WO2000059488A2 - Enhanced tissue and subcellular delivery of vitamin e compounds - Google Patents

Abstract

The present invention provides a method for enhancing the delivery of antioxidant Vitamin E compounds to tissues, cells and subcellular sites (both in vivo and in vitro) and in particular to mitochondria, thereby increasing the antioxidative capacity of mitochondria. The Vitamin E compounds that are administered may be cleaved by cellular esterases to release antioxidant α-T, β-T, η-T, and/or δ-T. Further, the administration of Vitamin E compounds may have a coordinate action with respect to treating tumors in that the intact, uncleaved form of the Vitamin E compound has antitumor activity while the cleaved form exerts antioxidant effects.

Description

ENHANCED TISSUE AND SUBCELLULAR DELIVERY OF VITAMIN E COMPOUNDS

DESCRIPTION

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention generally relates to an improved method for delivery and retention of antitumor and antioxidant Vitamin E compounds to tissues and subcellular sites. Specifically, the present invention provides a method to enhance the antioxidant capacity of normal cells and subcellular sites such as mitochondria and to inhibit the growth of and kill tumor cells by administering an aqueous suspension of tris salts of Vitamin E compounds.

Background Description It is well established that reactive oxygen intermediates (oxidative stress) generated from both endogenous and exogenous insults (e.g. drug, chemical, hyperoxia, hypoxia, radiation, ischemia/reperfusion, aging, and inflammation) play an important role in the toxic injury/disease process (1,2). Recent studies suggest that mitochondria are the most important endogenous source of these reactive oxygen species (ROS), generating approximately 85% of all ROS produced endogenously in the cell. The overproduction of mitochondrial ROS can result in the inhibition of energy production and mitochondrial ROS production is thought to be the cause of numerous human diseases, including neurodegenerative diseases, cancer, cardiovascular disease, and drug-induced disease, to name a few. To prevent the ROS-induced oxidation of important cellular lipids, proteins, and nucleic acids, cells normally contain a battery of endogenous protective systems (antioxidants) to insure the maintenance of viability as well as metabolic and functional performance (2,3). An example of such a protective system is unesterified d-α- tocopherol (α-T), also known as vitamin E. Though α-T appears to function as the predominant chain-breaking antioxidant in cellular membranes (4-6), this lipophilic compound is not synthesized by mammalian cells but rather is derived solely from exogenous sources. The antioxidant properties of α-T result from its ability to trap reactive peroxyl radicals by donating a hydrogen atom, becoming a tocopherol radical in the process. In order to preserve cellular α-T and its membrane antioxidant activity, other cellular hydrophilic reductants such as ascorbate, glutathione and possibly NADPH can regenerate active α-T by donating a hydrogen atom to the tocopheroxyl radical (7-9). However, if the tocopherol radical is attacked by another peroxyl radical resulting in a 2-electron oxidation of α-T (e.g. tocopherylquinone formation), the cell's ability to regenerate active α-T is apparently lost (7,8). The continual need in cellular membranes for the replacement of consumed oxidized α-T with dietary active α-T suggests that the cellular uptake and subcellular distribution of this important antioxidant is crucial to its ability to protect membrane constituents and cellular integrity (especially during an oxidative challenge), thus limiting cell injury and disease.

Previous studies have demonstrated that α-T is an extremely lipophilic molecule and as such is absorbed from the intestine in chylomicrons through the lymphatic system and is transported in plasma by a tocopherol-binding protein incorporated in lipoproteins (10). The cellular uptake of α-T has been reported to be mediated by both lipoprotein receptor-dependent and -independent pathways (11). It is generally accepted that in lipid bilayers and biomembranes, α-T intercalates between phospholipids with the chroman head group (phenolic hydrogen) toward the surface (in close proximity to water-soluble reducing agents for regeneration) and with the hydrophobic phytyl chain buried in the hydrocarbon region (12). Interestingly, in biological membranes only one tocopherol molecule is present for every 500-1000 polyunsaturated fatty acids (PUFA) (13). This concentration of membrane-bound α-T is thought to be close to the threshold of α-T required to effectively protect phospholipid bilayers [0.2 mol %, (14)] and biomembranes [0.4 mol % (15)] against oxidative damage, thus again emphasizing the importance of replenishing oxidized α-T by regenerating α-T with hydrophilic reductants or by incorporating new active α-T into the membrane.

Biomembranes or lipid bilayers are not limited to this ratio of α-T to PUFA. In fact, Lai et al. (16) have shown that lecithin liposomes can be prepared with up to 40 mol % α-T, while numerous reports indicate that increasing the α-T content of biomembranes decreases the susceptibility of these membranes to lipid peroxidation (13, 17). At present, it is not understood why the concentration of α-T in biomembranes is kept at such a low mol % (close to the threshold). The amount of α-T embedded in intracellular membranes appears to result from the concentration of α-T available from the diet and its intracellular transport as well as from the rate of consumption by oxidation and by transport out of the cell.

During an oxidative challenge when membrane-bound α-T is being rapidly consumed, a rate limiting factor in providing intracellular membranes with additional active α-T may be the requirement for a tocopherol transporting protein. Niki et al. (18) have demonstrated using artificial phospholipid membranes that the extreme water insolubility conferred on α-T by the phytyl tail greatly inhibits its ability to exchange between membranes in the absence of any transporting factors. Other investigators (19-21) have also suggested that the intracellular transport of α-T requires a tocopherol transport protein that can carry α-T to subcellular locations. Such a protein has been identified in rat and rabbit liver (19, 20) and heart (21) for the transfer of α-T to the nucleus (22), mitochondria (23) and microsomes (24) and the binding of α-T to this protein is saturable. However, the ability of this tocopherol transfer protein to rapidly supplement intracellular membranes with active α-T remains unclear and seems doubtful based on our limited knowledge of α-T transport. In fact, the observed inability of α-T to freely exchange between intracellular membranes may limit the ability of acute α-T administration, given in vitro or in vivo, to protect cells, organs and organisms from the toxic effects of oxidative stress.

In light of the enormous cost of health care in the U.S. and the considerable role of oxidative stress in causing and exacerbating human disease, it is clear that therapeutic strategies to combat oxidative injury are required. To develop pharmaceuticals that protect us from the adverse effects of oxidative stress, one strategy is to rapidly augment the intracellular content of α-T at the appropriate concentration, time and subcellular site (particularly mitochondria) to diminish or eliminate oxidative stress-mediated injury and disease.

The in vivo administration of vitamin E (and related compounds) as antioxidants and antitumor agents is hampered by insolubility in the aqueous solutions which are required for parenteral injection. Thus, these agents must be solubilized with additional additives that may also cause undesirable and even toxic effects in the patient. It would be highly advantageous to have a means of increasing the solubilization of these compounds in a non-toxic vehicle thus making them amenable to parenteral injection.

Numerous studies have demonstrated that the succinate derivative of vitamin E (vitamin E succinate, d-α-tocopheryl succinate, TS) administration protects experimental animals, tissues, cells and subcellular organelles from toxic cell death (25, 27, 31). These cytoprotective effects of TS do not appear to be selective for a particular toxic insult, cell type, or species. Interestingly, the mechanism for TS cytoprotection appears to be indirect: cellular esterases cleave TS, releasing antioxidant d-α-tocopherol (α-T). It is the released α-T which confers cytoprotection.

In a curious contrast to the results that are obtained with normal cells, the incubation of tumor cells with TS results not in protection but rather in growth inhibition and cell death (26). The antitumor activity of TS has been reported for a wide variety of tumor cells regardless of species or cell type. It appears that the antitumor activity of TS is the result of the intact compound, TS, and not the release of αT. A plausible explanation is suggested by the lack of esterase activity in tumor cells. Tumor cells often exhibit little or no esterase activity; therefore, the intact TS compound is taken up by and persists in tumor cells, leading to tumor cell death. It is also of interest that antitumor agents may trigger apoptosis (cell death) in tumor cells through their interactions with mitochondria. It would be beneficial to have available a method that takes advantage of both the cytoprotective and the tumor killing properties of TS and related vitamin E compounds for the prevention and treatment of cancer.

SUMMARY

The present invention provides a method for the delivery of vitamin E compounds to tissues, cells and subcellular sites (including mitochondria) in order to 1) increase the antioxidant capacity and protect normal (non-tumor) tissue, cells, mitochondria, and other subcellular organelles or substances 2) inhibit the growth of and kill tumor cells, and 3) both protect normal tissue while killing tumor cells by administering vitamin E compounds. According to the invention, the delivery of vitamin E compounds (either a single vitamin E compound or a plurality of vitamin E compounds) is achieved by making an aqueous suspension of the tris salts of those compounds by sonication. The aqueous suspension may be administered intravenously, transdermally, parenterally, by inhalation of an aerosol, orally, or by other delivery routes. In addition, the cells to which the vitamin E compound is delivered may be either in vivo or in vitro. Further, the subcellular sites to which the vitamin E compounds are delivered are, in particular, the outer and inner mitochondrial membranes.

