Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
1998-12-04
2001-11-06
Allen, Marianne P. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S028000, C435S007910, C435S007950
Reexamination Certificate
active
06312917
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of screening compounds which are candidates for use as therapeutic agents for susceptibility to metabolic enzyme activity. More particularly, the present invention relates to a method of screening such candidate compounds for susceptibility to cytochrome P450-mediated metabolism.
Table of Abbreviations
ANOVA
analysis of variance
Cl
in
intrinsic clearance
CYP
cytochrome P450
CYP1A2
cytochrome P450 1A2 reductase
CYP2C9
cytochrome P450 2C9 reductase
CYP2C19
cytochrome P450 2C19 reductase
CYP2D6
cytochrome P450 2D6 reductase
CYP3A4
cytochrome P450 3A4 reductase
CYP3A4OR
cytochrome P450 3A4 and human
cytochrome P450 reductase
DCFH-DA
2′,7′-dichlorodihydrofluorescin
diacetate
DCF
2′,7′-dicholorofluorescein
EDTA
ethylenediamine tetraacetate
11&agr;-OH progesterone
11&agr;-hydroxy progesterone
HP
Hewlett Packard
HPLC
high performance liquid
chromatography
K
m
Michaelis-Menten constant for
enzyme-substrate reaction
&lgr; em
maximal emission wavelength
&lgr; ex
maximal excitation wavelength
mg pr.
milligrams protein
min
minute
NADPH
nicotinamide adenine dinucleotide
phosphate (reduced)
ROS
reactive oxygen species
6&bgr;-OH testosterone
6&bgr;-hydroxy testosterone
SD
standard deviation
TAO
troleadomycin
UV
ultraviolet
V
max
maximum velocity of enzyme-
catalyzed reaction
BACKGROUND ART
The advent of combinatorial chemistry techniques has enabled the identification of extremely high numbers of compounds that have potential as therapeutic agents. However, assays for drug metabolism that can rapidly identify those candidate compounds which have a lower potential for rapid metabolic degradation (i.e. short biological half-life) or drug-drug interaction have lagged behind the pace of synthesis and screening of pharmalogical activities. Thus, there is a long-felt need for high throughput assays to assess susceptability of candidate compounds to metabolic degradation, particularly oxidative metabolism, that can rapidly identify suitable candidate compounds (i.e. metabolically stable) for further testing as therapeutic agents.
Of particular interest is the cytochrome P450 (CYP) superfamily of enzymes. The CYP enzymes catalyze reactions which have profound effects on the biological activities of drugs, environmental chemicals and endogenous compounds (Guengerich,
FASEB J
6:667-668 (1992); Eastabrook,
FASEB J
10:202-204 (1996); and Rendic and Di Carlo,
Drug Metab. Rev.
29:413-580 (1997)). In recent years, advances in the study of CYP's by enzyme purification and characterization, gene cloning, and heterologous expression have indicated that five CYP isoforms, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, appear to be most commonly responsible for the metabolism of drugs in humans (Spatnegger and Jaeger,
Drug Metab. Rev.
27:397-417 (1995)). Moreover, a number of CYP superfamily enzymes have been successfully expressed in bacterial, yeast, insect and mammalian cells, and have been used to identify substrates and/or inhibitors of these major CYP enzymes (Guengerich and Shimada,
Chem. Res. Toxicol.
4:391-407 (1991); Birkett et al.,
Trends Pharmacol. Sci.
14:151-185 (1993); and Wrighton et al.,
Drug Metab. Rev.
25:453-484 (1993)).
To date, CYP metabolic activity, and/or inhibition thereof, has been assessed in most cases by performing in vitro incubation using cDNA-expressed enzymes or human liver microsomes (Parkinson,
Toxicol. Pathol.
24:45-57 (1996)). Such assessments have required the development and use of assays for quantitative analysis of the parent drug molecules, or the metabolites thereof, which is time-consuming, labor-intensive and costly.
