Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2000-08-10
2002-07-16
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C549S391000, C435S004000, C435S006120, C435S007400, C435S189000, C435S191000, C435S968000
Reexamination Certificate
active
06420131
ABSTRACT:
FIELD OF INVENTION
This invention relates to the field of drug and xenobiotic metabolism. The invention includes novel cytochrome P450 fluorescent probe substrates and reaction products, methods for their preparation and their use as assay reagents.
BACKGROUND OF THE INVENTION
Cytochromes P450 (CYP) are the principal enzymes for the oxidative metabolism of many drugs, procarcinogens, promutagens, and environmental pollutants. Cytochrome P450 is a heme-containing, membrane-bound, multienzyme system that is present in many tissues in vivo but is present at the highest level in liver. In human liver, it is estimated that there are 15-20 different xenobiotic-metabolizing cytochrome P450 forms. A standard nomenclature based on relatedness of amino acid sequences has been developed. A relatively limited subset of these enzymes, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 appear to be most commonly responsible for the metabolism of drugs and associated drug-drug interactions (
Spatzenegger and Jaeger,
1995). The relative importance of this subset of enzymes is due to: the mass abundance of these enzymes (e.g. CYP3A4 is the most abundant P450 in human liver at ~30% of total P450), the preference of these enzymes to bind and/or metabolize chemical structures commonly found in drugs (e.g. CYP2D6 preferentially binds and metabolizes drugs with basic amine functionalities), enzyme polymorphisms (such as CYP3A4 and CYP2C19), and enzyme regulation in response to environmental chemicals some (such as CYP1A2 and CYP3A4). Competition for metabolism by a particular cytochrome P450 form is a principal mechanism of some clinically significant drug-drug interactions.
Identification of the enzymes responsible for metabolism is becoming an important aspect of drug development. Such identifications consider both the metabolism of the new drug as well as inhibition by the new drug. The identification of enzymes involved in metabolism of the new drug allows prediction, based on knowledge of the ability of coadministered drugs to inhibit the same enzymes, of which coadministered drugs may inhibit the metabolism of the new drug. This information can also be used to predict individual variability based on known metabolic polymorphisms. The identification of the enzymes most sensitive to inhibition by the new drug allows prediction, based on knowledge of which coadministered drugs are metabolized by the same enzyme, of which coadministered drug's metabolism may be inhibited by the new drug. Obtaining information for a series of drug candidates early in the drug discovery process can assist in the choice of the best drug candidate for further development.
Most cytochrome P450 assays have focused on the metabolism of drug molecules or drug candidates. While these chemicals are effective in assessing cytochrome P450 activity and inhibition, they are not amenable to high throughput screening assay technology (they require time consuming separation of enzyme reaction products using HPLC). Also, most of these substrates do not have the necessary fluorescent properties that make the substrate useful for in situ fluorescent plate analysis.
Sensitive fluorescent assays have been developed for human CYP1A1 and CYP1A2 enzymes. Members of the CYP1A family preferentially oxidize planar aromatic molecules not unlike the structure of common fluorescent chromophores. Microtiter plate based assays have been developed based on the O-dealkylation of alkyl ethers of resorufin (Donato, M. T. et. al., Anal. Biochem. 213, 29-33 (1993); Kennedy, S. W. and S. P. Jones, Anal. Biochem., 222, 217-223 (1994)) and coumarin (C. L. Crespi et al. Anal Biochem. 248, 188-190, (1997)). An intact cell assay using an alkyl ether of fluorescein (Quan, T. et al., Carcinogenesis 15, 1827-32, (1994)) has been described.
