) shows a significantly better response than hydrocortisone (HC) 1% cream or no treatment. Our 0.05%> formulation creams also show a better response than their emollient base. In terms of moisturizing effects, there was no difference in trans epidermal water loss (TEWL) between 0.05% formulation creams and their emollient base. In terms of skin blanching activity, the steroid-based creams achieve lower colorimetric values than the emollient base cream. Results from experimentally induced skin inflammation indicate that our 0.05% formulation creams have both more effective anti-inflammatory activity and better moisturizing properties than hydrocortisone 1% cream.

EXPERIMENTAL EXAMPLES

Carrageenan-induced inflammation in the air-pouch model was performed and evaluated substantially as previously described (14). Alpha-T and gamma-T were measured in plasma and exudate as previously described (12). Gamma-CEHC was quantified essentially as previously described (13). For the measurement of PGE2, LTB4 and 8-isoprostane, the exudate fluid was mixed vigorously with 2 mL methanol to precipitate proteins, and with 5 mL hexane to remove lipids. Following a brief centrifugation and aspiration of the hexane layer, the methanol layer was removed and evaporated under N2. PGE2, LTB4 and 8-isoprostane were measured using the corresponding ELISA kits from Cayman Chemicals (Ann Arbor, MI). TNF-a and lactate dehydrogenase (LDH) in the exudate were measured directly using an ELISA kit from R&D (Minneapolis, MN) and an analytical kit from Roche (Indianapolis, IN), respectively.

The total amount of nitrate and nitrite in the exudate was measured using the Model 280 nitric oxide analyzer (NO A™) (Sievers Instruments, Inc.; Boulder, CO). Nitrite and nitrate in the exudate were reduced by vanadium (III) to nitric oxide, which then was measured by a red-sensitive photomultiplier tube after being converted by ozone to chemiluminescent reactive nitrogen dioxide. Nitrite and nitrate were quantified based on a standard curve from nitrate that was established under the identical conditions.

Administration of gamma-T and its major metabolite, gamma-CEHC, but not alpha-T, significantly inhibits pro-inflammatory eicosanoids at the site of inflammation. Carrageenan- induced inflammation in the air-pouch model is commonly used to evaluate the pharmaceutical potency of anti-inflammatory drugs (14). In this model, an injection of air into the intrascapular area resulted in the formation of a connective tissue cavity mainly lined with macrophages and fibroblasts (11, 15). These cells play a key role in the inflammatory response (11, 14, 15). A single injection of carrageenan caused a potent localized inflammation, as indicated by a marked increase in white cell infiltration, eicosanoid formation and tissue damages (11). To study the effect of gamma-CEHC on eicosanoid synthesis and neutrophil infiltration, it (1 mg/ l, 2 ml in PBS) was injected directly into the air pouch right before the injection of carrageenan. Since the retention of gamma-CEHC is relatively short (16), its effect was evaluated at 6 h after the induction of inflammation. Administration of gamma-CEHC significantly reduced PGE2 (-29%, P < 0.05) in the pouch, which is consistent with our previous observations in vitro (10). At this dose, gamma-CEHC also inhibited LTB4 and neutrophil infiltration, though non-significantly.

To test the effects of tocopherols, alpha-T or gamma-T, dissolved in tocopherol- stripped corn oil, was continuously administrated by gavage for three days before the induction of inflammation. Effects were evaluated at 20 h after carrageenan injection, when cell infiltration had reached a maximum in the pouch. At a dose of 33 mg/kg, gamma-T, but not alpha-T, significantly reduced the pro-inflammatory PGE2 (46%, P < 0.05) and LTB4 (70%, P < 0.05), a potent chemotactic eicosanoid produced by the 5-lipoxygenase in neutrophils. At a higher dose, i.e. 100 mg/kg, gamma-T showed an inhibitory potency against PGE2 (51%, P < 0.05) and LTB4 similar to the lower dose. Despite the inhibitory effects on PGE2 and LTB4, gamma-T did not affect neutrophil infiltration. Gamma-T reduced TNF-a and total nitrite and nitrate at the higher dose of 100 mg/kg.

In addition to the eicosanoids, we have investigated the effects of gamma-T on TNF-a, an important inflammation mediator, and total nitrite and nitrate, an index of the generation of reactive nitrogen oxides. At the lower dose of 33mg/kg, gamma-T non-significantly inhibited TNF-a (~51%, P = 0.29), but had no effect on total nitrate and nitrite. At the higher dose of 100 mg/kg, gamma-T reduced TNF-a (65%, P = 0.069) and total nitrite and nitrate (40%, P *= 0.1).