ABBREVIATIONS

ALT:Alanine aminotransferase; LDH: lactate dehydrogenase; CC1₄: carbon tetrachloride; CYP2E1 : cytochrome P450, 2E1 form; G6Pase: glucose-6-phosphatase; ip: intraperitoneal; iv- :intavenous; PNP: p -nitrophenol; α-T: -α-tocopherol; TA: d-α-tocopheryl acetate; TS: d- - tocopheryl hemisuccinate; TS-FA: d-α-tocopheryl hemisuccinate free acid; TS-tris: d- - tocopheryl hemisuccinate tris salt; TSE: d-α-tocopheryloxybutyrate; TSE-tris: d- - tocopheryloxybutyrate tris salt; TS-2,2-dimethyl: d-α-tocopheryl 2,2,-dimethylsuccinate; TG-2,2- dimethyl: d-α-tocopheryl 2,2-dimethylglutarate, TS-3-monomethyl d-α-tocopheryl 3 methyl succinate; PUFA: polyunsaturated fatty acids; OCM-1 : cell line derived from ocular melanoma; TRF: tocotrienol rich traction; TRF-succinate-tris salt: tris salt of the hemisuccinate ester of TRF; NADPH: nicotinamide adenine dinucleotide phosphate (reduced form); NADP⁺ : nicotinamide adenine dinucleotide phosphate (oxidized form); NaOH: sodium hydroxide; KC1: potassium chloride.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Graph showing the effect of oxidative stress on the induction of lipid peroxidation in mitochondria isolated from the livers of rats treated with TS-tris and TSE-tris. A d-α-tocopheryl hemisuccinate tris salt (TS-tris) and a d-α-tocopheryloxybutyrate tris salt (TSE-tris) were administered intraperitoneally to rats at a dose of 0.19 mmol/kg 18 h prior to isolation. An increase in fluorescence indicates an increase in lipid peroxidation. Figure 2A and 2B. 2A. Graph showing the effect of TS-tris suspension in water (after sonication) (#)on the growth and viability of OCM-1 cells. 2B. Graph showing the effect of taxol dissolved in ethanol (•), and taxol combined with TS-tris and sonicated in water (O) on the viability of OCM-1 cells.

Figure 3. Graph showing the effect of an 18 hour pretreatment of rats (n=3) with a single ip injection of αT or TS-tris (TS-T) (0.19 mmol/kg) on tocopherol and tocopherol succinate levels in liver homogenates. Data points represent the mean ± SE.

Figure 4. Graph showing the effect of an 18 hour pretreatment of rats (n=3) a single ip injection of αT or TS-tris (TS-T) (0.19 mmol/kg) on tocopherol and tocopherol succinate levels in liver mitochondria. Data points represent the mean ± SE.

Figure 5. Graph showing the effect of an 18 hour pretreatment of rats (n=3) a single ip injection of αT or TS-tris (TS-T) (0.19 mmol/kg) on tocopherol and tocopherol succinate levels in liver mitochondrial outer membranes. Data points represent the mean ± SE.

Figure 6. Graph showing the effect of an 18 hour pretreatment of rats (n=3) with a single ip injection of αT or TS-tris (TS-T) (0.19 mmol/kg) on tocopherol and tocopherol succinate levels in liver mitochondrial inner membranes. Data points represent the mean ± SE.

Figure 7. Graph showing the effect of an 18 hour pretreatment of rats (n=3) with a single ip injection of αT or TS-tris (TS-T) (0.19 mmol/kg) on the antioxidant capacity of inner and outer mitochondrial membranes. Antioxidant capacity was determined by examining the susceptibility of these membranes to lipid peroxidation using the plate reader assay of Tirmenstein et al. (49). Lag time indicates the amount of time prior to the onset of lipid peroxidation. A lag time of 300 indicates complete protection against lipid peroxidation (or maximal antioxidant capacity). Data points represent the mean ± SE.

Figure 8. Graph showing the effect of an 18 hour pretreatment of rats (n=3) with a single ip injection of αT (■) or TS-tris (TS-T) (*) (0.19 mmol/kg) on the susceptibility of hepatocytes

(isolated from these rats) to iron (ferrous ammonium sulfate) induced cell death. Hepatocytes were exposed to a variety of iron concentrations for one hour and cell death was measured as %

LDH leakage. Data points represent the mean ± SE. DETAILED DESCRIPTION OF THE INVENTION

The cytoprotective effects of vitamin E compounds such as TS appear to be the result of uptake of TS by cells, followed by cleavage of the compound by endogenous esterases. This results in the release of tissue, cellular, and subcellular T from TS, providing an increased antioxidant capacity. In contrast, the antitumor activity of TS appears to be the result of the intact, uncleaved compound. Thus, the administration of TS or related vitamin E compounds has the potential to provide a two-fold, coordinate result: 1) the killing of tumor cells via accumulation of the intact compound (e.g. TS); and 2) cytoprotective, antioxidant activity via hydrolysis of TS to release the antioxidant αT.

The present invention provides a method for the administration of TS (or related vitamin E compounds) in order to both individually and in coordination 1) treat or prevent tumor cell growth and/or formation, and 2) provide increased cytoprotective, antioxidant capacity to normal cells and mitochondria. By "coordinate" or "coordinately" we mean that the administration of a single form of the vitamin E compound has more that one beneficial effect on the tissue or cells to which it is administered. The beneficial effects occurs in concert via two related but distinct mechanisms. For example, traditional anticancer agents often cause extensive oxidative damage to normal cells even as they are killing cancer cells. According to the method of the present invention, the TS or related vitamin E compound enhances the antioxidant capability of normal tissue to prevent or attenuate such damage. Further, intact TS compound will be taken up by and selectively persist in tumor cells, augmenting tumor killing by the anticancer agent.

Thus, this dual antioxidant and cytotoxic activity for TS (and related vitamin E compounds) in normal and tumor cells, respectively, should prove useful in the development of more effective therapies for the prevention and treatment of oxidative stress-related diseases including cancer.

In this application, we present data in support of the use of an aqueous formulation of vitamin E (the tris salts of anionic vitamin E esters and ethers) which provides enhanced tissue, cellular and subcellular delivery and retention of these vitamin E esters and unesterified vitamin E. Our data suggest that once this formulation of vitamin E (the tris salts of anionic vitamin E esters) has accumulated in normal tissue, cells or subcellular sites (mitochondria), endogenous cellular esterases can cleave these vitamin E esters, thereby releasing the potent antioxidant α-T, thus providing enhanced antioxidant protection at this site. In addition, a single administration of Vitamin E ester-tris salts results in elevated blood, tissue, and mitochondrial levels of α-T and TS for a longer period of time (i.e. α-T and TS have a longer half-life) when administered in this manner, compared to the administration of α-T alone. TS-tris salt provides a tissue, cellular and subcellular reservoir of T (in the form of TS) that can be slowly released over time.

In addition, we demonstrate that the tris salts of anionic vitamin E esters and ethers are a formulation that provides an aqueous solution for parenteral injection. In fact, the administration of such a preparation results in an enhanced concentration of the antitumor agent, vitamin E succinate (TS), in tissue, cells and subcellular fractions (e.g. mitochondria) for an extended length of time.

The present invention provides a method for enhancing the delivery of antitumor and antioxidant Vitamin E compounds to tissues and subcellular organelles (mitochondria). The invention is based on the discovery that, upon sonication in saline (0.9% NaCl in water) or in water, the Tris salts of vitamin E compounds, (especially succinate derivatives of Vitamin E), form a suspension that can be administered parenterally. Further, as will be seen in the Examples, parenteral administration of the suspension results in high levels of the Vitamin E compound being present and sustained in the normal tissues and in subcellular organelles (mitochondria), and affords protection against oxidative stress-induced lipid peroxidation and cell death.

The method described herein can be useful for treating cancer in mammals (for example liver cancer, prostate cancer, ocular melanoma, cutaneous melanoma, colon cancer, lung cancer and the like) by delivering TS and related vitamin E derivatives. The method of the present invention can also be used to protect tissue, cells and subcellular organelles (e.g. mitochondria) against oxidative stress-induced injury or disease in mammals. Such injuries or diseases may include but are not limited to: neurodegenerative diseases such as Alzheimer's and Parkinson's disease; vascular disease; heart disease (atherosclerosis and ischemic damage); carcinogenesis; aging; cigarette smoking-induced diseases; smog-induced pathologies; diabetes-induced tissue damage; and many other diseases for which the inception and progression of the disease is thought to be, at least in part, due to oxidative stress. It will be understood by those of skill in the art that the present invention can be practiced in the treatment of any condition for which protection of the tissue, cells, and subcellular organelles (e.g. mitochondria) against oxidative stress is desirable. Those of skill in the art will also recognize that the present invention can be practiced in both human and veterinary applications.