A journal article by Crespi et al. entitled “Microtiter Plate Assays for Inhibition for Human, Drug-metabolizing Cytochrome P450
”, Anal. Biochem.
248:188-190 (1997) describes a CYP inhibitor assay which utilizes microtiter plate-based fluorometric methods for several major xenobiotic-metabolising CYP isoenzymes, such as CYP3A4 and CYP1A2. Similar assays are also described in Kennedy and Jones,
Anal. Biochem.
222:217-223 (1994) and in Donato et al.,
Anal. Biochem.
213:29-33 (1993). These assays utilize a known substrate for each of the major CYP isoenzymes as a model substrate. The ability of potential drug candidates to interact with CYP enzymes is ranked based on the relative inhibitory effect on metabolism of the model substrates. These assays are relatively fast as compared to other known methods, such as HPLC. However, these assays suffer from significant limitations in that they cannot directly assess the metabolic stability of candidate compounds.
In summary, prior art assays for susceptibility to oxidative metabolism, particularly CYP-mediated metabolism, rely on the measurement of the substrate and/or metabolite(s) in a reaction mixture. Such assays measure the presence of substrate and/or metabolite as a function of time and are thus limited to screening of one compound (or a best a few compounds) at a time. Therefore, what is needed is a rapid, high throughput assay which enables the screening of many compounds for susceptibility to oxidative metabolism in a single effort.
SUMMARY OF THE INVENTION
A method of screening a candidate compound for susceptibility to metabolism by a selected enzyme is disclosed herein. The method comprises the steps of reacting the candidate compound, an indicator compound precursor and the selected enzyme, the enzyme characterized as having a side reaction associated with metabolic activity of the enzyme wherein a chemical species capable of reacting with the indicator compound precursor is produced; and detecting as well as measuring an indicator compound, the indicator compound produced from the indicator compound precursor by reaction with the chemical species generated from the side reaction associated with metabolic activity of the enzyme, the presence of the indicator compound indicating the susceptibility of the candidate compound to metabolism by the enzyme.
Accordingly, it is an object of the present invention to provide a high throughput method of screening of candidate compounds for susceptibility to oxidative metabolism.
It is a further object of the present invention to provide a method of screening candidate compounds for susceptibility to oxidative metabolism which does not rely on the measurement or detection of the candidate compounds or their metabolites.
It is yet a further object of the present invention to provide a method of screening candidate compounds for susceptibility to oxidative metabolism which facilitates the screening of many candidate compounds in a single effort.
It is still a further object of the present invention to provide a method of screening candidate compounds for susceptibility to oxidative metabolism that is particularly suited to screening for susceptibility to cytochrome P450-mediated metabolism.
Some of the objects of the invention having been stated herein above, other objects will become evident as the description proceeds, when taken in connection with the accompanying Laboratory Examples and drawings as best described herein below.
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Ding et al. Purification and characterization of cytochrome P450 2E2 from hepatic microsomes of neonatal rabbits. Arch. Biochem. Biophys. 291 (2), pp. 270-276, (Dec. 1991).*
Nutter et al. Cellular biochemical determinants modulating the metabolism of estrone 3,4-quinone. Chem. Res. Toxicology. 7 (5), pp. 609-613, (Sep. Oct./1994).*
Shet et al. The effects of cytochrome b5, NADPH-P450 reductase, and lipid on the rate of 6&bgr;-hydroxylation of testosterone as catalyzed by a human P450 3A4 fusion protein. Arch. Biochem. Biophys. 318 (2), pp. 314-321, (Apr. 1995).*
Ghosh et al., “NADPH-Initiated Cytochrome P450-Dependent Free Iron-Independent Microsomal Lipid Peroxidation: Specific Prevention by Ascorbic Acid,” Mol Cell Biochem.,
Chen Cuiping
Thakker Dhiren R.
Allen Marianne P.
Jenkins & Wilson, P.A.
Moran Marjorle A.
The University of North Carolina at Chapel Hill
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