Organic compounds -- part of the class 532-570 series – Organic compounds – Carboxylic acid esters
Reexamination Certificate
2000-02-08
2001-09-18
O'Sullivan, Peter (Department: 1621)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carboxylic acid esters
C560S008000, C560S010000, C560S035000, C562S011000, C562S030000, C562S427000, C562S440000, C562S462000, C564S001000, C564S015000, C564S123000, C564S253000, C564S257000, C564S297000, C564S298000, C564S299000, C564S300000, C564S435000, C564S440000, C568S305000, C568S423000, C568S924000, C568S949000
Reexamination Certificate
active
06291702
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to chromotropic azulenyl nitrone spin trapping agents, methods of making these agents, compositions comprising same, and methods of their use. In particular, azulenyl nitrones of the present invention are effective agents for trapping and identifying free radical species and find use as efficient antioxidants in physicochemical and biological systems.
BACKGROUND OF THE INVENTION
2.1 General Considerations
The technique of spin trapping is an important method for garnering, information on free radicals difficult or impossible to detect by direct spectroscopic observation due to their exceedingly short lifetimes and low concentrations. To date, two classes of spin trapping agents have received the most attention, namely nitroso compounds and nitrones. Of these, the latter have been more frequently used, especially in biological systems.
The most commonly cited drawbacks to the application of spin trapping agents bearing nitroso functionality are instability and toxicity. On account of these undesirable characteristics, researchers often opt for nitrone spin traps despite the fact that their nitroxide spin adducts generally provide less structural information from ESR than do those from nitros based spin traps Furthermore, the nitroxides obtained from the addition of certain carbon-centered radicals (tertary alkyl and aryl) to the most widely used nitrone spin traps alpha-phenyl-N-tert-butyinitrone (PBN), pyridine N-oxide-4-N-tert-butylnitrone (POBN) and dimethylpyrroline N-oxide (DMPO) are, due primarily to disproportionation, less persistent than those obtained from addition of such radicals to nitroso compounds.
Several groups have described the use of isotopically labeled spin traps or the application of special equipment consisting of GC/MS or HPLC-interfaced ESR spectrometers designed to detect, isolate and characterize free radical adducts of nitrone spin traps in biological systems with varied success.
2.2. Detection and Characterization of Free Radicals
Nitrones behave as spin trapping agents when a diamagnetic nitrone compound (the “spin trap”) reacts with a transient free radical species (having the “spin”) to provide a relatively more stable radical species (referred to as the “spin adduct”). The spin adduct may be detectable by electron paramagnetic resonance (EPR) spectroscopy if the spin adduct has a reasonable lifetime. Thus, information about the spin can be gleaned from a study of the structure and spectroscopic characteristics of the spin adduct. For example, the toxicity of synthetic beta-amyloid peptide preparations toward glutamine synthetase could be correlated with the characteristics of the EPR signal generated by the spin adduct formed from each batch of synthetic beta-amyloid peptide and the spin trap PBN. See, Hensley, K. et al., in
NeuroReport
(1995) 6:489-492. Beta-amyloid peptides are neurotoxic substances that are postulated to be involved in the etiology of Alzheimer's disease.
2.3. Methods of Diagnoses
Low molecular weight nitroxides are non-immunogenic. Moreover, they are typically cell permeable and can exist as a non-toxic, stable free radical capable of partitioning among various cellular compartments. Being paramagnetic, nitroxides are detectable by electron paramagnetic resonance (EPR) spectrometry and may serve as contrast agents in magnetic resonance imaging (MRI). See, Brasch, R. C., in
Radiology
(1983) 147:781; Keana, J. F. and Van, N. F., in
Physiol. Chem. Phys. Med. NMR
(1984) 16:477. Nitroxides have also been used as biophysical markers to probe cellular metabolism, oxygen level, intracellular pH, protein/lipid mobility and membrane structure. Hence, nitroxides find use in a number of diagnostic methods to determine the physiological/medical condition of a subject or the biophysical characteristics of a given sample, including samples obtained from a biological fluid.
