WO2006114275A1

WO2006114275A1 - Assay for cytochrome p450 1a2

Info

Publication number: WO2006114275A1
Application number: PCT/EP2006/003812
Authority: WO
Inventors: Annalise Di Marco; Ralph Laufer
Original assignee: Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
Current assignee: Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
Priority date: 2005-04-26
Filing date: 2006-04-21
Publication date: 2006-11-02
Anticipated expiration: 2007-10-26

Abstract

A rapid and sensitive radiometric assay for assessing the activity of cytochrome P-450 (CYP) 1A2 and the potential of an analyte to inhibit CYP3A1A2 activity or induce CYP1A2 expression is described. All the steps of the assay, including incubations, product separation, and radioactivity counting are preferably performed in a multiwell format, which can be automated.

Description

TITLE OF THE ESfVENTION

ASSAY FOR CYTOCHROME P450 1A2

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present invention relates to an assay for assessing the activity of CYPl A2 and the potential of an analyte to modulate CYP 1A2 activity, e.g., inhibitor of CYPl A2 activity or inducer of CYP1A2 expression. The assay determines CYP1A2 activity or expression by measuring CYPl A2- mediated O-deethylation of phenacetin in reactions comprising CYP 1A2 or hepatocytes using phenacetin labeled with 14c in the ethyl leaving group as a substrate and a sorbent which preferentially binds non-polar compounds such as the labeled phenacetin to separate the labeled phenacetin from the 14C-labeled acetaldehyde formed during the O-deethylation. The assay is useful for assessing CYP 1A2 enzymatic activity and CYP 1A2 inhibition or induction potential of drug candidates in order to exclude potent CYP inhibitors or inducers from further development.

(2) Description of Related Art

The pharmacokinetic and toxicokinetic properties of pharmaceuticals depend in great part on their biotransformation by drug metabolizing enzymes. The main drug metabolizing system in mammals is cytochrome P450 (CYP), a family of microsomal enzymes present predominantly in the liver. Multiple isoforms of CYP catalyze the oxidation of chemicals of endogenous and exogenous origin, including drugs, steroids, prostanoids, eicosanoids, fatty acids, and environmental toxins (Ioannides, In Cytochromes P450. Metabolic and Toxicological Aspects. CRC Press, Boca Raton. (1996)). If a drug that is metabolized by a particular CYP isozyme is co-administered with an inhibitor of that same enzyme, changes in its pharmacokinetics can occur, which can give rise to adverse effect (Bertz and Granneman, Clin. Pharmacokinet. 32: 210-258 (1997); Lin and Lu, Clin. Pharmacokinet. 35: 361-390 (1998); Thummel and Wilkinson, Ann. Rev. Pharmacol. Toxicol. 38: 389-430 (1998); von Moltke et ah, Biochem. Pharmacol. 55: 113-122 (1998)). It is therefore important to be able to predict and to prevent the occurrence of clearance changes due to metabolic inhibition. During the drug discovery process, it is routine practice in the pharmaceutical industry to assess CYP inhibition potential of drug candidates in order to exclude potent inhibitors from further development (Lin and Lu, ibid.

(1998); Crespi and Stresser, J. Pharmacol. Toxicol. Methods 44: 325-331 (2000); Bachmann and Ghosh, Curr. Drug Metab. 2: 299-314 (2001); Riley, Curr. Opin. Drug Disc. Dev. 4: 45-54 (2001)).

Many CYPs are also strongly inducible by xenobiotics, up to 50 to 100 fold. In drug therapy, there are two major concerns with respect to CYP induction. First, induction may cause a reduction in therapeutic efficacy by decreasing systemic exposure as a result of increased drug metabolism. Second, induction may create an undesirable imbalance between toxifϊcation and detoxification as a result of increased formation of reactive metabolites (Lin and Lu, Clin. Pharmacokinet. 35: 361-390 (1998)).

CYP1A2, a member of the Cytochrome P450 family, catalyses the metabolism of a number of clinically used drugs. CYP1A2 has also been found be induced in individuals consuming charred foods and in smokers which results in its ability to activate heterocyclic aromatic amines which are capable of acting as carcinogens or mutagens (Sesardic et al., Carcinogen. 11: 1183-1188 (1990)). Further, a number of compounds have been found to inhibit CYP 1A2 activity (Sesardic et al., Br. J. Clin. Pharmacol. 29:651-663 (1990); Brosen et al., Biochem. Pharmacol. 45: 1211-1214 (1993)). Therefore, there is considerable interest in drug development arenas for assays that can detect activation or inhibition of CYPl A2 activity. Thus, over the years, a number of assays that use human liver microsomes (HLM) have become available (Rodrigues Biochem. Pharmacol. 48: 2147-2156 (1994); Ramussen et al., Anal. Biochem. 222: 9-13 (1994); Bourrie et al., J. Pharmacol. Exp. Ther. 277: 321-332 (1996) Butler et al., Proc. Natl. Acad. Sci. USA 86: 7696-7700 (1989)). However, because many of these assays require use of HPLC, they are not particularly useful in a high throughput screening context. Fluorometric assays for CYP1A2 are available (See, for example, Moody et al., Xenobiotica 29: 53-75 (1999)); however, differences have been described in inhibitor potencies between fluorogenic assays vs. conventional CYP assays using classical drug substrates (Cohen et al., Drug Metab. Disp. 31: 1005-1015 (2003)). Moreover, assays using fluorogenic substrates suffer from the limitation that many test compounds can interfere with fluorescence readouts.