Administration of gamma-T attenuates the partial loss of food consumption induced by inflammation, and inhibits inflammation-mediated lipid peroxidation and cytotoxicity. The effect of gamma-T and alpha-T on inflammation-induced lipid peroxidation and inflammation-site tissue damage was assayed by the 8-isoprostane level (17) and by the release of lactate dehydrogenase (LDH), respectively (11). Carrageenan-induced inflammation resulted in a marked increase in 8-isoprostane and LDH in the pouch. In contrast to alpha-T (33 mg kg), gamma-T at the dose of 33 or 100 mg/kg significantly reduced 8-isoprostane (for both doses, ~57%, P < 0.05) in the pouch. gamma-T, at the higher dose of 100 mg/kg, lowered LDH (30%, P = 0.067), a marker of cytotoxicity and tissue damage. In addition, carrageenan-induced inflammation resulted in a marked reduction in food consumption (~40%, P < 0.01), which is likely caused by the discomfort associated with inflammation. Administration of gamma-T at 100 mg/kg significantly attenuated (30%, P < 0.03) the loss of food consumption, while a smaller, non-significant, effect was observed at the lower dose of 33 mg/kg (20%, P = 0.2).

Plasma and exudate concentrations of alpha-T, gamma-T and gamma-CEHC. To evaluate the relative bio-availability of the administrated compounds, we measured plasma and exudate concentrations of gamma-T and alpha-T as well as plasma gamma-CEHC. Administration of alpha-T or gamma-T led to the significant increases in both tocopherols in the plasma and exudate, while their relative increase in the exudate is more than that in the plasma. Thus, gamma-T-administrated (33 or lOOmg/kg) rats had nearly 10- or 20-fold elevation of gamma-T in exudates fluid, in contrast to 3- or 5-fold increase in the plasma, as compared with corn-oil fed controls. With alpha-T, a similar trend also was observed. In gamma-T-administrated rats (33 or lOOmg/kg), the ratio of gamma-T to alpha-T in the exudate (~ 0.3 or 0.7) is higher than that in the plasma (-0.15), consistent with the idea that tissues may have a higher gamma-T partition than does plasma (18). gamma-T administration did not significantly affect alpha-T, but alpha-T caused significant decreases of gamma-T both in the plasma (-55%, P < 0.05) and exudate (40%, P < 0.05). gamma- CEHC, the major metabolite of gamma-T, increased in response to gamma-T supplementation. Nano-molar concentrations of gamma-CEHC were found in the plasma, a level less than 10% that of plasma gamma-T. gamma-T administration caused a 2.5-4 fold elevation of gamma-CEHC in the plasma.

Carrageenan-induced inflammation in the rat air pouch model mimics the pathological process occurring in the joint diseases such as arthritis; the connective tissues formed along the air pouch are similar to those found in chronic joint diseases (11, 15). Carrageenan-induced inflammation and chronic joint diseases also share other features including markedly elevated PGE2, neutrophil infiltration, cytokine formation and tissue damage (11). Studies have established that in this model, COX-2, which is quickly induced in the lining macrophages and fibroblasts, is the primary enzyme responsible for the elevation of PGE2 (14). Thus, various COX-2 inhibitors have been shown to inhibit the formation of PGE2 in the pouch (14). We recently found that the major form of vitamin E in the diet, gamma-T, and its metabolite, but not alpha-T, the major form in supplements, effectively inhibited COX-2 activity in lipopolysacharide-activated macrophages and interlukin- lb- treated epithelial cells (10). In line with this in vitro observation, the present study shows that in the carrageenan air pouch model, gamma-T (33 or lOOmg/kg), in contrast to alpha-T (33mg/kg), significantly lowered PGE2 elevation at the site of inflammation. Local delivery of gamma-CEHC into the air pouch also led to a significant inhibition of PGE2. These results therefore demonstrate that gamma-T and gamma-CEHC show in vivo anti-inflammatory properties that appear to be similar to those of NSAIDs. In addition, gamma-T but not alpha- T significantly inhibits LTB4, a potent chemotactic agent that is synthesized by 5- lipoxygenase of neutrophils (8).

In addition to the inhibitory effects on the pro-inflammatory eicosanoids, gamma-T administration reduced inflammation-mediated damage as shown by its reduction of lipid peroxidation and LDH activity. gamma-T attenuated the marked loss of food consumption that is likely caused by the inflammation-associated discomfort. Because PGE2 is known to play a key role in causing pain and fever, a reduction of this eicosanoid may in part explain gamma-T' s effect on the food consumption. In addition, other unique properties of gamma-T may also contribute to the observed beneficial effects (18). Because of the unsubstituted 5- position, compared with alpha-T, gamma-T is capable of trapping reactive nitrogen oxide to form a stable nitrated adduct (19). Thus, gamma-T is better than alpha-T in the protection of peroxynitrite-induced lipid peroxidation (19) and enzyme inactivation (20). We recently found that in the zymosan-induced inflammation model, gamma-T supplementation consistently inhibited protein nitration and ascorbate oxidation (12).