It will be recognized by those of skill in the relevant arts that the suspensions and methods of the present invention will also be useful for the protection of tissue and cells in vitro, in addition to the in vivo uses outlined above. For example, the methods of the present invention may be used to increase the antioxidant capacity of tissue and cells during storage, such as tissue, cells and organs to be used for transplants or for other uses. This may include, for example, liver cells or tissue, kidney cells and tissue, whole organs, sperm cells, cells in blood, etc. In addition, the methods of the present invention may also be used in such procedures as bone marrow transplants, wherein the patient's bone marrow is removed and could be treated with TS to selectively kill tumor cells, and then replaced in the patient. Those skilled in the art will recognize that the treatment of cells and tissue according to the methods of the present invention can be used in any procedure in vivo, in vitro, or both combined, in which it is desirable to confer protection from oxidative stress, or to kill tumor cells. The methods may also be useful for research purposes in tissue culture procedures, for example, for the passage, maintenance, or storage of immortal or primary tissue culture cells, or subcellular fractions.

In a preferred embodiment, the suspension of a Vitamin E compound is made by sonicating the salt of the compound in saline or water. However, those skilled in the art will recognize that other suitable aqueous suspensions may also be used in the practice of the present invention. In addition, any dispersal technique that results in a suitable suspension of the compound(s) may be utilized in the practice of the invention (for example, vigorous vortexing).

In a preferred embodiment of the invention, the Vitamin E compounds which are used in the practice of the present invention include the tris salts of the anionic esters or ethers of Vitamin E (tocopherol and its various forms) prepared individually or in combination, for example: the tris salts of TS, TSE, and TS-2,2-dimethyl and TRF-succinate, including the d and dl isomers of α, β, γ and δ forms of tocopherol related compounds. However, it will be readily understood by those of skill in the art that other derivatives of other Vitamin E compounds ( e.g. TG-2,2-dimethyl and TS-3-monomethyl) can also be used in the practice of the present invention.

In a preferred embodiment of the invention, the tris salts of the Vitamin E compounds are utilized. However, it will be readily understood by those of skill in the art that other pharmaceutically acceptable salts of a Vitamin E compound which is capable of forming an aqueous suspension suitable for administration may be used in the practice of the present invention.

In a preferred embodiment of the invention, the suspension of the salt of a Vitamin E compound may be administered by injection either intravenously or parenterally. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispensing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.

Alternatively, the suspension of the salt of a Vitamin E compound may be administered by inhalation of an aerosol. This method has the advantage of delivering the antitumor or antioxidant compound directly to the lungs where it could, for example, provide protection against carcinogens and oxidants such as those found in cigarette smoke and atmospheric pollutants, or effectively kill cancer cells located at this site. Those skilled in the art will recognize that a variety of inhalers appropriate for the practice of the invention are available, including those with various dose metering chambers, various plastic actuators and mouthpieces, and various aerosol holding chambers (e.g. spacer and reservoir devices) so that an appropriate dose of the Vitamin E compound can be delivered. Also, several non-ozone depleting (non-chlorofluorocarbon) propellants, such as various hydrofiuoroalkanes (e.g. HFA 134a and HFA 227) are available.

Administration may also be achieved transdermally using a patch impregnated with the aqueous solution of the salt of the Vitamin E compound, by ocular administration (eye drops), sublingual administration, nasal spray administration and rectal administration (suppository).

Administration may also be oral. As demonstrated in Example 4, the oral administration of TSE-tris resulted in high (9.2 nmol/ml) plasma TSE levels (18 hours following administration) that were similar to the levels observed for the same dose of TSE-tris given intraperitoneally (11.3 nmol/ml) Thus, these data indicate that vitamin E ethers and vitamin E ester compounds that are not hydrolyzable (eg. TS-2,2 dimethyl, data not shown) can be absorbed following ingestion or oral administration. In the case of oral administration of hydrolyzable vitamin E esters, absorption may be accomplished by coating the tris salt vitamin E compounds (liposomes) with an impermeable polymer membrane that is not susceptible to the action of digestive enzymes (duodenal esterases) or is biodegraded very slowly. In addition, amino acid polymers such as polylysine could be used. Impermeable polymer films would be degraded by microflora found in the colon. Thus, the vitamin E ester compound would be released in a part of the intestine devoid of secreted digestive enzymes. However, it will be readily understood by those of skill in the art that other methods for preventing the hydrolysis of esters and promoting vitamin E ester or ether absorption following oral administration can also be used in the practice of the present invention. For oral administration, the Vitamin E compounds may be administered in any of several forms, including tablets, pills, powders, lozenges, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosol, soft or hard gelatin capsules, or sterile packaged powders.

The Vitamin E compound may be administered as a composition which also includes a pharmaceutically acceptable carrier. The Vitamin E compound may be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier. When the carrier is a diluent, it may be a solid, semisolid or liquid material which acts as a vehicle, excipient or medium for the Vitamin E compound. Some examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphates, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations can also include lubricating agents, wetting agents, emulsifying agents, preservatives, and sweetening or flavoring agents.

The dose of the Vitamin E compound to be delivered will vary depending on a variety of factors including the route of administration, the particular condition being treated, the condition of the individual patient, the patient's age, gender, weight, etc., and other various factors that will vary from situation to situation. The exact dosage will thus be determined on a case by case basis by the attending physician or other appropriate professional, but will generally be in the range of 1 to 100 mg/kg of body weight. For administration via inhalation, the dose may be less and will vary according to the exact delivery technology that is employed.

The Vitamin E compounds may be used alone (i.e. one Vitamin E compound per suspension) or as mixtures of Vitamin E compounds (i.e. more than one Vitamin E compound per suspension). For example, TRF-succinate tris salt forms liposomes, indicating that the five different tocopherol and tocotrienol isomers that are contained in TRF are contained together in the liposome. It may be advantageous to deliver to tissue more than one isomer or form of vitamin E compound, especially if different forms of α-T are able to distribute to different subcellular sites, or protect against different types of oxidative stress, or are hydrolyzed at different rates.

The Vitamin E compounds may be used by themselves or in combination with other drugs. For example, the Vitamin E compounds may be used with other antitumor drugs such as taxol, or with other antitumor agents such as doxorubicin, and with other tocopherol derivatives. When used in combination with other drugs, the Vitamin E compounds may be administered prior to, after, concomitant with, or in the same preparation as the other drugs.

MATERIALS AND METHODS

Chemicals

Absolute ethanol was obtained from J. T. Baker Inc. (Phillipsburg, NJ). Ascorbic acid, HPLC- grade methanol, chloroform and hexane were obtained from Fisher Scientific (Pittsburgh, PA). The tocopherol compounds α-T (96%) and d-δ-tocopherol (96%) (internal standard) were a generous gift from Henkel (La Grange, IL). TS-tris (99%) was prepared as described by Fariss et al. (7). The compound TSE-tris (>95%) was synthesized according to the procedures reported by Fariss et al. (9). CC1₄ (99.9+%) was of the highest purity available and was obtained from Aldrich (Milwaukee, WI). Hemin, bilirubin, TS-FA (99%), -nitrophenol and all other chemicals used for this study were obtained from Sigma (St. Louis, MO). Taxol was a gift from Dr. David Bailey at Hauser, Inc. γ-TS-tris and TRF-S-tris were synthesized by Dr. Doyle Smith. Cell lines

The cell line OCM1, derived from human ocular melanoma , was used for the taxol studies and was a gift from Dr. June Kan-Mitchell at the University of California, San Diego, CA.

Animals

Male Sprague-Dawley rats from Simonsen Labs (Gilroy, CA) weighing 175-225 g were used throughout the course of this study. Animals received water and food (Purina Rat Chow 5001, Ralston Purina, St. Louis, MO) ad libitum for at least three days prior to the onset of the experiment. α-T was dissolved in olive oil (approx. 100 mg/ml) and administered by ip injection at a dose of 0.19 mmol/kg body weight. The vehicle, olive oil, was administered at a dose of 1 ml/kg body weight. Powdered TS-tris and TSE-tris were suspended in saline with brief sonication (30 sec) and were given intraperitoneally at a dose of 0.19 mmol/kg body weight (approximately 100 mg/kg). Saline was given to rats at a dose of 4 ml/kg. In most cases, after the administration of the tocopherol compounds animals were sacrificed 18, 72 or 120 hours later for tissue procurement (frozen in liquid nitrogen), liver homogenization and subcellular fractionation. In the tissue distribution studies, animals were fasted 18 hours prior to sacrifice. In experiments in which rats were sacrificed 6 h after receiving TS-tris, rats were fasted for a 12 h period prior to receiving TS-tris and for an additional 6 h until sacrifice.

