Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2001-02-27
2002-02-12
Henley, III, Raymond (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
Reexamination Certificate
active
06346544
ABSTRACT:
BACKGROUND
The present invention relates to concentrated preparations of desmethyl tocopherols, including but not restricted to gamma tocopherol (&ggr;T), which localize to lipid environments in cardiovascular tissue and scavenge reactive nitrogen species (RNS) by virtue of a phenolic structural element lacking one or more methyl substituents on the phenolic ring system. The capability to scavenge RNS imparts cardiovascular protective properties to the compound.
Tocopherols are a class of lipophilic, phenolic compounds of plant origin. The major tocopherol found in mammalian tissue is alpha tocopherol (&agr;-tocopherol or &agr;T or vitamin E)
FIG. 1
, although significant quantities of demethylated (desmethyl) forms (particularly &ggr;-tocopherol or &ggr;T)
FIG. 1
, are also present. &agr;-Tocopherol acts as a free radical scavenger (i.e., a chain-breaking antioxidant) when the phenolic head group encounters a free radical:
Toc-OH+L.→Toc-O.+LH Toc-OH=tocopherol L.=lipid radical
The phenoxyl radical Toc-O. is much more stable, and less reactive, than L.. The aromatic nature of the tocopherol ring system, combined with steric and electronic influences from the methyl
The phenoxyl radical Toc-O. is much more stable, and less reactive, than L.. The aromatic nature of the tocopherol ring system, combined with steric and electronic influences from the methyl substituents, stabilizes the tocopheroxyl radical and thereby ends the lipid peroxidation process. Eventually, Toc-O. is reduced back to Toc-OH by ascorbate acting in conjunction with NADPH reductase. While &agr;-tocopherol is the major tocopherol in the body, other tocopherols exist. The second major tocopherol in the human body is &ggr;-tocopherol (&ggr;T), which, like &agr;-tocopherol, is made by plants and taken into the human diet with foodstuffs.
Recently, it has become appreciated that reactive nitrogen species (RNS) are significant to many diseases including coronary artery disease (CAD), hypertension, and other forms of cardiovascular disease where localized inflammatory reactions occur. RNS are derived from the enzymatic oxidation of arginine via the intermediate nitric oxide free radical (FIG.
2
). Unlike oxygen-centered free radicals, reactive nitrogen species are not scavenged effectively by &agr;-tocopherol. On the other hand, &ggr;-tocopherol can react easily with RNS because of the presence of an open space on the chromanol head of the molecule (FIG.
1
). The major product of &ggr;-tocopherol reaction with RNS is 5-nitro-&ggr;-tocopherol (5N&ggr;T, FIG.
1
). Recent discoveries indicate that: (A) &ggr;T protects biological systems from RNS much more effectively than &agr;T; (B) &ggr;T is extensively nitrated in human plasma, particularly among smokers and hypertensive individuals; (C) &ggr;T inhibits RNS toxicity to a critical enzyme (&agr;-ketoglutarate dehydrogenase, or &agr;KGDH) which is severely damaged in injured vascular tissue; and (D) &ggr;T protects cultured endothelial cells from RNS. Thus, &ggr;T possesses unique biochemical functions from &agr;T that suggest &ggr;T may be a superior dietary supplement, cardioprotectant, cardioplegia additive, or a preservative in cardiovascular tissue exposed to RNS. Other desmethyl tocopherols likewise should be cardioprotective by this mechanism or another.
Chemistry of tocopherol reaction with oxidizing agents. &ggr;-Tocopherol is a natural product (a desmethyl tocopherol) of plant origin, present in many vegetable oils, especially soybean oil (1-2). &ggr;-Tocopherol is normally taken into the body through consumption of foodstuffs. Human plasma &ggr;T concentration is variously reported as 5-30% of &agr;T (3). The &ggr;T&agr;T ratio varies markedly among individuals; plasma &ggr;T/&agr;T proportionalities may be as low as 0.2% and as high as 30% (inventors' observations). Both &agr;T and &ggr;T are absorbed equally well by the gut, but &ggr;T is packaged into lipoproteins less effectively than &agr;T (4). For this reason, &agr;T supplementation decreases systemic &ggr;T concentration (3-4).