At 100 mg/kg but not 33mg/kg, gamma-T lowers the accumulation of total nitrate and nitrite. Although some studies show that reactive nitric oxide stimulates PGE2 formation (21, 22), in the current model, however, these two events appear to be independent because gamma-T decreases PGE2 at both doses. In LPS-stimulated macrophages, we previously found that gamma-T moderately inhibits nitrite accumulation via a moderate inhibition of the induction of inducible nitric oxide synthase (10). It is possible that the currently observed reduction of total nitrate and nitrite is caused by gamma-T's suppression of this enzyme. Alternatively, gamma-T's capability of trapping reactive nitrogen oxides may also result in lowered levels of total nitrate and nitrite. However, we did not observe an apparent increase in 5-nitro-gamma-tocopherol in the exudate fluid. Nevertheless, this possibility cannot be ruled out due to the lack of measuring 5-nitro-gamma-CEHC, a putative breakdown product of 5-nitro-gamma-tocopherol (18).

Gamma-T can decrease TNF-a, a key proinflammatory cytokine known to activate macrophages and provoke the inflammatory response (23). Studies have shown that inhibition of TNF-a provides beneficial effects on inflammatory diseases (24). The utilization of antibody against TNF-a has been proven to be an effective therapy for inflammatory disease (25).

Although gamma-T significantly inhibits the pro-inflammatory eicosanoids, it has no effect on the neutrophil infiltration. It has been shown that in the carrageenan air-pouch model, there is no casual correlation between the inhibition of PGE2 and neutrophil infiltration (11). For example, aspirin, at doses of 100-150mg/kg, caused 50-70% reduction of PGE2, but yet it did not affect neutrophil infiltration (26). Although at higher doses, i.e. >200-300 mg/kg, aspirin inhibits cell infiltration, the mechanisms may include the inhibition of NFkB signal transduction (27) or the activation of adenosine formation (28). The relatively high bio-availability of gamma-T contributes to its in vivo inhibition of eicosanoids in the pouch. gamma-T administration resulted in a more pronounced elevation of this tocopherol in the exudate than that in the plasma. When gamma-T showed significant inhibitory effects on PGE2 at 20-h after the injection of carrageenan, its concentration in the collected exudate was estimated to be 93.3 (33mg/kg) to 193 nmol/gprotein (lOOmg/kg), corresponding to -10 to 20 mM, which is comparable to the apparent IC50 (~5-10mM) estimated in the cell culture (10). An increase in gamma-T administration from 33 to lOOmg/kg did not significantly improve the inhibitory potency, probably due to the saturation effect. Although gamma-CEHC, when applied directly into the pouch, significantly inhibited PGE2, the effect of gamma-T cannot be attributed to this metabolite. This is because the concentration of gamma-CEHC is in the nanomolar range, which is much lower than the estimated IC50 (30-40mM) for g-CEHC to inhibit COX-2 activity (10). Though as much as 50% of gamma-T may be converted to gamma-CEHC (29), this metabolite is not likely to be accumulated in the plasma or tissues (except the kidney) because of its short retention time

(16). In the present study, although administration of alpha-T (33mg/kg) led to an approximately 2-fold increased level in the plasma and exudate, alpha-T did not show any significant effects with respect to the generation of eicosanoids and 8-isoprostane, in line with our in vitro observations (10). It may be also relevant that alpha-T has a high baseline in tissues as a result of the high content of alpha-T in the diet. However, administration of the same dose of gamma-T did show significant effects. This observation indicates that gamma-

T possesses unique properties that are not shared alpha-T (18). Because alpha-T is preferentially retained by the body and is the strongest antioxidant in the vitamin E family, supplementation of both gamma-T and alpha-T (especially when alpha-T is relatively low) may result in better outcomes. Not only does gamma-T reduce PGE2, but it also inhibits lipoxygenase-catalyzed synthesis of LTB4 as well as decreases TNF-a, an activity most NSAIDs do not have (26). These findings indicate a potentially superior pharmaceutical use of gamma-T compared with traditional NSAIDs. Gamma-T may be useful in cancer prevention. It is known that COX-2 and PGE2 are elevated in inflammation-associated diseases including cancer (30). Frequent intake of NSAIDs such as aspirin is known to reduce the risk of certain cancers (31, 32). Recently, Helzlsouer et al. (33) reported that in a nested case-control study, men in the highest quintile of plasma gamma-T levels had a 5-fold reduction in the risk of prostate cancer compared to those in the lowest quintile. We recently found that gamma-T but not alpha-T showed anti-proliferative effects on prostate and lung cancer cell lines, but had no effect on normal epithelial cells. The anti-inflammatory effects of gamma-T may be beneficial in cardiovascular disease, another inflammation-associated disorder (34). Several studies (35, 36) reported that plasma concentrations of gamma-T, but not alpha-T, are inversely associated with the incidence of coronary heart diseases.

This experimental example shows that gamma-T inhibits the pro-inflammatory eicosanoids, suppresses pro-inflammatory cytokines and attenuates inflammation-caused damage in an in vivo rat inflammation model.