Rats received CC1₄ by oral lavage 6 or 18 h after tocopherol administration. CC1₄ was dissolved in peanut oil (0.5 g /ml) and given at a dose of 1.0 g/kg. Food was restored 1 h after receiving CC1₄. In some experiments, rats were sacrificed 4 h after receiving CC1₄ and plasma and liver samples were collected for hepatotoxicity determinations. Rats were anesthetized with diethyl ether, and blood samples (4-5 ml) were withdrawn from the inferior aorta. Blood samples were immediately mixed with 15 mg tripotassium EDTA, and aliquots were centrifuged at low speed to prepare plasma samples. Liver microsomes were also isolated after 4 h for the determination of lipid peroxides, tocopherol, G6Pase activity and j-»-nitrophenol (PNP) hydroxylase activity levels. All procedures were approved by the Washington State University Animal Care and Use Committee. Tumor cell methods and viability assay

Preparation of Taxol and TS-tris Suspensions and Liposomes:

TS-tris suspensions were prepared by adding 1 ml of water or saline to 30 mg of TS-tris in a microfuge tube and sonicating for 15 sec, twice. For taxol-TS-tris suspensions, 1 mg of taxol was added to 30 mg of TS-tris prior to the addition of water or saline, and the mixture was sonicated for 15 se , twice. Alamar Blue Assay:

Alamar blue dye was used to evaluate cell survival and proliferation. Living cells metabolize the non-fluorescent dye to a fluorescent metabolite which can be detected by a fluorescence plate reader. There is a positive correlation between the level of fluorescence and the number of living cells. The fluorescence intensity of the cells treated with a test compound was compared to that of a control group which has no added test compound (vehicle only). The result was expressed as "Cell number (% control)". A reduction in the cell number indicates inhibition of cell growth, or an increase in cell death. PROCEDURE:

1) On day one, OCM-1 cells were plated at a density of 2.5 or 5 x 10³ cells/well, depending on the cell type, in a 96- well flat-bottomed plate in DMEM (10% fetal bovine serum) medium. On the second day, the medium was replaced with 200 μL RPMI 1640 (10% fetal bovine serum) medium containing the desired concentration of test compound. The concentration of test compound used ranged from 0 to 50μM. 200μL of medium without cells was plated as a blank.

2) Cells were maintained in a humidified atmosphere in 5% CO₂ at 37°C for 42-70 hours, depending on the cell type. 2-6 hours prior to the end of exposure to the test compound, 10-20μL of Alamar blue stock solution was added to each well. After incubation at 37°C for an additional 2-6 hours, the plate was maintained at room temperature for 30 minutes. The exact time of addition of Alamar blue, the precise amount of Alamar blue added, and the length of incubation with Alamar blue varied depending on the cell type and level of activity of the compound being tested. After 30 minutes at room temperature, the fluorescence intensity of each well was measured using a CytoFluor series 4000 multiplate reader (excitation wavelength, 530nm; emission, 590nm; gain, 40). Fluroescent values from blank cells were subtracted from fluorescent values of cells treated with test compounds. The resulting values were then divided by the corresponding values obtained from the control samples to give the number of viable cells (% control). The IC₅₀ values (concentration required to inhibit cell growth by 50%) were extrapolated from the plot of cell number (%control) versus compound concentration. The cell number (% control) plotted was the mean ± SD (n=6).

Rat liver homogenization and subcellular fractionation

Livers were excised from rats and minced in ice cold homogenization buffer (250 mM sucrose, 10 mM tris and 1 mM EDTA, pH 7.4). The minced liver was subsequently rinsed several times with ice cold homogenization buffer and finally suspended in 2.5 volumes of homogenization buffer. The liver mince was then homogenized with five strokes of a Potter-El vehj em tissue grinder. Aliquots of the homogenate were retained for tocopherol and protein determinations. The remainder of the liver homogenate was used for subcellular fractionation according to previously described procedures (27, 28). In all cases, the subcellular markers corresponding to the appropriate fractions were substantially enriched and were similar to previously reported values (28). Following isolation, subcellular fractions were resuspended in phosphate buffered saline and aliquots were withdrawn for tocopherol and protein determinations. Proteins were measured according to the procedures of Lowry et al.(29) as modified by Peterson (30).

Preparation of respiring mitochondria

Mitochondria were prepared from either naive rats, or following pretreatment with T or TS as outlined in the Animals section above. Rats were anaesthetized with 0.6 mg/kg pentobarbitone, and livers rapidly excised, weighed and placed in ice-cold 0.25M sucrose, ImM EDTA in lOmM Tris Hcl, pH 7.4 containing 0.5% BSA. The liver was lightly minced, then homogenized by 3 passes of a drill-assisted Dounce homogenizer. The homogenate was centrifuged at 3,000 xg, 4°C for 10 minutes with the pellet containing debris and nuclei being discarded. The supernatant was centrifuged at 10,000 xg for 10 minutes, supernatant aspirated and mitochondrial pellet collected. The pellet was resuspended in ice cold 0.25M sucrose in lOmM Tris HCI, pH 7.4 containing 0.5% BSA, and centrifuged as before. The mitochondrial pellet was washed one further time before being resuspended to a volume being 1 gram wet weight liver per ml. Mitochondria were stored on ice and used immediately for swelling experiments.

Measurement of Mitochondrial Swelling

The swelling of freshly isolated mitochondria, (lg wet weight liver/ml) during exposure to ferrous ammonium sulfate was measured spectrophotometrically as a decrease in absorbance at 540nm. In these experiments, analysis buffer comprised of 0.25M sucrose, 1 rnM potassium dihydrogen orthophosphate and 2 μM rotenone in lOmM MOPS buffer, pH 7.4. At 1 minute prior to the start of the experiment, a 0.1 ml aliquot of 60mM succinate, prepared in analysis buffer was added to 0.9 ml of analysis buffer in a 1.5 ml cuvette. A 0.1 ml aliquot of mitochondria was added, inverted to mix and left at room temperature for a further minute. The swelling agent was added and the monitoring of change in absorbance commenced immediately and continued for up to 10 minutes. Prior to investigating mitochondrial swelling to ferrous ammonium sulfate (0-2500μM), the ability to swell in the presence of calcium (20μM) was used as a positive control.

Preparation of inner and outer mitochondrial membranes.

Mitochondria were prepared as outlined above with the absence of BSA in the homogenization and washing solutions. The two washings remove the majority of residual microsomal contamination. After the final wash, mitochondria were resuspended in 5 ml of 0.25M sucrose and 1 mM EDTA in lOmM TrisHCI buffer, pH 7.4. Mitochondria were snap frozen in liquid nitrogen and stored at 80°C until use. After thawing, a 1 ml aliquot of mitochondria were diluted with 1 ml of phosphate buffered saline, pH 7.4 and sonicated on ice for 3 seconds. The mitochondria were then placed on a sucrose gradient of three 1 ml layers of 51%, 37.7% and 25.3% and centrifuged at 140,000 xg for 3 hours 15 minutes. The outer membranes of the mitochondria form a band at the 25.3:37.7 interface. The inner mitochondrial membranes form a band at the 37.7:51 interface. Both bands are individually collected, resuspended in 40ml of PBS and centrifuged at 12,000 xg for 20 minutes. The inner mitochondrial membranes form a smoky red pellet, which is resuspended in 1 ml of PBS and snap frozen in liquid nitrogen until use. The pellet from the outer membrane sample is discarded, and supernatant centrifuged at 120,000 xg for 90 minutes. The clear red pellet is resuspended in 1 ml of PBS and snap frozen until use. Protein content of the cellular and subcellular fractions were determined by the BCA protein kit (Pierce, Rockford, U,).

Susceptibility of Mitochondria to Lipid Peroxidation:

The mitochondria (as well as the inner and outer mitochondrial membranes) isolated as described above from TS-tris and TSE-tris animals (18 hour treatment) were examined for their susceptibility to lipid peroxidation by the fluorescence plate reader assay of Tirmenstein et al.

(49).

Preparation of Rat Hepatocyte Suspensions and Cytotoxicity Studies: Adult male Sprague-Dawley rats (200-3 OOg) were obtained from Simonsen Laboratories Inc. (Gilroy, CA). Rats were housed in small groups with food and water ab lib. in a standard 12 hour light/dark cycle for at least 1 week prior to use. Rats were administered either 0.19 mmol/kg α-tocopherol dissolved in olive oil or α-tocopherol succinate tris by intraperitoneal injection 18 hours prior to cell isolation. Tocopherol succinate (Tris salt) was prepared in saline with two 15 second sonications on ice to form a fine aqueous suspension.

Hepatocytes were isolated using the 2 step collagenase perfusion method described in Fariss et al. (50). A yield of 5-7 x 10⁸ cells was routinely obtained with viability of >94%, as determined viatrypan blue exclusion. Hepatocyte suspensions (2 x 10⁶ cells/mL, 12 mL, total) were prepared in modified Waymouth's medium. After a 15 minute equilibration time, an aliquot of cells was taken as the 0 time point. After collection of the 0 time point, the cytotoxicant under investigation was immediately added. Ferrous ammonium sulphate (0.1 to 5 mM) was prepared in water immediately before addition. A 0.5 mL aliquot of cell suspension was collected at half-hourly or hourly intervals for up to 6 hours. The aliquot was centrifuged at 12,000 rpm for 5 seconds, supernatant collected and stored at 4°C until lactate dehydrogenase (LDH) activity analysis.

