Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – C-o-group doai
Patent
1997-08-22
2000-02-29
Jones, Dwayne C.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
C-o-group doai
A01N 3108
Patent
active
060310081
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The method of the present invention is derived from a complex screening method (1) which relates to the oxidation and co-antioxidation of low density lipoprotein (LDL) lipids by a tocopherol-mediated mechanism (TMP, tocopherol-mediated peroxidation) (2). The latter mechanism summarizes a novel approach to explain the activity of .alpha.-tocopherol (.alpha.-TOH) in LDL in terms of both its ability to act as a phase transfer agent and also its role in the peroxidation of the lipid components of the lipoprotein. This earlier study (1) indicated that an effective co-antioxidant (XH) for .alpha.-TOH acted in three specific modes: the co-antioxidant must associate with an oxidizing LDL particle, reduce the lipid peroxidation chain carrying .alpha.-tocopheroxyl radical (.alpha.-TO.cndot.) [reaction 1], and the ensuing, co-antioxidant-derived radical (X.cndot.) must escape the lipoprotein particle (so as to minimize the possibility of regeneration of .alpha.-TO.cndot.)
The co-antioxidant efficacy was evaluated previously by the corresponding anti-TMP index (1), where anti-TMP indices of approximately 0 referred to highly effective co-antioxidants, and anti-TMP indices of approximately 100 indicated poor co-antioxidant activities. As this methodology required the isolation and labour-intensive work-up of biological material we sought to develop a simple and rapid screen to identify potential co-antioxidants for .alpha.-TOH, based upon the ability of a test compound to reduce .alpha.-TO.cndot. which was compartmentalized from the surrounding aqueous medium.
BRIEF SUMMARY OF THE INVENTION
The invention is directed to methods for screening and/or testing, in vitro, synthetic or natural compounds for co-antioxidant potency in eliminating the .alpha.-tocopheroxyl radical. The invention is further directed to methods for inhibiting lipid peroxidation and restoring endothelial function employing compounds identified by the claimed screening and/or testing methods.
DETAILED DESCRIPTION OF THE INVENTION
Description of Tocopheroxyl Radical Attenuating Ability (TRAA) Test
The method of the present invention is based on the direct observation of .alpha.-TO.cndot. by electron paramagnetic resonance (EPR, a technique used specifically for the direct detection of free radical species) spectroscopy and the subsequent effect of the addition of a potential co-antioxidant which will reduce this radical. In brief, the method we have developed makes use of .alpha.-TO.cndot. generated in micellar suspensions of .alpha.-TOH using a commercially available mercury-vapour lamp. The .alpha.-TO.cndot. generated under the test conditions was found to decay with a half life of approximately 25 min in the absence of an added co-antioxidant. Measurement of the observed rate of decay of .alpha.-TO.cndot. in both the absence and presence of a potential co-antioxidant permitted the determination of a relative rate index which then allowed the comparison of the tocopheroxyl radical attenuating ability (TRAA) of each test compound to be determined. We defined an efficient inhibitor of the .alpha.-TO.cndot. (i.e. co-antioxidant) as a compound which caused a greater than 5-fold increase in the rate of decay of the .alpha.-TO.cndot. radical as compared with the rate in the absence of the test compound.
Thus, the present invention provides a method for screening and/or testing in vitro synthetic or natural compounds for co-antioxidant potency in eliminating the radical, characterised by measuring the decay of this radical in the presence or absence of the test compound using EPR spectroscopy and identifying co-antioxidants which exhibit a greater than 5-fold increase in the rate of decay of the radical.
Details of the TRAA Test
Preparation of micelles--100 mM Stock solutions of cetyltrimethyl ammonium chloride (HTAC) and sodium dodecyl sulfate (SDS, both obtained from Aldrich) were prepared in phosphate buffer principally as described in (3). Micellar dispersions of .alpha.-TOH were prepared by diluting an ethanoli
REFERENCES:
patent: 4115590 (1978-09-01), Lerner
Witting et al., "A rapid and simple screening test for potential inhibitors of tocopherol-mediated peroxidation of LDL lipids", J. Lipid Res., vol. 37, No. 4, 1996, pp. 853-867.
Biochim. Biophys. Acta, vol. 801, 1984, M. Scarpa et al., "Formation of alpha-tocopherol radical and recycling of alphatocopherol by ascorbate during peroxidation of phpsphatidylcholine liposomes", pp. 215-219.
FEBS Lett., vol. 290, No. 1,2, 1991, R. H. Bisby et al., "Reactions of the alpha-tocopheroxyl radical in micellar solutions studied by nanosecond laser flash photolysis", pp. 205-208.
Arterioscler. Throm., vol. 11, No. 5, 1991, S.J.T. Mao et al., "Attenuation of atherosclerosis in a modified strain of hypercholesterolemic Watanabe rabbits with use of a probucol analog (MDL 29,311) that does not lower serum cholesterol", pp. 1266-1275.
Lindqvist Ann-Margret
Pettersson Knut
Stocker Roland
Westerlund Christer
Witting Paul
Jones Dwayne C.
The Heart Research Institute Ltd.
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