PARENTHETICAL REFERENCES l.Balkwill, F., and Mantovani, A. (2001) Lancet 357, 539-545.

2.Libby, P., Ridker, P. M., and Maseri, A. (2002) Circulation 105, 1135-1143. 3.McGeer, P. L., and McGeer, E. G. (2001). Neurobiol Aging 22, 799-809.

4.Vane, J. R. (1976). Adv Prostaglandin Thromboxane Res 2, 791-801

5.Samad, T. A„ et al. (2001). Nature 410, 471-475.

6.Williams, J. A., and Shacter, E. (1997) 2. JBiol Chem 272, 25693-25699

7Nane, J. R., and Botting, R. M. (1998). Int J Tissue React 20, 3-15 δ.Yokomizo, T., Izumi, T., and Shimizu, T. (2001) Arch Biochem Biophys 385, 231-241.

9.Wynne, H. A., and Campbell, M. (1993) Pharmacoeconomics 3, 107-123.

10. Jiang, Q., Elson-Schwab, I., Courtemanche, C, and Ames, B. N. (2000) Proc NatlAcad

Sci USA 91, 11494-11499. ll.Sedgwick, A. D., and Lees, P. (1986) Agents Actions 18, 429-438. 12. Jiang, Q., Lykkesffeldt, J., Shigenaga, M. K, Shigeno, E. T., Christen, S., and Ames, B.

N. (2002) Gamma-tocopherol inhibits protein nitration and ascorbate oxidation in rats with inflammation. Eree Rad Biol Med 33, in press

13.Stahl, W., et al. (1999) AnalBiochem 275, 254-259

KMasferrer, J. L., et al. (1994) Proc NatlAcadSci USA 91, 3228-3232.

15.Sedgwick, A. D., et al. . (1983) JPathol 141, 483-495. 16.Murray, E. D., Jr., et al. (1997) J Pharmacol Exp Ther 282, 657-662.

17.Morrow, J. D. (2000) DrugMetab Rev 32, 377-385.

18Jiang, Q., Christen, S., Shigenaga, M. K., and Ames, B. N. (2001) Am J Clin Nutr 74, 714-

722.

19.Christen, S., Woodall, A. A., Shigenaga, M. K., Southwell-Keely, P. T., Duncan, M. W., and Ames, B. N. (1997) Proc NatlAcad Sci US A 94, 3217-3222.

20.Williamson, K. S., et al. (2002) Nitric Oxide 6, 221-227.

21.Beharka, A. A., et al. (2002) Free Radic Biol Med 32, 503-511

22.Landino, L. M., et al. (1996). Proc NatlAcadSci USA 93, 15069-15074

23.Belardelli, F. (\995) Apmis 103, 161-179. 24.Criscione, L. G., and St Clair, E. W. (2002) Curr Opin Rheumatol 14, 204-211.

25.Lorenz, H. M., and Kalden, J. R. (2002) Arthritis Res 4, SI 7-24.

26.Kirchner, T., et al. (1997) J Pharmacol Exp Ther 282, 1094-1101.

27.Yin, M. J., Yamamoto, Y., and Gaynor, R. B. (1998) Nature 396, 77-80.

28.Cronstein, B. N., et al. (1999) Proc NatlAcad Sci USA 96, 6377-6381. 29.Swanson, J. E., et al. (1999) J Lipid Res 40, 665-671.

30.Marnett, L. J. (1992) Cancer Res 52, 5575-5589.

31.Smalley, W. E., and DuBois, R. N. (1997) Adv Pharmacol 39, 1-20

32.Thun, M. J., et al. (1993) Cancer Res 53, 1322-1327

33.Helzlsouer, K. J., et al. (2000) JNatl Cancer Inst 92, 2018-2023. 34.Plutzky, J. (2001) Am J Cardiol 88, 10K-15K.

35.Kristenson, M., et al. (1997) Bmj 314, 629-633

36.Ohrvall, M., Sundlof, G., and Vessby, B. (1996) JLntern Med 239, 111-117

I. Gamma-T and delta-T, and tocotrienols, particularly, alpha-, gamma-, and delta-, show dose-dependent anti-adipogenesis activity.

We have found that gamma-T and delta-T, and tocotrienols, particularly, alpha-, gamma-, and delta-, show dose-dependent anti-adipogenesis activity inhibit embryonic cell (C3H10T1/2) differentiation to adipocytes, and decrease intracellular triglyceride accumulation. In contrast, alpa-T was much less effective. For these example, differentiation was induced in the presence of insulin with drugs, including indomethacin, and troglitazone, one of the anti-diabetes drags of the thiazolidinedione family, as well as 15-deoxy-delta (12,

14)-prostaglandin-J2, a putative endogenous ligand for the peroxisome proliferator-activated receptor-gamma. Our results clearly indicate that tocopherols and tocotrienols inhibit adipogenesis.

II. Gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, gamma-tocotrienol and delta- tocotrienol reduce triglyceride accumulation in adipocytes from a high-fat diet.

We evaluate weight gain and triglyceride accumulation in adipocytes in mice fed a high-fat diet. Six-week-old BALB/c mice are fed powdered chow with various tocopherols and tocotrienols and mixtures thereof, as 0.1%, 3%, 0.02%, or 0.01% food admixtures. For histological analysis of adipose and hepatic tissues and determination of adipocyte size, adipose tissue is removed from each animal and fixed in 10% formaldehyde/PBS and maintained at 4°C until use. Fixed specimens are dehydrated, embedded in tissue-freezing medium and frozen in dry ice and acetone. White adipose tissue is cut into 10-μnι sections, and the sections mounted on silanized slides. The adipose tissue is stained with hematoxylin and eosin (H&E). Mature white adipocytes are identified by their characteristic multilocular appearance. Total adipocyte areas are traced manually and analyzed with Win ROOF software (Mitani Co. Ltd., Chiba, Japan). White adipocyte areas are measured in 400 or more cells per mouse in each group according to methods previously described (Kubota, N. et al.1999, Mol. Cell. 4:597-609). We find that gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, gamma- tocotrienol and delta-tocotrienol, and various combinations effectively reduce triglyceride exert antiobesity effects in vivo as measured by reduced weight gain and reduced triglyceride accumulation in adipocytes roughly in proportion to their anti-adipogenetic potencies in vitro. Untreated mice on a high-fat diet gained significantly more weight than the mice on the high-carbohydrate diet. In contrast, treatment with tocopherols and tocotrienols reduce the time-dependent increase in weight on the high-fat diet. Tocopherol and tocotrienols treatments also reduce high-fat diet-induced hyperglycemia and hyperinsulinemia. On the high-fat diet, the glucose-lowering effect of insulin is greater in tocopherol and tocotrienol- treated mice treated than in untreated mice.

III. Formulations 1-3, 7-10 and 20 (supra) reduce triglyceride accumulation in adipocytes in mice fed a low-fat, high-carbohydrate diet and treated with corticosteroids.

We evaluate weight gain and triglyceride accumulation in adipocytes in mice fed a low-fat, high-carbohydrate diet and treated with corticosteroids. Six-week-old BALB/c mice are fed powdered chow with formulations given as 0.1%, 3%, 0.02%, or 0.01% food admixtures. Control animals are identically treated except that the corresponding steroid is provided in tocopherol-stripped corn oil. For histological analysis of adipose and hepatic tissues and determination of adipocyte size, adipose tissue is removed from each animal and fixed in 10% formaldehyde/PBS and maintained at 4°C until use. Fixed specimens are dehydrated, embedded in tissue-freezing medium and frozen in dry ice and acetone. White adipose tissue is cut into 10-μm sections, and the sections mounted on silanized slides. The adipose tissue is stained with hematoxylin and eosin (H&E). Mature white adipocytes are identified by their characteristic multilocular appearance. Total adipocyte areas are traced manually and analyzed with Win ROOF software (Mitani Co. Ltd., Chiba, Japan). White adipocyte areas are measured in 400 or more cells per mouse in each group according to methods previously described (Kubota, N. et al.1999, Mol. Cell. 4:597-609).

We find that our formulations exert antiobesity effects in vivo as measured by reduced weight gain and reduced triglyceride accumulation in adipocytes roughly in proportion to their anti-adipogenetic potencies in vitro. Control mice treated with corticosteroids alone gained significantly more weight than either the no-treament controls or the formulation-treated mice. Treatment with each tested formulation reduces the time-dependent increase in weight associated with the corresponding corticosteroid.

IV. Formulations 4-6, 11-19 and 21-24 (supra) reduce triglyceride accumulation in adipocytes in mice fed a low-fat, high-carbohydrate diet and treated with anti-diabetic drugs. We evaluate weight gain and triglyceride accumulation in adipocytes in db/db diabetic mice fed a low-fat, high-carbohydrate diet and treated with various anti-diabetic drugs. Six-week-old mice are fed powdered chow with formulations 4-6, 11-19 and 21-24 given as 0.1%, 3%, 0.02%, or 0.01% food admixtures. Control animals are identically treated except that the corresponding drag is provided in tocopherol-stripped corn oil. For histological analysis of adipose and hepatic tissues and determination of adipocyte size, adipose tissue is removed from each animal and fixed in 10% formaldehyde/PBS and maintained at 4°C until use. Fixed specimens are dehydrated, embedded in tissue-freezing medium and frozen in dry ice and acetone. White adipose tissue is cut into 10-μm sections, and the sections mounted on silanized slides. The adipose tissue is stamed with hematoxylin and eosin (H&E). Mature white adipocytes are identified by their characteristic multilocular appearance. Total adipocyte areas are traced manually and analyzed with Win ROOF software (Mitani Co. Ltd., Chiba, Japan). White adipocyte areas are measured in 400 or more cells per mouse in each group according to methods previously described (Kubota, N. et al.1999, Mol. Cell. 4:597-609).