Lactate dehydrogenase activity:

LDH activity was determined by monitoring the enzymatic formation of NADH for NAD+ in the presence of L-lactic acid. Post-centrifugation supematants were diluted 1:80 with PBS. A 100 μL aliquot was added to a well of a 96 well plate and mixed with 100 μL of reagent to give a final concentration of 3.75 mM NAD+ and 25 mM L-lactic acid in 125mM Tris-HCl buffer, pH 8.9. The increase in fluorescense was immediately monitored at room temperature (gain 70) using a CytoFluor 4000, Perceptive Biosystems (Framingham, MA). The percent LDH leakage was calculated by comparing values to total LDH activity. Total LDH was measured from a sample of hepatocytes collected at 0 time and lysed with a final concentration of 0.2% Triton X-100.

Tocopherol determinations

Tocopherol, tocotrienol, and tocopherol ester levels were measured according to the methods described by Fariss et al. (31). TSE levels were measured according to the procedures of Tirmenstein et al. (32). Samples were analyzed by reversed-phase high-performance liquid chromatography equipped with fluorimetric detection. Retention times for J-δ-tocopherol (internal standard), α-T and TSE were 8.0, 11.5 and 13.4 min respectively.

Microsomal enzyme assays

Microsomes isolated from vehicle and tocopherol-treated animals were washed and resuspended in 0.154 M KC1, 50 mM tris, pH 7.4 buffer and were stored frozen at -80° C prior to assays. PNP hydroxylase activity was measured according to the procedures of Koop (33) as modified by Speerschneider and Dekant (34). Aliquots of microsomes were resuspended in 0.1 M potassium phosphate buffer, pH 7.4 at a concentration of 0.75 mg protein/ml with a final volume of 1 ml. The NADPH-generating system consisted of 1 mM NADP+, 9 mM glucose-6-phosphate and 0.2 units/ml glucose-6-phosphate dehydrogenase. Microsomes were preincubated with the NADPH generating system at 37° C for 5 min. Incubations were started by adding PNP to a final concentration of 450 μM. After 20 min at 37° C with shaking, incubations were stopped by adding 0.5 ml 0.6 N perchloric acid to 1 ml of samples. Samples were centrifuged at 16,000g for 2 min, and 1 ml of the supernatant was mixed with 0.1 ml of 10 N NaOH. The amount of the reaction product, 4-nitrocatechol, formed was determined by measuring the absorbance at 546 nm (extinction coefficient of 9.53 mM-lcm-1). Microsomal lipid peroxide and GόPase activity determinations

In the studies described in Table 3, rats were sacrificed 4 h after CC1₄ administration and rat liver microsomes were prepared as previously described (27,28). Microsomes were washed and resuspended in 0.154 M KC1, 50 mM tris HC1, pH 7.4 buffer. After isolation, microsomes were stored frozen at -80° C. Lipid peroxide levels in microsomes were determined according to the procedures of Ohkawa et al. (35). Aliquots of the washed microsomes were also assayed for GόPase activity as described by Aronson and Touster (36).

Statistics

Results are presented as means ± SD. Analysis of variance was performed with the InStat 2.03 (GraphPad Software, Inc., San Diego, CA) statistical package. Differences between groups were determined using the Dunnett multiple comparison post test.

EXAMPLES

The foregoing are Examples which represent preferred embodiments of the present invention, but should not be construed so as to limit the invention in any way.

EXAMPLE 1.

Subcellular distribution of tocopherol analogs

The subcellular and liver homogenate levels of α-T, TS and TSE measured at either 6 h or 18 h after the ip administration of tocopherol analogs (0.19 mmol/kg) to rats are reported in Table 1, and the values are normalized per mg protein. The administration of the tris salts of TS and TSE yielded much greater incorporation of TS and TSE, respectively, into the liver than an equimolar dose of T. TS-tris administration increased the total tocopherol (TS + α-T) levels found in liver homogenates and subcellular fractions by a factor of 8-36 fold over those seen in vehicle controls. The highest total tocopherol values expressed per mg of protein were observed in mitochondria and in plasma membranes. If tocopherol values were not normalized per mg protein, the majority of the tocopherol found in hepatocytes was associated with mitochondria and microsomes. Our data indicate that these fractions consistently contained the majority of the cell's tocopherol levels, regardless of the tocopherol treatment. Supplementation of rats with α-T was much less effective in increasing total tocopherol levels in rat liver homogenates and subcellular fractions were increased only about 2 fold in rats receiving α-T. α-T is insoluble in water and was dissolved in olive oil prior to administration. In contrast, powdered TS-tris and TSE-tris can be placed in saline and suspended by sonication. Light microscopic analysis of these TS-tris and TSE-tris suspensions suggested that they consisted of lipsomes of variable size. The presence of TS-tris and TSE-tris liposomes was confirmed by negative-stain electron microscopy (data not shown). Previous findings by Lai et al. (51) have shown that TS is capable of forming liposomes, and Janoff et al. (52) have characterized α-tocopherol hemisuccinate vesicles. In agreement with our findings, Juzmoto et al. (45) have previously shown that powdered TS shaken in tris buffer forms liposomes at pH 7.4.

TS-tris administration to rats also proved to be the most effective means of increasing α-T levels in the liver and exceeded the ability of TS administration to increase hepatic α-T levels (Table 1 and reference 27). The administration of α-T produced a modest but significant increase in α-T levels in all of the fractions except microsomes. In the microsomal fraction, α- T administration produced a nonsignificant 1.7 fold increase in α-T levels. In contrast, TS-tris administration increased microsomal α-T levels 7.6 fold. These findings suggest that TS-tris administration is the most effective means for increasing the antioxidant capacity of tissue, cells and subcellular fractions. This was confirmed in studies demonstrating that hepatic mitochondria isolated from rats treated with TS-tris were less susceptible to lipid peroxidation than mitochondria isolated from control or TSE-tris treated rats (Fig. 1).

Large quantities of the intact TS molecule were also present in each of the subcellular fractions (except cytosol) and liver homogenates from animals supplemented with TS-tris. In these animals, high TS levels (expressed as nmol/mg protein) were found in mitochondria, plasma membranes, microsomes and nuclei. As compared with TS administration, TS-tris treatments resulted in an up to 10-fold increase in homogenate and subcellular TS levels (Table 1 and reference 27). This superior ability of TS-tris administration to increase cellular and subcellular TS levels was especially high in mitochondria. These findings suggest that this large accumulation of TS could serve as a α-T reservoir for the release of site-specific α-T over time (thus increasing the antioxidant capacity of the cell or cellular organelle) or could serve to enhance the antitumor abilities of TS administration.

The administration of TSE-tris to rats led to the uptake and incorporation of TSE into hepatocyte membranes that exceeded by up to 10-fold that observed following TSE administration, again with mitochondria showing the largest increase (Table 1 and reference 27). However, TSE-tris did not introduce as much TSE into liver homogenates and subcellular fractions as an equimolar dose of TS-tris. Supplementation of rats with TSE-tris did not significantly increase homogenate or subcellular levels of α-T supporting the observation that TSE is a non-hydroylzable form of TS that will not produce an increase in the antioxidant capacity of a tissue or subcellular fraction (as noted in Example 3, Fig. 1). However, the administration of TSE-tris may prove useful in enhancing the antitumor activity of this compound (26).

TABLE 1. Tocopherol analog levels in liver homogenates and subcellular fractions from rats treated with -α-tocopherol (α-T), d-α-tocopheryl hemisuccinate tris salt (TS-tris) and d-α-tocopheryloxybutyrate tris salt (TSE-tris).

a Rats received a 0.19 mmol/kg ip injection of the tocopherol analog, 18 h prior to liver homogenization and subcellular fractionation. Each rat was fasted during this 18 h period and the vehicle was saline. b Values expressed as the mean ± SD (n = 3 to 6). c ND, not detected. d Values are significantly different (p < 0.05) from vehicle treated rats.

EXAMPLE 2.

Tocopherol protection against CCl₄-mduced microsomal lipid peroxide formation and enzyme inactivation: increase in hepatic C-T in TS-treated rats following toxic insult.

The effects of CC1₄ on microsomal α-T, lipid peroxide and enzyme activity levels are reported in Table 2. The administration of CC1₄ decreased microsomal α-T levels by about 20% after 4 h. This decrease could not be explained by CCl₄-induced liver cell death. Four hours following CC1₄ treatment, animals had plasma ALT values (89 ± 30 units/L) near controls (53 ± 11 units/L) and no liver injury as judged by histopathology (data not shown). Microsomal α-T levels in rats supplemented with TS-tris at 6 and 18 h prior to CC1₄ administration were 7.5 and 16 times higher than controls, respectively. In contrast, the microsomal α-T levels in rats pretreated with α-T and then administered CC1₄ were only about 1.7 times higher than the microsomal α-T levels measured in control animals. Interestingly, during a toxic insult (CC1₄) it appears that the hepatic microsomal α-T levels in TS-tris treated animals increases significantly from 1.68 (no toxic insult) to 3.83 (toxic insult) nmol/mg protein. These findings suggest that during a toxic insult, subcellular stores of TS can be converted to α-T at an accelerated rate, possibly as a means for increasing the antioxidant capacity of the organelle. Microsomal lipid peroxide levels were significantly increased only in CC1₄ treated animals. Supplementation of rats with α-T or TS-tris at either 6 or 18 h prevented CCl₄-induced increases in microsomal lipid peroxides.

