Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2001-03-28
2003-10-14
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S375000, C435S968000
Reexamination Certificate
active
06632630
ABSTRACT:
FIELD OF THE INVENTION
The field of this invention is monooxygenase assays.
BACKGROUND OF THE INVENTION
Monooxygenase include the numerous isoforms of the cytochrome P-450 enzymes. Because of the importance of these enzymes, particularly their activity in the liver, there is substantial interest in being able to assay for their activity and identify compounds that can modulate that activity. These enzymes serve to clear the blood of foreign factors. Unfortunately, in many cases these foreign factors are drugs, whose half-life is substantially diminished by virtue of being processed by the P-450 enzymes into inactive products. Also, reduced or modified activity of the P-450 enzymes may lead to poisoning or sensitivity to various agents, which in the normal person would be rapidly detoxified. The enzyme preparations are very expensive and each enzyme has multiple binding sites. Thus, multiple assays have to be performed to screen the enzyme(s).
With the advent of nanotechnology, there is an increased ability to perform numerous chemical and physical operations with very small volumes. This opportunity comes with the requirement that determinations have enhanced sensitivity to detect the fewer molecules that are present to provide the detectable signal. Part of the increased sensitivity may come from more sensitive detectors, but these are usually more expensive and are not readily available in most laboratories. The other opportunity is to provide assays that are more efficient in providing for detectable products, uses compounds that are readily accepted by the enzymes as substrates, and provide products with a strong signal, for fluorescent compounds, a high emission efficiency.
There is, therefore, substantial interest in providing P-450 assays that are rapid, accurate and can be performed in small volumes with low levels of enzyme and expensive reagents.
BRIEF DESCRIPTION OF THE PRIOR ART
U.S. Pat. No. 5,179,013 and references cited therein describe assaying for novel cytochrome P-450 enyzmes. Assays for P-450 enzymes are also described in Schwaneberg, et al., Anal Biochem 1999, 1:269:359-66; Tremblay, et al., Anal Biochem 1999, 276:215-26; Jansen, et al., J Chromatogr B Biomed Appl 1996, 684:133-45 and Eguchi et al., Xenobiotica 1996, 26:755-63. Other references that may be of interest include Hartmann and Frotscher, Arch Pharm 1999, 332:358-62; Ubeaud, et al., Eur J. Pharm Sci 1999, 8:255-60; Ertl, et al., Toxicol Appl Pharmcol 1999, 157:157-65; and Sanderson, et al., Toxicol Appl Pharmacol 1996, 137:316-25.
SUMMARY OF THE INVENTION
Methods and compositions are provided for determinations of monooxygenase enzymes, particularly P-450 enzymes, using a substrate having an ether group comprising an oxidizable &agr;-hydrogen, resulting in an aldehyde product. The aldehyde is reacted in situ with a fluorescent hydrazine and the resulting reaction mixture separated by capillary electrophoresis. The hydrazone product indicates the reaction occurrence and the area under the peak may be used for quantitation. Kits can be provided for performing the assay.
REFERENCES:
patent: 5179013 (1993-01-01), Matsuoka et al.
patent: 6312917 (2001-11-01), Thakker et al.
patent: WO 98/22502 (1998-05-01), None
patent: WO 99/65924 (1999-12-01), None
patent: WO 00/08169 (2000-02-01), None
patent: WO 00/12709 (2000-03-01), None
patent: WO 01/18046 (2001-03-01), None
Crespi C. Microtiter Plate Assays for Inhibition of Human, Drug Metabolizing Cytochromes P450. Anal biochem 248(1)188-190, 1997.*
Nererkar P. Methoxyresorufin and Benzyloxyresorufin. Biochemical Pharmacology 46(5)933-943, 1993.*
Renauld A. Identification of in vitro Cytochrom P450 Modulators to Detect Induction by Prototype Inducers in the Mallard Duckling. Comparative Biochemistry and Physiology Part C 122(2)273-281, 1999.*
Mayer, R. T., et al., 7-Alkoxyquinolines: New Fluorescent Substrates for Cytochrome P450 Monooxygenases,Biochemical Pharmacology, vol. 40, No. 7, pp. 1645-1655, 1990.
Genbank Accession No. AL139324, Feb. 15, 2000.
Genbank Accession No. AI756825, Jun. 23, 1999.
Meden and Kuhn, “Overexpression of the oncogene c-crbB-2 (HER2
eu) in ovarian cancer: a new prognostic factor,”European Journal of Obstetrics&Gynecology and Reproductive Biology 71:173-179, 1997.
Mayer, R. T., et al., “7-Alkoxyquinolines: New Fluorescent Substrates for Cytochrome P450 Monooxygenases,Biochemical Pharmacology, vol. 40, No. 7, pp. 1645-1655, 1990.
Crespi, C. L., et al., “Microtiter Plate Assays for Inhibition of Human, drug-metabolizing Cytochromes P450”,Analytical Biochemistry, vol. 248, pp. 188-190, 1997.
Burke, M.D., et al., “Differential Effects of Phenobarbitone and 3 Methylcholanthrene Induction on the Hepatic Microsomal Metabolism and Cytochrome P-450 Binding of Phenoxazone and a Homologous Series of its n-Alkyl Ethers (Alkoxyresorufins)”,Chemico-Biological Interactions, vol. 45, No. 2, pp. 243-258, 1983.
Anderson, J. M., “Fluorescent Hydrazides for the High-Performance Liquid Chromatographic Determination of Biological Carbonyls”,Analytical Biochemistry, vol. 152, No. 1, pp. 146-153, 1986.
Guebitz, G., et al., “Fluorogenic Labelling of Carbonylcompounds with 7-Hydrazino-4-Nitrobenzo-2 Oxa-1, 3-Diazole”,Journal of Liquid Chromatography, vol. 7, No. 4, pp. 839-854, 1984.
Copy of International Searh Report from Intl. Application No. PCT/US01/10155.
Chenna Ahmed
Gibbons Ian
Salimi-Moosari Hossein
Singh Sharat
Aclara Biosciences, Inc.
Dehlinger Peter J.
Gitomer Ralph
Mahoney Jacqueline F.
Perkins Coie LLP
LandOfFree
Monooxygenase assays does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Monooxygenase assays, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Monooxygenase assays will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3161013