We find that our formulations exert antiobesity effects in vivo as measured by reduced weight gain and reduced triglyceride accumulation in adipocytes roughly in proportion to their anti-adipogenetic potencies in vitro. Control mice treated with anti- diabetic drags alone gained significantly more weight than either the no-treament controls or the formulation-treated mice. Treatment with each tested formulation reduces the time-dependent increase in weight associated with the corresponding drug.

V. Formulations 4-6, 11-19 and 21-24 (supra) reduce weight gain in patients with non- insulin-dependent diabetes mellitus (NIDDM).

For these experimental protocols, we adapted the methods of DeFronzo, et al, N Engl J Med 1995 Aug 31;333(9):541-9, to perform randomized, parallel-group, double-blind, controlled studies in which Formulations 1-24, the corresponding anti-diabetic drugs alone, or placebos are given for 29 weeks to moderately obese patients with NIDDM whose diabetes was inadequately controlled by diet. In addition to weight gain, plasma glucose, lactate, lipids, insulin, and glycosylated hemoglobin are evaluated before, during, and at the end of the study. At the end of the study patients in the treatment groups, as compared with patients in the placebo group, have lower mean (+/- SE) fasting plasma glucose concentrations and glycosylated hemoglobin values. The effect of the fomulation treatments alone is similar to that of anti-diabetic drugs alone. Treatment groups have decreases in plasma total and low-density lipoprotein cholesterol and triglyceride concentrations, whereas the values in the respective control groups are unchanged. No significant changes are observed in fasting plasma lactate concentrations in any of the groups. No significant weight gain is observed in either the control or formulation treatment groups, whereas anti-diabetic drug treatment groups present significant weight gain.

The foregoing descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

All publications and patent applications cited in this specification and all references cited therein are herein incorporated by reference as if each individual publication or patent application or reference were specifically and individually indicated to be incorporated by reference. Any material accompanying this application on compact disc or other recorded medium is incorporated by reference.