The administration of CC1₄ to rats is known to inactivate microsomal enzymes such as CYP2E1 (48) and GόPase (37). These enzymes were assayed in our study to determine if α- T or TS-tris supplementation could protect against CCl₄-dependent enzyme inactivation. As expected, CC1₄ administration significantly decreased both GόPase and PNP hydroxylase activities (Table 2). PNP hydroxylase activity was reduced by 86% 4 h after CC1₄ administration. Supplementation of rats with either α-T or TS-tris provided no protection against CCl₄-induced inactivation of PNP hydroxylase activity.

CC1₄ administration also decreased GόPase activity levels by about 60% as compared to controls after 4 h. Pretreatment of rats with α-T did not significantly protect against GόPase inactivation by CC1₄. In contrast, supplementation of rats with TS-tris provided partial but significant protection against GόPase inactivation by CC1₄ GόPase activity levels were decreased by only 23% 4 h after CC1₄ administration in animals pretreated with TS-tris 18 h prior to CC1₄ and by 36% in animals pretreated with TS-tris 6 h prior to CC1₄.

CC1₄ is known to be metabolized to the trichloromethyl free radical (CC1₃) by CYP2E1. Once formed, CC1₃ can initiate the peroxidation of polyunsaturated fatty acids or covalently bind to cellular proteins. In addition, CC1₃ can react with molecular oxygen and generate secondary radicals such as the trichloromethylperoxyl free radical (CC1₃ O₂) which can also initiate free radical reactions. Since α-T effectively inhibits lipid peroxidation, we examined the relative capacity of tocopherol compounds to protect against CCl₄-induced lipid peroxide formation (Table 2). As expected, 4 hours after CC1₄ administration there was a significant decrease in microsomal α-T levels and an increase in lipid peroxide levels. Rats pretreated with either α-T or TS-tris (6 or 18 h pretreatment) , and then administered CC1₄ had microsomal α-T levels above those found in control animals and were protected against microsomal lipid peroxidation. However, despite this attenuation of microsomal lipid peroxidation, rats pretreated with α-T were not protected against CCl₄-induced hepatic necrosis. These results suggest that early microsomal lipid peroxidation can be dissociated from CCl₄-induced hepatic necrosis. This dissociation between CCl₄-induced lipid peroxidation has also been reported by other researchers (38-41). It is important to note, however, that in our study microsomal lipid peroxides were only measured 4 hours after CC1₄ administration. Lipid peroxidation may continue at later time points in response to oxidative stress induced by CC1₄ . Morrow et al. (42) concluded that lipid peroxidation continued for at least 24 h following CC1₄ administration.

Lipid peroxidation at these later time points may not be due to CC1₄ radical formation, but instead may involve activation of Kupffer cells and infiltration of neutrophils. Activated Kupffer cells produce reactive oxygen species and blocking Kupffer cell function has been shown to be protective against CC1₄ -induced hepatotoxicity (43). The amount of α-T introduced into hepatocytes following α-T administration may be sufficient to protect against the initial lipid peroxidation generated by CC1₄ radicals but may eventually be depleted by the generation of reactive oxygen species at later time points. TS-tris in contrast may continue to be hydrolyzed to free α-T by cellular esterases and inhibit lipid peroxidation over a longer period of time. Studies by Kagan et al. (44) demonstrated that esterases located in microsomes can hydrolyze tocopherol esters. These results may explain why we found TS- tris especially effective in increasing microsomal α-T levels 18 h after administration.

TABLE 2. Effects of carbon tetrachloride (CC1₄) administration on rat liver microsomal d-α-tocopherol (α-T), lipid peroxide and enzyme activity levels: protective effects of α-T and the tris salt of d-α-tocopheryl hemisuccinate (TS-tris).

a Rats received a 0.19 mmol/kg ip injection of the tocopherol analog, 6 or 18 h prior to receiving CC1₄ (1 g/kg). Each rat was fasted for an 18 h period prior to sacrifice, and the vehicle was saline. Rats were sacrificed and microsomes were isolated 4 h after CC1₄ administration. Following isolation, microsomes were washed and resuspended in 0.154 M KC1, 50 mM tris, pH 7.4 buffer. b Units are expressed as μmol phosphate formed per h at 37° C. c Units are expressed as nmol/. -nitrocatechol formed per min at 37° C. d Values expressed as the mean ± SD (n = 3 or 4). e Values are significantly different (p < 0.05) from vehicle treated rats. f Values are significantly different (p < 0.05) from CC1₄ treated rats.

EXAMPLE 3.

Effect of the Administration of TS-tris and TSE-tris on Mitochondrial Lipid Peroxidation Using an ADP/Fe system to generate an oxidative stress, Fig. 1 shows that hepatic mitochondria isolated from TS-tris treated rats are protected against oxidative stress-lipid peroxidation. (Lipid peroxidation is measured by an increase in fluorescence.) By contrast, hepatic mitochondria isolated from TSE-tris treated or vehicle (saline) treated rats (data not shown) are susceptible to oxidative stress-induced lipid peroxidation. From Table 1 we know that TS-tris administration results in an elevation of mitochondrial α-T and TS levels as compared to the levels measured in TSE-tris and control mitochondrial. Thus these findings support the hypothesis that an elevation in tissue, cellular or subcellular α-T and TS concentrations result in an enhanced antioxidant capacity and prevention of oxidative-stress mediated damage.

EXAMPLE 4.

The Effect of d-CC-Tocopherol ((X-T), d-CC-Tocopherol Hemisuccinate Tris Salt (TS-Tris), and d-OC -Tocopheryloxybutyrate Tris Salt (TSE-tris) Administration on Tocopherol Analog Levels in Tissue from Rats, 18 Hours after a Single Dose.

In rats receiving a single ip dose of α-T, the tissue concentrations of α-T were significantly elevated (as compared with control rats) in all tissues measured (liver, kidney, heart, lung, plasma, and blood), except for the brain. In TS-tris (ip) treated rats, the amount of T-equivalents (α-T + TS) measured in each tissue (except brain) exceeded that observed for α-T treated animals. The greatest concentration of TS and T-equivalents was found in liver. However, the tissue α-T concentration in TS-tris (ip) treated rats rarely exceeded that of α-T treated animals, 18 h following vitamin E administration. These data suggest that TS-tris administration can deliver more vitamin E to tissues (than α-T) but 18 hr following administration may not be sufficient time for adequate release of α-T from the TS tissue reservoirs. The observation that TS-tris treated animals do not have significantly greater tissue α-T levels, as compared with α-T administration, may result from tissue esterase activities that limit the rate of α-T release from TS or the rate of elimination of tissue TS or TS-released α-T from each tissue may be accelerated (transport out of the tissue or utilization). These possibilities will be examined in future studies (Tables 4 and 5) by measuring tissue levels of vitamin E analogs in tissue, 72 and 120 h following a single administration of these vitamin E compounds. The iv administration of TS-tris resulted in a tissue distribution of TS and α-T that was nearly identical to that achieved with TS-tris administered intraperitoneally. The only exception was in the lung, where TS levels of intravenously treated animals were almost 5 -fold the levels observed in the lungs of rats which had received TS-tris intraperitoneally. These results are most likely due to larger liposomes being trapped by the first capillary bed which with iv administration would be the lung (whereas with ip injections the large liposomes may not leave the intraperitoneal space).

The administration of TSE-tris (intraperitoneally) resulted in TSE accumulation in all tissues measured except the brain. Tissue TSE accumulation was considerably lower than that observed for TS-tris (ip) and the liver contained the highest concentration of TSE. Interestingly, the oral administration of TSE-tris by oral administration resulted in plasma levels that were nearly identical to that observed with ip administration. These findings indicate that the oral absorption of TS-tris is excellent and that oral administration is a viable route for this anti-tumor agent. These data also suggest that other non-hydrolyzable vitamin E derivatives (such as TS-2,2 dimethyl) can also be administered orally. TABLE 3. Tocopherol Analog Levels in Tissue from Rats, 18 Hours after single Treatment with d-α-Tocopherol (α-T), d-α-Tocopherol Hemisuccinate Tris Salt (TS-Tris), and d-α- Tocopheryloxybutyrate Tris Salt (TSE-tris).