To date, only three well-disseminated studies have compared &agr;T and &ggr;T with respect to their ability to inhibit nitrative stress specifically (5-7). These studies generally investigated the in vitro reaction of nitrating equivalents with target substrates in “pure” chemical systems, and two of the three studies reached very different conclusions. The first investigation from Cooney's lab (5) reported that &ggr;T reaction with NO
2
gas was 6 times more rapid than the corresponding reaction of &agr;T. Furthermore, exposure of &agr;T (but not &ggr;T) to NO
2
caused the formation of a secondary nitrating species which could nitrate the target compound morpholine (5). In the same manuscript, Cooney et al. showed that &ggr;T was 4-fold more effective than &agr;T at inhibiting neoplastic transformation of methylcholanthrene-treated C3H/10T1/2 fibroblasts, a process which the authors suggest might involve nitrative stress (5). The second study (Christen et al. 1997; reference 6) incorporated either &agr;T or &ggr;T, or both, into liposomes which were then exposed to synthetic peroxynitrite (ONOO
−
). Christen and colleagues found that &ggr;T was twice as effective as &agr;T at inhibiting lipid hydroperoxide formation in liposomes exposed to ONOO
−
. Moreover, these researchers found that &ggr;T nitration rates were not influenced by the presence of &agr;T. This latter finding suggests that nitration of &ggr;T may occur preferentially to reaction with &agr;T when both tocopherols are simultaneously exposed to a nitrating species. In the third study (7), Goss et al. take issue with the findings of Christen et al. and report that &agr;T does spare &ggr;T in liposomes exposed to the superoxide and NO-generating compound SIN-1 [5-amino-2-(4-morpholinyl)-1,2,3-oxadiazolium].
A search of the literature revealed only two studies in which &agr;T and &ggr;T were compared for efficacy using in vivo models of cardiovascular stress (no studies were found investigating neurological stress). In the first study (c. 1983), tocopherol-depleted rats were fed &agr;T or &ggr;T for two weeks after chronic exposure to iron-dextran as an inducer of oxyradical stress (8). While both &agr;T and &ggr;T inhibited systemic lipid oxidation in the animals, &ggr;T was approximately 35% as effective as &agr;T. Lipid nitration was not an endpoint of this investigation, and physiologic parameters were not recorded. In a second, very recent study (reference 9; Saldeen et al.,
J. Am. Coll. Card
.,Oct. 1999), rats on an otherwise normal diet were fed &agr;T or &ggr;T (100 mg/kg/day) for 10 days after which the abdominal aorta was exposed to patch soaked in 29% FeCl
3
(9). This stress induced obstructive thrombus within 20 minutes. Saldeen et al. found that &ggr;T supplementation was significantly more effective than &agr;T supplementation at inhibiting iron-induced lipid peroxidation and occlusive thrombus (9). Time to occlusive thrombus was delayed by 25% in the &agr;T supplemented animals while the same parameter was increased by 65% in &ggr;T supplemented animals (9). Platelet aggregation kinetics were similarly inhibited, with &ggr;T supplementation being 2-fold more efficacious than &agr;T supplementation (9). Most importantly, the &ggr;T concentration in the plasma of the &ggr;T supplemented rats never exceeded 10% of the &agr;T concentration although the feeding paradigm did increase &ggr;T levels 6-fold above baseline (9). By comparison, &agr;T supplementation increased &agr;T plasma concentration only 2-fold (9). When treatment effects were considered in reference to plasma tocopherol concentrations, the Saldeen study found &ggr;T to be 20-30 times more potent than &agr;T at inhibition of throbogenic correlates. No conclusive explanation for the &ggr;T effect was offered by the Saldeen study, though superoxide dismutase activity increased significantly in the aortas of &ggr;T treated animals as compared to the &agr;T treated group (9). T
Floyd Robert A.
Hensley Kenneth L.
Head Johnson & Kachigian
Henley III Raymond
Oklahoma Medical Research Foundation
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