Claims

WHAT IS CLAIMED IS:
1. A medicament comprising predeteπnined amounts of a phytyl substituted chromanol and an inhibitor of prostaglandin E2 (PGE2) formation, wherein: said medicament is in unit dosage form suitable for pharmaceutical administration; said phytyl substituted chromanol is selected from the group consisting of gamma- tocopherol, delta-tocopherol, gamma-tocotrienol and delta-tocotrienol; and said PGE2 inhibitor is selected from the group consisting of an omega-3 fatty acid cyclooxygenase substrate and a non-steroidal anti-inflammatory drug (NSAID) cyclooxygenase inhibitor.
2. The medicament of claim 1, wherein the medicament comprises less than 5% alpha- tocopherol.
3. The medicament of claim 1, wherein the medicament comprises less than 0.5% alpha- tocopherol.
4. The medicament of claim 1, wherein the medicament comprises less than 0.05% alpha- tocopherol.
5. The medicament of claim 1, wherein the medicament comprises a mixture of gamma- tocopherol and delta-tocopherol.
6. The medicament of claim 1, wherein the medicament comprises a mixture of gamma- tocopherol and gamma-tocotrienol.
7. The medicament of claim 1, wherein the phytyl substituted chromanol is purified to at least 95% homogeneity.
8. The medicament of claim 1, wherein the PGE2 inhibitor is at a dosage that is suboptimally therapeutical when administered alone.
9. The medicament of claim 1, wherein the PGE2 inhibitor is at a dosage that is subtherapeutic when administered alone.
10. The medicament of claim 1 , wherein the PGE2 inhibitor is at a dosage that is at least 50%o subtherapeutic when administered alone.
11. The medicament of claim 1, wherein the PGE2 inhibitor is cyclooxygenase-2 (COX2) selective inhibitor.
12. The medicament of claim 1, wherein the fatty acid is selected from the group consisting of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).
13. A method of inhibiting inflammation, comprising administering to a patient a medicament according to claim 1.
14. A medicament comprising predetermined amounts of a phytyl substituted chromanol and an obesity-promoting drug, wherein: said medicament is in unit dosage form suitable for pharmaceutical administration; said phytyl substituted chromanol is selected from the group consisting of gamma- tocopherol, delta-tocopherol, alpha-tocotrienol, gamma-tocotrienol and delta-tocotrienol; and said obesity-promoting drug is selected from the group consisting of a corticosteroid and an anti-diabetes drag selected from the group consisting of hypoglycemic drags, starch blockers, glucose production blockers, and insulin sensitizers.
15. The medicament of claim 14, wherein the medicament comprises less than 5% alpha- tocopherol.
16. The medicament of claim 14, wherein the medicament comprises less than 0.5% alpha- tocopherol.
17. The medicament of claim 14, wherein the medicament comprises less than 0.05%o alpha- tocopherol.
18. The medicament of claim 14, wherein the medicament comprises a mixture of gamma- tocopherol and delta-tocopherol.
19. The medicament of claim 14, wherein the medicament comprises a mixture of gamma- tocopherol and gamma-tocotrienol.
20. The medicament of claim 14, wherein the medicament comprises a mixture of gamma- tocopherol and alpha-tocotrienol.
21. The medicament of claim 14, wherein the medicament comprises a mixture of delta- tocopherol and alpha-tocotrienol.
22. The medicament of claim 14, wherein the medicament comprises a mixture of gamma- tocopherol, delta-tocopherol and gamma-tocotrienol.
23. The medicament of claim 14, wherein the phytyl substituted chromanol is purified to at least 95% homogeneity.
24. The medicament of claim 14, wherein the obesity-promoting drag is a glucocorticosteroid selected from the group consisting of prednisone (Deltasone®, Orasone®), methylprednisolone (Medrol®), prednisolone (Prelone®, Pediapred®), dexamethasone (Decadron®), and triamcinolone (Aristocort®).
25. The medicament of claim 14, wherein the obesity-promoting drug is a hypoglycemic drug selected from the group consisting of glyburide (DiaBeta®, Micronase®), Amaryl®, Glucotrol®, repaglinide (Prandin®), and nateglinide (starlix®).
26. The medicament of claim 14, wherein the obesity-promoting drug is a starch blocker selected from the group consisting of acarbose (Precose®), and miglitol (Glyset®).
27. The medicament of claim 14, wherein the obesity-promoting drag is a glucose production blocker selected from the group consisting of metformin (Glucophage®).
28. The medicament of claim 14, wherein the obesity-promoting drug is an insulin sensitizer that is a thiazolindinedion drag selected from the group consisting of rosiglitazone
(Avandia®) and pioglitazone (Actos®)
29. A method of reducing obesity-promotion, comprising administering to a patient a medicament according to claim 14.
30. A method for reducing triglyceride accumulation in adipocytes, said method comprising the steps of contacting a patient predetermined to have or be predisposed to undersirably high triglyceride accumulation in adipocytes with an effective amount of a phytyl substituted chromanol selected from the group consisting of gamma-tocopherol, delta-tocopherol, alpha- tocotrienol, gamma-tocotrienol and delta-tocotrienol, and detecting in the patient a resultant reduction in adipocyte triglyceride accumulation.
31. The method of claim 30, wherein the medicament comprises less than 5% alpha- tocopherol.
32. The method of claim 30, wherein the medicament comprises a mixture of gamma- tocopherol and delta-tocopherol.
33. The method of claim 30, wherein the phytyl substituted chromanol is purified to at least 95% homogeneity.
PCT/US2003/037135 2002-11-21 2003-11-19 Tocopherol and tocotrienol medicaments WO2004047732A3 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US10/301,211 2002-11-21
US10301211 US20040102421A1 (en) 2002-11-21 2002-11-21 Tocopherol and tocotrienol anti-inflammatory medicaments
US10304918 US7399784B2 (en) 2002-11-26 2002-11-26 Tocopherol and tocotrienol anti-obesity medicaments
US10/304,918 2002-11-26

Publications (2)

Publication Number Publication Date
WO2004047732A2 true true WO2004047732A2 (en) 2004-06-10
WO2004047732A3 true WO2004047732A3 (en) 2004-08-19

Family

ID=32396699

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/037135 WO2004047732A3 (en) 2002-11-21 2003-11-19 Tocopherol and tocotrienol medicaments

Country Status (1)

Country Link
WO (1) WO2004047732A3 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8951514B2 (en) 2011-02-16 2015-02-10 Pivotal Therapeutics Inc. Statin and omega 3 fatty acids for reduction of apolipoprotein-B levels
US8952000B2 (en) 2011-02-16 2015-02-10 Pivotal Therapeutics Inc. Cholesterol absorption inhibitor and omega 3 fatty acids for the reduction of cholesterol and for the prevention or reduction of cardiovascular, cardiac and vascular events
US9119826B2 (en) 2011-02-16 2015-09-01 Pivotal Therapeutics, Inc. Omega 3 fatty acid for use as a prescription medical food and omega 3 fatty acid diagniostic assay for the dietary management of cardiovascular patients with cardiovascular disease (CVD) who are deficient in blood EPA and DHA levels

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8715648B2 (en) 2011-02-16 2014-05-06 Pivotal Therapeutics Inc. Method for treating obesity with anti-obesity formulations and omega 3 fatty acids for the reduction of body weight in cardiovascular disease patients (CVD) and diabetics