^a Rats received a 0.19 mmol/kg dose (ip, iv, or oral) of the tocopherol analog, 18 hours prior to sacrifice. Immediately following sacrifice, tissues were obtained by freeze-clamp method. ^b Values expressed as the mean ± SD (n=3-6). ^c ND, not detected. ^d T-equiv. : total T and/or T derivatives detected

EXAMPLE 5.

The Effect of d- (X -Tocopherol ((X-T), and d- (X -Tocopherol Hemisuccinate Tris Salt (TS- Tris) Administration on Tocopherol Analog Levels in Tissue from Rats, 72 Hours after a Single Dose.

Seventy two hours following a single injection of α-T, tissue α-T levels continued to be elevated (as observed after 18 hrs) except for brain, plasma and blood levels which were now back to control levels. By contrast, TS-tris treated animals continued to show plasma and blood α-T levels that were markedly elevated and similar to the levels observed at 18h (2x control). Furthermore substantial levels of TS were detected in all tissues except brain. Plasma, blood and liver TS levels at 72 h had declined by approx. 50% from the 18 h time point. Interestingly, though approximately 200 nmol/g of TS was lost between the 18 and 72 hr measurements, liver α-T levels did not change appreciably. These results suggest that the liver eliminates hepatic stores of TS and α-T. Our findings also suggest that TS-tris administration results in the dramatic maintenance of plasma and blood α-T levels, thus providing a continual source of α-T for other tissues. In the kidney, heart and lung, T- equivalents (TS + α-T) are maintained at levels similar to that observed at 18 hrs and the α-T level in each of these tissues is, if anything, on the rise.

TABLE 4. Tocopherol Analog Levels in Tissue from Rats, 72 Hours Following Single Treatment with d-α-Tocopherol (α-T) and d-α-Tocopherol Hemisuccinate Tris Salt (TS-tris)

^a Rats received a 0.19 mmol/kg ip injection of the tocopherol analog, 72 hours prior to sacrifice. Immediately following sacrifice, tissues were obtained by freeze-clamp method. ^b Values expressed as the mean ± SD (n=3-5).

^CND, not detected. ^d T-equiv. : total T and/or T derivatives detected EXAMPLE 6.

The Effect of d- ( -Tocopherol ((X-T), and d-(X -Tocopherol Hemisuccinate Tris Salt (TS- Tris) Administration on Tocopherol Analog Levels in Tissue from Rats, 120 Hours after a Single Dose.

One hundred and twenty hours following a single dose of α-T, the plasma and blood α-T levels are at control levels and the α-T level in the remaining tissues are declining as compared with the 72 hour time point. In contrast, the total α-T equivalents (α-T + TS) found in kidney, heart, lung, plasma and blood obtained from TS-tris (ip) treated animals is similar to that observed at 72 h. In these animals, the plasma and blood α-T and TS levels at 72 and 120 h have not changed. The kidney, heart and lung TS levels [from TS-tris (ip) treated rats] are declining at 120 h (as compared with 72 h) but the loss in TS appears to result in an concomitant increase in α-T levels in these tissues. These results suggest that TS- tris administration offers the advantage of maintaining tissue levels of α-T and TS for an extended length of time and at an enhanced level. Importantly, the loss of TS in many tissues appears to be related to its hydrolysis to α-T which is retained by the tissue. These findings suggest that this enhanced maintenance of tissue α-T levels will provide an increased antioxidant capacity which may protect these tissues from oxidative stress-induced damage and disease. In the liver, the α-T concentration is maintained at approx. 70-80 nmol/g, a level observed at 18, 72 and 120 h., even though hepatic stores of TS are continuing to be lost during this time period.

These results suggest that the liver has a limited capacity to store high levels of α-T and/or TS. In one rat administered TS-tris (intravenously), the tissue distribution of α-T and TS was nearly identical to that described above for TS-tris (ip). This again suggests that TS- tris administered intraperitoneally and intravenously result in similar tissue distributions of TS and α-T.

TABLE 5. Tocopherol Analog Levels in Tissue from Rats, 120 Hours after Single Treatment with d-α-Tocopherol (α-T), d-α-Tocopherol Hemisuccinate Tris Salt (TS-tris) or d-α- Tocopheryloxybutyrate (TSE-tris).

^a Rats received a 0.19 mmol/kg ip or iv injection of the tocopherol analog, 102 hours prior to sacrifice. Immediately following sacrifice, tissues were obtained by a freeze-clamp method. ^b Values expressed as the mean ± SD (n=3-5), except iv treatment (n=l). ^c ND, not detected. ^d T-equiv. : total T and/or T derivatives detected

EXAMPLE 7.

Effect of TS-TRIS Suspension in the Presence and Absence of Taxol on the Viability of Ocular Melanoma Tumor (OCM-1) Cells .

These experiments were conducted to examine the antitumor properties of TS-tris and the ability of TS-tris treatment to enhance the ability of other traditional antitumor agents such as taxol to induce tumor cell death. As can be seen in Figure 2 A, suspensions of TS-tris (sonicated in water) added to a human ocular melanoma tumor cell line resulted in significant tumor cell killing at a medium concentration of 25 micromolar. The addition of T at similar concentrations is not cytotoxic to these tumor cells (data not shown). The effect of taxol and taxol in combination with TS-tris on tumor cell viability was also examined. As can be seen in Figure 2B, taxol alone is a potent antitumor agent with approximately 95% cell kill observed with a 10 nanomolar concentration in the medium. However, even at 10 micromolar concentrations of taxol in the medium, 100% cell kill was not observed. Interestingly, the addition of TS-tris and taxol allows for 100% kill of tumor cells that normally is not observed with taxol alone (see Fig. 2B). These findings suggest that both taxol and TS-tris are killing tumor cells by different mechanisms and that the administration of both of these compounds in combination confers a distinct advantage over administration of taxol alone.

EXAMPLE 8.

The Effect ofd- γ-Tocopherol Hemisuccinate Tris Salt (γ-TS-tris) and Tocotrienol-rich Fraction Hemisuccinate Tris Salt (TRF-S-tris) Administration on Tocopherol and Tocotrienol Analog Levels in Tissues from Rats, 18 and 120 Hours after a Single Dose

In previous examples in this application we have clearly demonstrated that α-TS-tris has a distinct and significant advantage (as compared to unesterified α-tocopherol) in terms of delivering to tissue, cells and subcellular fractions large amounts of TS that can serve as a α-T reservoir for the release of α-T over time. In addition, we have shown that α-TS-tris also has an advantage in terms of providing a dramatic maintenance of plasma and blood α-T levels (in concentration and over time), thus providing a continual source of α-T for blood (lipoproteins and formed elements) and other tissues. In the present example, we investigated whether a change in the structure of the tocopherol molecule would have an effect on the tissue delivery properties of α-TS-tris mentioned above. Vitamin E is a generic term that includes, in nature, eight substances, d-α-, d-β-, d-γ-, d-δ-tocopherol and d-α-, d-β-, d-γ-, d-δ-tocotrienol. Thus, in the present example we examined the tissue distribution of d-α- and d-γ -tocopherol and d-α-, d-γ- and d-δ-tocotrienol in rats administered in a single dose (0.19 mmol/kg ip injection) of γ-TS-tris or TRF-S-tris. For administration, an aqueous suspension was prepared for each compound as previously described for α-TS-tris.

The findings from the studies examining the tissue distribution following γ-TS-tris administration showed that γ-TS-tris has tissue distribution properties similar to those described in previous examples for α-TS-tris. This is a significant finding since it is well known that although γ-T is the most abundant form of tocopherol found in nature (plants) and has excellent antioxidant properties, it is poorly retained by our tissues. As a result of these properties, γ-T is found in low concentrations in our cells and tissues (approx. 1-3 nmol/g of tissue) as compared to α-T which has a 10-fold higher tissue concentration (20-40 nmol/g tissue) (see Tables 5 and 6). Since γ-T has recently been shown to be a more effective antioxidant than α-T against some oxidative insults, the ability to enhance γ-T tissue accumulation may provide important therapeutic benefits. To briefly summarize the results of our studies (as shown in Table 6), 18 h following the single administration of rats with γ- TS-tris, significant γ-TS levels were detected in all tissues (except brain) with the liver containing 363 nmol/g tissue (a similar concentration as observed following α-TS-tris administration (see Table 3). The in vivo hydrolysis rate for γ-TS appears to be faster than for α-TS as noted by the near absence of tissue γ-TS, 120 h following administration (Table 6). This finding agrees with that predicted from the structure of γ-TS where the absence of a methyl group surrounding the ester linkage would be expected to promote an accelerated esterase hydrolysis rate. As with α-TS-tris administration, the injection of γ-TS-tris suspensions in rats resulted in a dramatic increase and maintenance of plasma and blood levels of γ-T. These data suggest the enhanced delivery of tocopherols (regardless of the number of methyl groups on the chromanol ring) to tissue can be accomplished by administering an aqueous suspension of the succinate ester tris salt. The second substance examined, TRF-S-tris, is a mixture of vitamin E compounds containing the succinate ester tris salt of d-α- tocopherol (28%) and d-α- (35%), d-γ-(22%), and d-δ-tocotrienol (16%). The presence of the succinate ester of each of these vitamin E derivatives was confirmed by HPLC analysis using base hydrolysis. In naive rat tissues, these tocotrienol compounds were not detectable using our standard analytical methods. In rats, 18h following the single injection of TRF-S-tris (0.19 nmol/kg intraperitoneally) significant vitamin E levels in tissues were only observed for α-TS. In contrast, each tocotrienol succinate compound did not appear to accumulate in tissues to any appreciable degree including the blood (approximately less than 1 nmol/g tissue was detected). Thus, these results agree with our previous experiments using α-TS-tris administration (both had similar tissue distributions regardless of administering alone or in combination with other vitamin E succinate ester tris salts). However, the absence of tissue tocotrienol accumulation indicates that changing the phytyl tail of the tocopherol molecule to an isoprenoid side chain (as with tocotrienol compounds) dramatically alters the transport and/or retention of these compounds by tissues in vivo.

TABLE 6. Tocopherol Analog Levels in Tissue from Rats 18 or 120 Hours after Single Treatment with d-γ -Tocopherol (γ-T) or d-γ -Tocopherol Hemisuccinate Tris Salt (γ- TS-

^a Rats received a 0.19 mmol/kg, intraperitoneally at the indicated number of hours prior to sacrifice. Immediately following sacrifice, tissues were obtained by a freeze-clamp method. ^b Values expressed as the mean ± SD (n=3-6). ^c ND, not detected. ^d T-equiv. : total T and/or T derivatives detected

EXAMPLE 9. Effect of TS-TRIS administration on the delivery ofTS and T to liver, hepatocytes, and mitochondria and protection against iron-induced lipid peroxidation and cell death.

Our previous studies (Examples 1 and 3) demonstrate that the administration of α-TS-tris, results in the accumulation of TS and T in mitochondria and that these mitochondria are protected against iron-induced lipid peroxidation. To further characterize this apparent directed subcellular transport and accumulation of α-TS, we developed an improved method for the purification of isolated mitochondria (respiring mitochondria method) to reduce the contamination of lysosomes in the preparation (it is well known that αT accumulates in lysosomes). Using this improved isolation method, we measured the amount of αT and αTS in liver homogenates, liver mitochondria, liver outer mitochondrial membranes and liver inner mitochondrial membranes in rats, 18 h following the administration of a single dose (0.19 mmol/kg, intraperitoneally) of αT or αTS-tris. As shown in Figure 3, αTS-tris administration resulted in a 10 fold increase in αT equivalents (αT + αTS) and a 2 fold increase in αT levels as compared to homogenate from αT-treated rats. In purified hepatic mitochondria isolated from αT-treated rats (Figure 4), the αT concentration did not significantly differ from that observed in mitochondria obtained from control (naive-no treatment) rats. In contrast, TS-tris administration resulted in dramatic increases in both αT (10-fold higher than αT-treated) and αTS levels, indicating that αTS has a unique ability to selectively concentrate in mitochondria (as compared to homogenate and αT-treatment data). Next, respiring mitochondria were isolated from the treated rats (as described above) and exposed to high levels of iron (a well known oxidative challenge). Using mitochondrial swelling as an indicator of mitochondria damage and dysfunction, we found that only mitochondria isolated from the livers of TS-tris treated rats were completely protected against iron-induced damage (data not shown). Since mitochondria are considered the most important cellular site for the production of reactive oxygen species and thus a potential cause for numerous oxidative-stress related diseases, the ability of αTS-tris administration to load mitochondria with αTS and then release large amounts of the antioxidant αT has tremendous implications in terms of a novel therapeutic strategy for the prevention and treatment of oxidative stress-related diseases. To insure that these mitochondrial stores of αT and αTS (observed following αTS-tris administration) do indeed accumulate at submitochondrial sites (inner and outer membranes) where most ROS are generated, we measured αT and αTS levels in isolated inner and outer mitochondrial membranes from the mitochondria described above, and then measured their susceptibility to oxidative damage (lipid peroxidation) following exposure to iron (an endogenous metal thought to be responsible for the propagation of ROS in many human diseases). The results from these studies are shown in Figures 5-7 and clearly demonstrate that αTS-tris administration results in large accumulations of αTS and αT in the inner and outer membrane of hepatic mirochondria, and this dramatic accumulation (as compared with αT-treatment) results in complete protection of these membranes against a strong oxidative challenge (a lag time of 300 indicates complete protection against membrane lipid peroxidation in Figure 7). These findings (Figures 5-7) also demonstrate the inability of T, administered acutely, to be rapidly transported and retained by mitochondria and provide antioxidant protection. Though it is clear from these studies that αTS-tris administration protects mitochondria from iron-mediated damage, the implications of this treatment for cell viability were unknown. Thus we investigated whether viable cells isolated from the livers of rats treated with αTS-tris (as described above) were also protected against the toxic effects of iron exposure. As shown in Figure 8, αTS-tris treatment does indeed protect hepatocytes from cell death induced by the oxidative challenge of iron, unlike control cells or cells isolated from αT-treated rats. These findings support the conclusion that oxidative stress-mediated toxicity can be prevented by enhancing the antioxidant capacity of mitochondria and α-TS-tris administration appears to accomplish this remarkably well.

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Claims

CLAIMSHaving thus described my invention, what I desire to secure by Letters Patent is the following:

1. A method for the delivery of a vitamin E compound to tissues, cells and subcellular sites in order to promote an effect selected from the group consisting of the protection of normal cells; increasing the antioxidant capacity of tissue, cells and subcellular sites; inhibiting the growth of and killing tumor cells; the coordinate action of protecting normal cells and inhibiting the growth of and killing tumor cells; and the coordinate action of increasing the antioxidant capacity of tissue, cells and subcellular sites and inhibiting the growth of and killing tumor cells, in a patient in need thereof, comprising, administering a sufficient quantity of an aqueous suspension of a tris salt of said Vitamin E compound to protect said normal cells and inhibit the growth of and kill said tumor cells.

2. The method of claim 1 wherein said effect is increasing the antioxidant capacity of tissue, cells and subcellular sites.

3. The method of claim 1 wherein said effect is inhibiting the growth of and killing tumor cells.

4. The method of claim 1 wherein said effect is the coordinate action of protecting normal cells and inhibiting the growth of and killing tumor cells.

5. The method of claim 1 wherein said aqueous suspension comprises the tris salts of a plurality of Vitamin E compounds.

6. The method of claim 1 wherein said aqueous suspension further comprises an antitumor agent other than a vitamin E compound.

7. The method of claim 6 wherein said antitumor agent is taxol.

8. The method of claim 1 wherein said tissue and said cells are in vitro.

9. The method of claim 1 wherein said subcellular sites are selected from the group consisting of mitochondria, the outer mitochondrial membrane, and the inner mitochondrial membrane.

10. A method for providing tocopherol to mitochondrial membranes, comprising administering to a mammal an aqueous suspension of a tris salt of a esterified tocopherol compound, said esterified tocopherol compound releasing tocopherol in mitochondrial membranes upon cleavage by cellular esterases.

11. The method of claim 10 wherein said aqueous suspension comprises the tris salts of a plurality of esterified tocopherol compounds.

12. The method of claim 10 wherein said tocopherol is in a form selected from the group consisting of d-α-tocopherol, dl-α-tocopherol, d-β-tocopherol, dl-β-tocopherol, d-γ- tocopherol, dl-γ-tocopherol, d-δ-tocopherol, and dl-δ-tocopherol.

13. The method of claim 10 wherein said aqueous suspension is provided in said administering step in a sufficient quantity to enhance antioxidant capacity.

14. The method of claim 10 wherein said aqueous suspension is provided in said administering step in a sufficient quantity to protect said mitochondria.

15. The method of claim 10 wherein said esterified tocopherol compound is selected from the group consisting of d-α-TS, dl-α-TS, d-β-TS, dl-β-TS, d-δ-TS, dl-δ-TS, d-γ-TS and dl- γ-TS.

16. A method for providing a tocopherol ester or ether compound to mitochondria, comprising administering to a mammal an aqueous suspension of a tris salt of said tocopherol ester or ether compound.

17. The method of claim 16 wherein said aqueous suspension comprises the tris salts of a plurality of tocopherol ester or ether compounds.

18. The method of claim 16 wherein said aqueous suspension is provided in said administering step in a sufficient quantity to inhibit the growth of and kill tumor cells.

19. The method of claim 16 wherein said tocopherol compound ester or ether is selected from the group consisting of d-α-TS, dl-α-TS, d-β-TS,dl-β-TS, d-δ-TS, dl-δ-TS, d-γ-TS and dl-γ-TS.

20. The method of claim 1 wherein said effect is the coordinate action of increasing the antioxidant capacity of tissue, cells and subcellular sites and inhibiting the growth of and killing tumor cells.