Patent Application: US-79429201-A

Abstract:
the present invention involves the use of desmethyl tocopherols such as gamma tocopherol for the protection of cardiovascular tissue from nitrative stress . while mechanisms other than scavenging of reactive nitrogen species may be involved , desmethyl tocopherols exhibit significant protection and may be utilized to treat or help prevent cardiovascular particularly arterial vascular disease . the desmethyl tocopherols may be administered dietarily or parenterally when a more direct dosage is desired . both routes may be utilized together or separately to optimize therapeutic and prophylactic benefits . the lessening of damage induced by reactive nitrogen species leads to the lessening of arterial blockage in thrombosis .

Description:
the present application demonstrates the superiority of desmethyl tocopherols , exemplified by gamma tocopherol , as protectors against nitrative 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 ( α - tocopherol or αt ) ( 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 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 ofcardiovascular or neurodegenerative disease ( 15 ). in clinical studies performed to date , α - tocopherol has failed to provide a consistent beneficial effect on outcome parameters ( 32 - 33 , 25 - 38 ). in point of fact , oral supplementation of humans with α - tocopherol actually depletes the human body of γ - tocopherol ( reference 3 and the present inventors &# 39 ; observations ). 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 orally 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 or other mucosal 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 a protectant against nitrative stress in neurodegenerative conditions ; intraperitioneal or intravascular administration in appropriate media may also be used when desired . as a cardiprotectant or neuroproctectant , oral γ - tocopherol supplements could be taken at a dose of 100 - 4000 mg / day by individuals suffering from or at risk for cardiovascular or neurological diseases . the γ - tocopherol supplements would consist of γ - tocopherol alone or as a predominant component mixed with other tocopherols , medications 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 / or other tocopherols . in these applications , effective in vivo concentrations would likely be from about 1 μm to about 10 mm , more preferably from about 1 nm to about 10 mm . after consideration of the data described below , these and other advantages and objects of the invention will be apparent to those skilled in the art . the following examples are intended for illustrative purpose only and are not to be construed as limiting the invention in sphere or scope . αkgdh is a rate - limiting enzyme of mitochondrial energy production which is diminished in conditions of vascular ischemia ( 53 - 58 ); augmentation of the enzyme with α - ketoglutarate has been found beneficial and α - ketoglutarate is now included as a component in cardioplegia fluid to improve recovery after heart surgery ( 59 ). we undertook to determine whether γt protects αkgdh against 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 ). fig3 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 - 1i 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 higher tocopherol 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 1 μm tocopherol in the case of both αt and γt ( fig3 ). 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 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 . demonstration of αkgdh protection against nitrative stress in vivo by gamma tocopherol rat pups were injected intraperitoneally ( i . p .) with αt or γt in an olive oil vehicle every other day for 30 days beginning 2 days after birth ; control animals received vehicle only . olive oil was chosen as a vehicle because of the low tocopherol content in this particular vegetable oil . a total of 15 mg tocopherol was delivered to each animal over the 30 day period ; animal weight at the end of the period was approximately 90 g for all three groups . one half of each animal group was injected with a septic dose of lps ( 2 . 5 mg / kg , i . p ). after 24 h , animals were killed and organs collected . αkgdh was assayed in heart tissue and tocopherols were measured by hplc - ecd / pda . the supplementation paradigm was sufficient to approximately double the heart tissue level of both αt and γt [ αt concentration = 23 ± 12 ng / mg protein in controls vs . 48 ± 13 ng / mg in αt supplemented animals ( n = 5 ); γt concentration = 1 . 8 ± 0 . 4 ng / mg protein in controls vs . 3 . 9 ± 0 . 7 ng / mg in γt supplemented animals ( n = 5 )]. similar changes were noted in plasma tocopherol concentrations ( not shown ). the effect of tocopherol supplementation on cardiac αkgdh activity is illustrated in fig4 . both αt and γt supplementation increased mean αkgdh activity by 40 % in animals not subjected to lps stress . this increase was significant at the 90 % confidence level ( p & lt ; 0 . 1 by student &# 39 ; s t - test ). in lps - treated animals , αkgdh activity decreased by 20 - 40 % in all groups . notably , the γt supplemented animals maintained the highest heart αkgdh activity among the several groups after lps challenge ( fig4 ). these data indicate that γt supplementation is at least as effective and probably more effective at maintaining mitochondrial homeostasis under a condition where nitrative stress is known to be relevant . cultured ecv304 human endothelial cells were exposed to sin - 1 in the presence of 10 μm α - t , 10 m γ - t or 5 μm of each ( fig5 ). tocopherols were incubated with the cells for 19 hours prior to addition of sin - 1 . viability was assayed 24 hours later using a standard tetrazolium ( mtt ) reduction assay . these cells proved very resistant to damage by sin - 1 ; however , a 5 mm initial concentration of sin - 1 produced approximately 26 % toxicity within 24 hours ( n = 5 ; fig5 ). while α - t had no apparent effect on sin - 1 toxicity , γ - t promoted viability somewhat and the combination of γ - t with α - t ( 1 : 1 molar ratio ) completely prevented sin - 1 toxicity ( fig5 ). the data suggest that γ - t may protect cells in a way that α - t does not . smoking is recognized as a major contributing factor to heart disease and γ - t reportedly decreases more than α - t in smokers . hypertension is also a strong risk factor for heart disease . we have begun collection of data from “ normal ” subjects who do not currently have cad , including smokers and nonsmokers , and hypertensive subjects . 