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020032171A1 (en) * 1999-06-30 2002-03-14 Feng-Jing Chen Clear oil-containing pharmaceutical compositions containing a therapeutic agent
US20030007961A1 (en) * 2001-06-22 2003-01-09 Wilburn Michael D. Orthomolecular vitamin E derivatives
US20030013693A1 (en) * 1998-02-11 2003-01-16 Rtp Pharma Inc. Method and composition for treatment of inflammatory conditions
US20030100603A1 (en) * 2001-08-21 2003-05-29 Peggy Beinlich Tocopherol enriched compositions and amelioration of inflammatory symptoms
US20030144219A1 (en) * 2001-11-15 2003-07-31 Phinney Stephen Dodge Formulations and methods for treatment or amelioration of inflammatory conditions

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030013693A1 (en) * 1998-02-11 2003-01-16 Rtp Pharma Inc. Method and composition for treatment of inflammatory conditions
US20020032171A1 (en) * 1999-06-30 2002-03-14 Feng-Jing Chen Clear oil-containing pharmaceutical compositions containing a therapeutic agent
US20030007961A1 (en) * 2001-06-22 2003-01-09 Wilburn Michael D. Orthomolecular vitamin E derivatives
US20030100603A1 (en) * 2001-08-21 2003-05-29 Peggy Beinlich Tocopherol enriched compositions and amelioration of inflammatory symptoms
US20030144219A1 (en) * 2001-11-15 2003-07-31 Phinney Stephen Dodge Formulations and methods for treatment or amelioration of inflammatory conditions

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8951514B2 (en) 2011-02-16 2015-02-10 Pivotal Therapeutics Inc. Statin and omega 3 fatty acids for reduction of apolipoprotein-B levels
US8952000B2 (en) 2011-02-16 2015-02-10 Pivotal Therapeutics Inc. Cholesterol absorption inhibitor and omega 3 fatty acids for the reduction of cholesterol and for the prevention or reduction of cardiovascular, cardiac and vascular events
US9119826B2 (en) 2011-02-16 2015-09-01 Pivotal Therapeutics, Inc. Omega 3 fatty acid for use as a prescription medical food and omega 3 fatty acid diagniostic assay for the dietary management of cardiovascular patients with cardiovascular disease (CVD) who are deficient in blood EPA and DHA levels

Also Published As

Publication number Publication date Type
WO2004047732A3 (en) 2004-08-19 application

Similar Documents

Publication Publication Date Title
Widlansky et al. Acute EGCG supplementation reverses endothelial dysfunction in patients with coronary artery disease
Patrick et al. Cardiovascular disease: C-reactive protein and the inflammatory disease paradigm: HMG-CoA reductase inhibitors, alpha-tocopherol, red yeast rice, and olive oil polyphenols. A review of the literature
JIANG et al. γ-Tocopherol, but not α-tocopherol, decreases proinflammatory eicosanoids and inflammation damage in rats
Shin et al. Enhanced bioavailability of tamoxifen after oral administration of tamoxifen with quercetin in rats
Gupta et al. Therapeutic roles of curcumin: lessons learned from clinical trials
Kunnumakkara et al. Curcumin, the golden nutraceutical: multitargeting for multiple chronic diseases
Wu et al. Caffeic acid phenethyl ester (CAPE), derived from a honeybee product propolis, exhibits a diversity of anti-tumor effects in pre-clinical models of human breast cancer
Naruszewicz et al. Combination therapy of statin with flavonoids rich extract from chokeberry fruits enhanced reduction in cardiovascular risk markers in patients after myocardial infraction (MI)
Hsu et al. Clinical studies with curcumin
Koh et al. Effects of alpha-lipoic acid on body weight in obese subjects
US20060003947A1 (en) Soft gel capsules containing polymethoxylated flavones and palm oil tocotrienols
Miquel et al. Menopause: a review on the role of oxygen stress and favorable effects of dietary antioxidants
US20010044462A1 (en) Desmethyl tocopherols for protecting cardiovascular tissue
Stan et al. Chemoprevention strategies for pancreatic cancer
Kidd Bioavailability and activity of phytosome complexes from botanical polyphenols: the silymarin, curcumin, green tea, and grape seed extracts
Joy et al. Anti-diabetic activity of Picrorrhiza kurroa extract
Kensler et al. Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas
Van Meeteren et al. Antioxidants and polyunsaturated fatty acids in multiple sclerosis
US6423742B1 (en) Compositions for reducing vascular plaque formation and methods of using same
WO2002058793A1 (en) Essential n-3 fatty acids in cardiac insufficiency and heart failure therapy
Santos et al. 1, 8-cineole (eucalyptol), a monoterpene oxide attenuates the colonic damage in rats on acute TNBS-colitis
Wang et al. Protocatechuic acid, a metabolite of anthocyanins, inhibits monocyte adhesion and reduces atherosclerosis in apolipoprotein E-deficient mice
US20080248129A1 (en) Compounds and methods for promoting cellular health and treatment of cancer
Fan et al. The clinical applications of curcumin: current state and the future
US20040229939A1 (en) Tetrahydrocannabinol compositions and methods of manufacture and use thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP