Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
1999-09-30
2001-03-27
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S004000, C435S968000, C549S283000, C514S457000
Reexamination Certificate
active
06207404
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of drug and xenobiotic metabolism. The invention includes novel cytochrome P450 fluorescent probe substrates, particularly novel P450 CYP3A fluorescent probe substrates, 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. Certain P450 forms (such as CYP3A4 and CYP2C19) are known to be polymorphic in humans and some (such as CYP1A2 and CYP3A4) are regulated in response to environmental chemicals. 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.
CYP3A4 is the most abundant cytochrome P450 in the human liver and intestine. CYP3A4 is responsible for the metabolism of many important drugs, for example: opioid analgesics, corticosteroids, immunosuppressants, and antiarrhythmics (Rendic, S. and F. J. Di Carlo (1997) Drug Metab Rev. 29, p.413-580). Inhibition of this enzyme by dietary compounds (for example, grapefruit juice) or drugs has been responsible for several clinical drug-drug interactions. The importance of this P450 in drug metabolism makes the screening for metabolism and inhibition of this enzyme important in drug development.
Assays for CYP3A have focused on the metabolism of drug molecules or drug candidates. Substrates such as testosterone or midazolam are effective in assessing CYP3A activity and inhibition, but are not amenable to high throughput screening assay technology (both require time consuming separation of CYP3A reaction products using HPLC). Also, neither of these substrates have the necessary fluorescent properties that make the substrate useful for in situ fluorescent plate analysis.
We have previously reported the use of the commercially available compound 7-benzyloxyresorufin (BzRes) as a fluorescent substrate for assessing CYP3A4 activity in a high throughput mode (See Crespi et al. Anal Biochem. 248, 188-190, (1997). However, the low enzymatic turnover and poor specificity of this substrate make it of limited utility.
The O-dealkylation of 7-alkoxycoumarins has been reported as a fluorometric assay for determining the metabolic differences between cytochrome P450 isoforms using microsomes from several rat tissues (Kobayashi, Y., Fang, X, Szklarz, G. D., and J. R. Halpert, (1998) Biochem. 37, pp6679-6688). The O-dealkylation of 7-pentoxy-, 7-hexoxy-, and 7-benzyloxycoumarin have been studied in rat liver microsomes (Mayer, R. T., Netter, K. J., Heubel, F., Hanemann, B, Buchheister, A., Mayer, G. K. and M. D. Burke, (1990) Biochem. Pharmacol. 40, pp1645-1655). 7-Ethoxy-4-trifluoromethylcoumarin is O-dealkylated by human CYP2B6 and other human P450s (Code et al., (1997) Drug Metab. Dispo. 25, pp985-993; Morse, M. A., and J. Lu (1998) J. Chrom. B, 708, pp290-293.). The first coumarin analog whose O-dealkylation is specific for a single human P450 was recently developed by us (U.S. Ser. No. 60/092,995, entitled Novel CYP2D Fluorescent Assay Reagents).
SUMMARY OF THE INVENTION
The present invention relates to novel fluorescent substrates of the human cytochrome P450 enzymes, particularly CYP3A4. These substrates are useful in assessing CYP3A4 enzyme activity and in selecting compounds which inhibit CYP3A4 enzyme activity. Accordingly, the compounds and methods of the invention are useful for identifying potential adverse drug interactions mediated by inhibition of CYP3A4 enzyme activity.
The compounds of the invention are substrates that are specific for CYP3A and are characterized in having properties which permit the sensitive quantitation of CYP3A activity using in situ fluorescence analysis. To satisfy these requirements, the compounds of the invention include: 1) a 7-hydroxycoumarin core for easy fluorescence detection, 2) an electron-withdrawing group on the coumarin core, and 3) an 0-alkyl group which can be easily O-dealkylated by the enzyme.
According to one aspect of the invention, compounds of Formula I are provided:
(a) wherein R1 is an aryl containing an aryl ring carbon and/or an aryl ring nitrogen and the (CH
2
)n is coupled via a covalent bond to the aryl ring carbon or the aryl ring nitrogen;
(b) wherein n is 0, 1, 2, or 3;
(c) wherein R2 is selected from the group consisting of an hydrido, CN, CH
3
, sulfomethyl, and a haloalkyl containing from 1 to 18 carbons;
(d) wherein R3 is selected from the group consisting of an hydrido, CN, and an aryl containing an aryl ring carbon and/or an aryl ring nitrogen, provided that R2 and R3 are not both hydrido, and wherein the aryl is coupled directly to the coumarin ring via a covalent bond between the coumarin ring carbon and the aryl ring carbon or the aryl ring nitrogen;
(e) wherein when R1 is phenyl and n is 1, R2 is not an hydrido when R3 is phenyl and R3 is not an hydrido when R2 is CH
3
; and
(f) wherein the compound is a cytochrome P450 substrate.
Preferably, R1 is a phenyl and n is 1 or R1 is a naphthyl and n is 1.
In certain preferred embodiments, the R1 aryl is substituted with an electron donating group, (e.g., at the para position of a phenyl group). Exemplary electron donating groups include any of the following: an o-methoxy, an hydroxy, a primary amine (NH
2
), a secondary amine (NR4R5, wherein R4 and R5 are independently selected from an alkyl containing from 1 to 5 carbon atoms); and a tertiary amine (NR4R5R6, wherein R4, R5, and R6 are independently selected from an alkyl containing from 1 to 5 carbon atoms).
In these and other embodiments, R2 preferably is selected from the group consisting of an hydrido, CN, CH
3
, sulfomethyl, and a haloalkyl containing from 1 to 18 carbons. The most preferred R2 is an alkyl which is substituted to contain an electron-withdrawing group, e.g., a perfluoroalkyl having 1-18 carbons (such as CF
3
) or CN. These substituted alkyl groups impart different fluorescent properties to the compounds of Formula I. Most notably, these electron-withdrawing groups increase the wavelengths for fluorescence excitation and emission. Thus, by altering the identity of the R2 group, one can predictably change the excitation and emission wavelengths of the fluorescent product which results from the action of a cytochrome P450 enzyme (e.g., CYP3A) on a compound of Formula I.
In these and other embodiments, R3 preferably is a hydrido, CN or an aryl selected from the group consisting of a phenyl, a benzoxazolyl, and a benzothiazolyl. Optionally, the R3 aryl is substituted with a halide (e.g., a chloro, a fluoro, a bromo, and an iodo) that may be the same or different from a halide contained in an
Crespi Charles L.
Miller Vaughn P.
Gentest Corporation
Leary Louise N.
Wolf Greenfield & Sacks P.C.
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