11 of 54 volunteers from the oklahoma city veteran &# 39 ; s administration hospital and the oklahoma medical research foundation indicated a current smoking habit . seven subjects indicated a chronic hypertensive condition . as outlined in table i , γ - tocopherol tended to decrease in hypertensive subjects while nitration products tended to increase in both smokers and hypertensive subjects . this was the trend regardless of whether the tocopherol concentrations were normalized to plasma triglycerides ( table i ). table i preliminary statistical data regarding plasma concentrations of α - tocopherol , γ - tocopherol and 5 - no 2 γ - tocopherol in a population of random volunteers from oklahoma city . population mean ± sem smokers mean ± sem hypertensives mean ± sem n = 54 n = 7 n = 7 α - tocopherol μg / ml plasma 18 . 3 ± 2 . 6 24 . 9 ± 9 . 2 23 . 1 ± 12 . 2 μg / mg triglyceride 34 . 2 ± 14 . 2 21 . 5 ± 4 . 5 21 . 9 ± 5 . 9 γ - tocopherol μg / ml plasma 0 . 713 ± 0 . 067 0 . 680 ± 0 . 153 0 . 463 ± 0 . 165 μg / mg triglyceride 0 . 813 ± 0 . 088 0 . 810 ± 0 . 213 0 . 606 ± 0 . 153 γ - toc / α - toc × 100 6 . 78 ± 1 . 13 5 . 54 ± 1 . 26 4 . 29 ± 1 . 40 5 - no 2 - γ - tocopherol ng / ml plasma 11 . 3 ± 1 . 6 20 . 0 ± 3 . 3 20 . 0 ± 3 . 7 ng / mg triglyceride 14 . 0 ± 2 . 5 23 . 5 ± 7 . 2 34 . 9 ± 10 . 9 5 - no 2 - γ - toc / γ - toc × 1000 3 . 49 ± 0 . 74 5 . 47 ± 1 . 96 8 . 20 ± 2 . 21 γt scavenges reactive nitrogen species during thrombosis in an animal model as discussed above , γt has been found much more effective than αt in the inhibition of thrombosis in a rat model ( saldeen et al . reference 9 ). consequently , we set forth to determine whether 5 - no 2 - γ - toc might be formed during thrombosis in vivo . in a first experiment , the saldeen model was used to induce occlusive thrombosis by application of an fecl 3 - laden patch to the descending aorta of anesthetized rats . 0 . 2 ml samples of blood were withdrawn into a 2 mm edta / saline solution by heart puncture at various time points after application of the fecl 3 - laden patch . control experiments were conducted using a saline - soaked patch , which does not induce thrombosis . plasma was isolated by centrifugation of the blood and was analyzed by hplc - ecd . as illustrated in fig6 - no 2 - γ - tocopherol was formed within minutes after initiation of thrombosis and increased steadily until the animal died ( approximately 20 min . after initiation of thrombosis ). plasma nitrite concentrations were not statistically altered in this model of thrombogenesis . the increase in 5 - no 2 - γ - tocopherol / γ - tocopherol was due both to a decrease in γ - tocopherol and an increase in 5 - no 2 - γ - tocopherol ; α - tocopherol was not substantially affected by fecl 3 - induced thrombosis ( data not illustrated ). γt inhibits platelet aggregation in vitro and in vivo , apparently through antagonism of protein kinase c ( pkc ) ( 60 - 63 ). concentrations of αt necessary to inhibit aggregation in vitro are on the order of 500 μm ( 60 - 63 ), or approximately 10 - fold higher than achieved in vivo . the concentration difference between in vitro and in vivo efficacy has been rationalized on the basis of incomplete tocopherol incorporation into membranes during in vitro incubation ( 63 ). nonetheless , subjects supplemented with oral αt have diminished platelet aggregation ( 63 ). we therefore sought to investigate the ability of γ - tocopherol to inhibit platelet aggregation . prp was obtained from two volunteers . platelets were stimulated to aggregate with adp , thrombin receptor activating peptide ( trap ) or the pkc agonist phorbol myristyl acetate ( pma ) and aggregation was measured using a 4 - channel clinical aggregometer . specific platelet samples were preincubated with vehicle , αt , γt or 5 - no 2 - γ - tocopherol for 1 h prior to stimulation . tocopherol concentrations were set at 10 - fold excess to average prp levels based on the precedent of previous in vitro aggregation experiments that used αt as an inhibitor ( 60 - 63 ). as shown in table ii , γt was similarly efficacious to αt when tested for ability to inhibit pkc - linked platelet aggregation ( e . g ., when trap or pma was used as the agonist ). this data suggests that γt has an activity similar to αt with respect to antagonism of pkc - dependent platelet aggregation and corresponding thrombogenic events , while other data ( see above ) indicates specifically enhanced rns scavenging ability inherent to the γt structure . table ii effect of tocopherols on platelet aggregation in vitro . % maximum = 100 % × ( aggregability with added tocopherol / aggregability without added tocopherol ). platelet aggregation (% maximum ) adp trap pma 20 μm 20 μm 200 nm subject 1 vehicle ( ethanol ) 100 100 100 500 μm α - tocopherol 85 44 74 50 μm γ - tocopherol 117 73 57 subject 2 vehicle ( ethanol ) 100 100 100 500 μm α - tocopherol 72 75 34 50 μm γ - tocopherol 90 88 29 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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