Patent Application: US-79429301-A

Abstract:
the present invention involves the use of desmethyl tocopherols such as gamma tocopherol for the prevention of and treatment of neurological disorders . dietary or parenteral administration of desmethyl tocopherols inhibits the undesired nitration of neurological components .

Description:
the present application demonstrates the superiority of desmethyl tocopherols , exemplified by gamma tocopherol , as protectors against nitrative and / or other damage to biological systems . the results described here are novel in several respects . particularly , the results demonstrate that gamma tocopherol ( γ - tocopherol or γt ) is superior to alpha tocopherol ( i . e ., vitamin e , a fully alkylated tocopherol ) in systems where nitrative stress is a relevant phenomenon . the invention of this utility for γ - tocopherol ( and other desmethyl tocopherols ) is not obvious to most ordinarily skilled practitioners of the art of antioxidant therapy . this contention is demonstrated by the fact that only α - tocopherol is currently being studied as a clinically relevant antioxidant in the treatment of neurodegenerative disease ( 15 ). one study has found that α - tocopherol decreases the progression of alzheimer &# 39 ; s disease ( ad ) marginally , and another large clinical trial is being planned ( 18 ); however , these plans only include the clinical assessment of α - tocopherol while desmethyl tocopherols are not being considered ( 18 ). moreover , efforts are underway to create crop plants overproducing α - tocopherol at the expense of γ - tocopherol ( 17 ). in point of fact , oral supplementation of humans with α - tocopherol actually depletes the human body of γ - tocopherol ( reference 3 and personal observations ). the γ - tocopherol and other desmethyl tocopherols are present in natural foods ( particularly soy and wheat ) in small amounts and are generally regarded as safe for human subjects . the biological activity of desmethyl tocopherols is associated with the chromanol head group of the molecule ( indicated by arabic numbers in the structure above ). this is to distinguish the tocopherols from tocotrienols , which inhibit cholesterol biosynthesis but whose activity is resident in the unsaturated lipid tail of the tocotrienol molecule . gamma tocopherol ( and other desmethyl tocopherols ) may be chemically synthesized or isolated from natural products . in practice , the γ - tocopherol ( or other desmethyl tocopherols ) would be formulated in a manner allowing safe delivery of effective doses to humans . the γ - tocopherol ( or other desmethyl tocopherols ) can be absorbed enterally by mammals and could be used by oral administration . the γ - tocopherol ( or other desmethyl tocopherols ) could be administered topically to inflamed skin or gum / mouth tissue as a cream or gel , or could be inhaled as an aerosol . the relative stability and lipophilicity of γ - tocopherol ( and other desmethyl tocopherols ) make these compounds amenable to delivery in numerous possible formulations . derivatives of γ - tocopherol ( or other desmethyl tocopherols ) which retain the structure of a phenolic ring lacking a h atom near the — oh group would also be useful as protectant against nitrative stress in neurodegenerative conditions . after consideration of the experimental data described below , these and other advantages and objects of the invention will be apparent to those skilled in the art . as a cardioprotectant or neuroprotectant , oral γ - tocopherol supplements are taken at a dose of 100 - 400 mg / day by individuals suffering from or at risk for these diseases . the γ - tocopherol supplements would consist of γ - tocopherol alone or as a predominant component mixed with other tocopherols , mediations or nutritive supplements . as a component of topical products or for intravenous administration , γ - tocopherol could be used alone or in combination with α - ketoglutarate and other tocopherols . in these applications , effective concentrations would likely be from about 0 . 1 μm to about 10 mm , more preferably from about 0 . 1 mm to about 10 mm . while the above enteral administration is preferred , parenteral administration may be desirable to more rapidly and consistently elevate γt levels or to more directly locate more optimal dosages . the following examples are intended for illustrative purpose only and are not to be construed as limiting the invention in spirit or scope . demonstration that γ - tocopherol scavenges rns ( reactive nitrogen species ) in the alzheimer &# 39 ; s diseased brain to study lipid - phase nitration chemistry , high performance liquid chromatography with electrochemical detection ( hplc - ecd ) has been applied by the present inventors to the study of tocopherol variants and their oxidation products ( 18 ). by connecting a photodiode array detector in - line with ( preceding ) the ecd , at least 7 discreet tocopherol variants can be simultaneously quantified in hexane - extracted human plasma ( 18 ). in studies with brain tissue taken from alzheimer &# 39 ; s diseased ( ad ) and normal brains by rapid postmortem protocol (& lt ; 3 h ), a significant 2 - 3 fold increase can be seen in the 5 - nitro - γ - tocopherol γ - tocopherol ratio in affected regions of the alzheimer &# 39 ; s diseased brain relative to age - matched normal brain ( fig3 a and 3b ). concomitantly , the γt content of the cortical tissue decreased by 20 - 50 % in ad depending on the brain region analyzed . the cerebellum , which is not severely affected in ad , was not severely nitrated ( fig3 b ). in a partial subcellular fractionation of these human brains , we found the highest level of tocopherol nitration in the mitochondrial fraction , where up to ⅔ of the γt appears to be nitrated . moreover , the 5nγt content of human brain mitochondria ( normal and ad combined ) correlates inversely { circumflex over ( r )}=− 0 . 41 ; p & lt ; 0 . 05 ; not illustrated ) with the marker enzyme α - ketoglutarate dehydrogenase ( αkgdh ), perhaps indicating that nitrative stress is a significant factor in age - related mitochondrial dysfunction . importantly , no significant decrease in α - tocopherol was observed in these studies , despite the profligate nitration of γ - tocopherol . using hplc - ecd techniques , we measured 2 - 7 fold increases in protein nitration and oxidation products in the alzheimer &# 39 ; s brain and published these results ( 11 ). as illustrated in fig4 nitrotyrosine ( 3 - no 2 - tyr ) was significantly elevated in regions of the alzheimer &# 39 ; s diseased brain that are histologically affected by the disease . the finding that human brain αkgdh activity correlates negatively with mitochondrial 5nγt stimulated us to ask whether γt could protect mitochondria from nitrative stress in vitro . mitochondria were isolated from adult rat brain then sonicated briefly in the presence of either αt or γt , or an ethanol vehicle . mitochondria were then exposed to sin - 1 , which generates no and superoxide simultaneously at a known rate ( 7 ). combination of no and superoxide yields onoo − in situ ( discussed above ). enzyme activity is destroyed by peroxynitrite and superoxide but not by no ( 19 ). predictably , αkgdh activity is diminished in lipopolysaccharide - treated cell culture in an rns - dependent manner ( 19 ). fig5 illustrates the protection of αkgdh by αt and γt present during exposure to the peroxynitrite ( rns )- generating compound sin - 1 . a 400 μm concentration of sin - 1 was sufficient to diminish αkgdh activity by approximately 50 % in one hour . under these conditions of nitrative stress , the αkgdh activity varied in a biphasic manner with respect to tocopherol concentration . at highertocopherol concentrations , the reaction medium became grossly turbid so that the apparent loss of enzyme activity might reflect a nonspecific physical consequence of the extreme lipid content . at all concentrations tested , γt was more protective than αt when tested in side - by - side comparisons . maximal protection was observed at about 1 μm tocopherol in the case of both αt and γt ( fig5 ). the maximal protection by γt was approximately 2 . 5 times greater than the maximal protection afforded by αt . at concentrations near 100 nm , γt was approximately 5 times more protective than the corresponding concentration of αt . moreover , 50 - 100 nm of γt offered as much protection as 1 - 10 μm of αt . thus , γt may be as important ( or more important ) an antioxidant as αt during nitrative stress , despite the lower intrinsic concentration of γt in most mammalian tissue . γ - tocopherol was further assessed for protective ability in a neurotoxicity assay where nitrative stress was imposed . primary cortical rat neurons were cultured and treated overnight with αt or γt at 10 μm . media was removed and cells were then washed and medium was replaced with fresh , tocopherol - free medium . neurons were then exposed to the nitric oxide - generating compound s - nitrosoprusside ( snp ) at 10 μm ( initial concentration ) for 24 hours and toxicity evaluated by release ofthe marker enzyme lactate dehydrogenase ( ldh ). as illustrated in fig6 a and 6b , γt significantly protected neurons from snp at a concentration of 200 nm while α - tocopherol was ineffective at a concentration of 10 μm . thus , in this particular cytotoxicity assay , γt may be orders of magnitude more protective against rns stress than is αt . the following references are incorporated in pertinent part by reference herein for the reasons cited . 1 . bieri , j . g ., evarts , r . p . gamma tocopherol : metabolism , biological activity and significance in human vitamin e nutrition . j . clin . nutr . 27 : 980 - 985 ; 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