2.4. Therapeutic Applications of Spin Trapping Agents
Free radicals and oxidative damage have been implicated in brain aging and several neurodegenerative diseases. See, Socci. D. J. et al., in
Brain Research
(1995) 693(1-2):88-91. Chronic treatment of aged rats with certain compounds, including the spin trapping agent alpha-phenyl N-tert-butylnitrone (PBN) and the antioxidant alpha-tocopherol (vitamin E), was found to benefit (i.e., improve) age-related changes in cognitive performance.
In vitro and in vivo evidence is mounting that the administration of antioxidants can strongly reduce the rate of progression of lesion formation associated with the process of atherosclerosis. Based on several experimental models, including low density lipoprotein (LDL)-receptor-deficient rabbits, cholesterol-fed rabbits and cholesterol-fed non-human primates, several antioxidants have manifested a 50-80% reduction in the rate of progression of lesions. The effectiveness of probucol, butylated hydroxytoluene (BHT), N,N′-diphenylphenylenediamine and vitamin E are attributed to their respective antioxidant potentials and to the proposition that oxidative modification of LDL contributes to the progression of atherosclerosis. See, Steinberg, D., in
Lancet
(1995) 346(8966):36-38. The one-electron oxidative potentials (vs. NHE) of vitamin E in an aqueous solution at pH 7 and 20 degrees C. is 0.48 V. The oxidative potentials of PBN, POBN and DMPO range from about 1.5-2.0 V.
Further, Downs, T. R. et al., in
Int'l J. Immunopharmacol.
(1995) 17(7):571-580, have shown that a cyclic nitrone antioxidant, MDL 101,002, reduces organ dysfunction and cytokine secretion induced by lipopolysaccharide (LPS) administered to rats. These authors also tested the ability of MDL 101,002 to prevent LPS-induced pulmonary edema, leukopenia and thrombocytopenia. They found that MDL 101,002 prevented pulmonary edema, partially reduced thrombocytopenia but failed to prevent leukopenia. These workers found that their results were consistent with the role that oxygen free radicals played in the development of endotoxin-induced organ dysfunction and shock. They further suggest that free radical scavengers could reduce the mortality consequent to sepsis by organ dysfunction, at least in part, through a reduction in free radical-stimulated cytokine secretion.
2.4.1. Radicals in Allergy and Allograft Rejection
Allergic reactions generate reactive oxygen species, including superoxide anions, which usher the influx of inflammatory cells to the site of allergen challenge and contribute to allergic inflammation. The inflammation may, in turn, lead to cell or tissue injury. For allergic reactions in the lung, these processes are also accompanied by increased vascular permeability and changes in airway mechanics. See, Sanders, S. P. et al. in
Am. J. Respir. Crit. Care Med.
(1995) 151:1725-1733. Thus, the administration of spin trapping agents to the site of challenge may reduce the inflammatory response and help reduce tissue or cell damage.
Separately, oxygen-derived free radicals are suspected in playing a role in cytotoxicity during episodes of allograft rejection/destruction following infiltration of the graft by mononuclear cells. The administration of radical scavengers may thus inhibit or reduce the incidence of allograft rejection. See, Roza, A. M. et al., in
Transplantation Proceedings
(1994) 26(2):544-545.
New reagents that could visually signal the formation of oxidative species would be extremely useful not only in skin tests or in cell culture but also in determining, for example, the compatibility of a patient's white blood cells with a particular kidney dialysis membrane. In vitro colorimetric assays would be of great utility.
2.5. Other Applications
PBN has been shown to offer protection in the cardiovascular disease area, in particular, by trapping free radicals generated during ischemia-reperfision-mediated injury to the heart See, e.g., Bolli, R. et al. J. Clin. Invest. (1988) 82:476. The benefits of trapping free radicals generated in similar types of injury to the brain of experimental animals has also been demonstrate
Florida International University
O'Sullivan Peter
Pepper Hamilton LLP
Pouliquen C. M.
Villacorta G. M.
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