The development of fluorescent assays for other cytochromes P450 has been more challenging. We have described microtiter plate based assays for CYP2D6 based on alkylcoumarin derivatives (C. L. Crespi et al. Anal Biochem. 248, 188-190, (1997); Miller, V. P. and C. L. Crespi, U.S. patent application Ser. No. 09/352,576, entitled “Novel CYP2D Fluorescent Assay Reagents,” filed Jul. 12, 1999.) We have also reported the use of commercial alkylcoumarins for the fluorescent assay of CYP2C9 and CYP2C19. However, the sensitivity (signal to noise ratio) of these CYP2C assays is limited. New substrates which increase the assay signal and minimize the required amount of enzyme reagent would be useful. No fluorescent substrate has been reported for the important drug metabolizing enzyme CYP2C8. CYP2C8 is the primary cytochrome P450 enzyme responsible for the metabolism of the anticancer drug paclitaxel (TAXOL™).
Among the different cytochromes P450 expressed in the liver, CYP3A4 is the most abundant. Important classes of substrates for this enzyme include steroids, macrolide antibiotics, antivirals, and polycyclic aromatic hydrocarbons. As the majority of commercially available drugs are metabolized by CYP3A4, the importance of screening for inhibition of this enzyme is essential in drug development. We have previously reported the use of the commercially available 7-benzyloxyresorufin (BzRes) as a fluorescent substrate for assessing CYP3A4 activity in a high throughput mode (Crespi et al. Anal Biochem. 248, 188-190, (1997)). We and others have demonstrated that the inhibition potential (e.g. IC
50
value) or activation potential for most compounds varies dramatically depending on the probe substrate used (K E Thummel and G R Wilkinson, Ann. Rev. Pharmacol. Toxicol. 38:389-430 (1998)). Additional fluorescent probe substrates, which differ significantly in structure and chemical properties from the current substrates, are needed to gain a better understanding of the potential for test compounds to inhibit CYP3A4.
Fluorescein ethers have been reported to be useful fluorescent cytochrome P450 assay reagents. A. G. Miller (A. G. Miller, Anal. Biochem. 133, 46-57 (1983)) described a series of ethylated fluoresceins. Ethoxyfluorescein ethyl ether, reportedly was the most useful for the flow cytometric analysis and sorting of intact, viable rat cells based on cytochrome P450 activity. Other laboratories have also reported using the diethoxy- or a dimethoxyflourescein derivative for the same purpose with rat or porcine cells (White et al., Biochem. J. 247, 23-28 (1987); Pan et al, Artif. Organs 20, 1173-1180 (1996); Anderson et al., Int. J. Artif. Organs 21, 360-364, (1998)). More recently, ethoxyfluorescein ethyl ester has been reported to monitor the activity of human skin fibroblasts transfected with human CYP1A1 cDNA (Quan, T. et al., Carcinogenesis 15, 1827-32, (1994)).
Aryl ether derivatives of fluorescein have not been reported as substrates for cytochrome P450 enzymes. A benzyl ether derivative of fluorescein, benzyloxyfluorescein benzyl ester, has been reported as an intermediate in the synthesis of fluorescein labeled polymers (Hargreaves and Webber, Can. J. Chem. 63, 1320-1327 (1985)), chiral prodyes (Tadic and Brossi, Heterocycles 31, 1975-1982, (1990)), and dye precursors (Dombrowski Jr. et al. U.S. Pat. No. 5,196,297).
SUMMARY OF THE INVENTION
The present invention relates to novel fluorescent substrates of cytochrome P450 enzymes. These substrates are useful in assessing cytochrome P450 enzyme activity and in selecting compounds which inhibit cytochrome P450 enzyme activity. Accordingly, the compounds and methods of the invention are useful for identifying potential adverse drug interactions which are mediated by inhibition of cytochrome P450 enzyme activity.
The compounds of the invention are substrates that are characterized in having properties which permit the sensitive quantitation of CYP2C8, CYP2C9, CYP2C19, and CYP3A activity using in situ fluorescence analysis. To satisfy these requirements, the compounds of the invention include: 1) An aryl ether group at position 6 of fluorescein which can be easily O-dealkylated by the enzyme, and 2) a fluorescein core for ease in fluorescence detection.
The enzyme reaction and a
Crespi Charles L.
Miller Vaughn P.
Streser David
Gentest Corporation
Lilling Herbert J.
Wolf Greenfield & Sacks P.C.
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