Rodrigues et al. (Drug Metab. Dispos. 25: 1097-1100 (1997) describe an assay that attempts to overcome the limitations of the previous assays. Rodrigues et al. describe a method for assessing the effect of a compound on CYP 1A2 activity in HLM by detecting the release of [14C]acetaldehyde which occurs upon CYPl A2-mediated deethylation of phenacetin labeled with 14c at the ethyl group ([O-ethyl-14C]phenacetin ) in the presence of the compound. The [14c]acetaldehyde is separated from unreacted [O-ethyl-14c]phenacetin by charcoal extraction.

Moody et al. (Xenobiotica 29: 53-75 (1999)) discloses an automated assay for high throughput screens for CYP2D6 inhibitors. The assay uses [O-methyl-14c]dextromethorphan as a substrate for the CYP2D6 and monitors demethylation of the substrate by the CYP2D6 to produce [14C]formaldehyde. The [14C] formaldehyde is separated from the substrate using SUPELCLEAN ENVI-CARB solid phase extraction columns (columns comprising a graphitized non-porous support available from Supelco, Inc., Bellefonte, PA). Di Marco et al., Eur. J. Biochem. 270: 3768-3777 (2003) disclose using OASIS 96-well plates for separating the [14C] formaldehyde from the substrate.

In light of the above, a non-HPLC assay for identifying modulators of CYP1A2 activity that could be adapted to high throughput screening format and which is based on use of a classical CYP 1A2 substrate such as phenacetin would be particularly desirable. Therefore, there remains a need for an assay for identifying CYP modulators that is based on using phenacetin as the substrate, is at least as sensitive and specific as the conventional assays, and is readily adaptable to a high throughput screening format. There is also a need for an assay for assessing CYP 1A2 activity in hepatocytes.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a rapid and sensitive radiometric assay for assessing the activity of cytochrome P-450 (CYP) 1A2 and the potential of an analyte to inhibit CYP 1A2 activity or induce CYP 1A2 expression. The assay uses phenacetin labeled with 14c at the ethyl group ([O-ethyl- 14c] phenacetin) as a substrate for CYP1A2 and detects CYP1A2 activity by measuring the release of [14C] radioactivity from the substrate, which occurs upon CYPlA2-mediated deethylation of the [O- ethyl-14c]phenacetin. CYP 1A2 activity is measured in the presence and absence of an analyte being tested for a modulating effect on CYPl A2 activity. An increase in the release of [14C] radioactivity over time in hepatocytes or the decrease in the release of [^C] radioactivity over time in reactions comprising CYPl A2 indicates that the analyte is a modulator of CYPl A2 activity or expression. The method further enables CYP 1A2 activity in hepatocyte preparations to be determined. The polar [14C] radioactivity products ([14c]acetaldehyde and/or [14c]acetic acid) are separated from the non-polar [O-ethyl- 14C]phenacetin in a solid-phase extraction process using a sorbent which preferentially binds non-polar compounds. All the steps of the assay, including incubations, product separation, and radioactivity counting are preferably performed in a multiwell format, which can be automated.

Therefore, in one embodiment, the present invention provides a method for identifying an analyte that inhibits activity of CYPl A2, which comprises providing a mixture comprising CYP 1A2, [O-ethyl-14C]phenacetin, NADPH, and the analyte; incubating the mixture for a time sufficient for the CYP1A2 to deethylate the [O-ethyl-14C]phenacetin; optionally removing the CYP1A2 from the mixture; applying the reaction mixture to a sorbent, which preferentially binds non-polar compounds, to remove any remaining [O-ethyl-14c]phenacetin from the mixture; and measuring [14C] radioactivity not bound to the sorbent (i.e., [^C] radioactivity in the mixture with the [O-ethyl-14c]phenacetin removed), wherein a decrease in the [^C] radioactivity in the mixture indicates that the analyte inhibits activity of the CYP 1A2. In a further aspect of the above embodiment, the sorbent comprises a water- wettable polymer formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon. In further aspects of the above embodiment, the lipophilic monomer comprises a phenyl, phenylene, ether, or C2-C18 alkyl group. In a further still aspect, the lipophilic monomer is divinylbenzene. In further aspects of the above embodiments, the hydrophilic monomer comprises a saturated, unsaturated, or aromatic heterocyclic group. In a further still aspect, the hydrophilic monomer is N-vinylpyrrolidone. In further still aspects of the above embodiment, the water wettable polymer is poly(vinylbenzene-co-N-vinylpyrτolidone, preferably, a polymer wherein the poly(vinylbenzene-co-N- vinylpyrrolidone comprises more than 12 mole percent N-vinylpyrrolidone, more preferably, a polymer wherein the poly(vinylbenzene-co-N-vinylpyrrolidone comprises from about 15 mole percent to about 30 mole percent N-vinylpyrrolidone.

In another aspect of the above embodiment, the sorbent comprises a non-polar group bonded to a silica substrate. In a further still aspect, the sorbent comprises one or more silanes selected from the group consisting phenyl silane, dimethylsilane, trimethylsilane, ethyl silane, butyl silane, hexyl silane, octyl silane, and octadecyl silane. In further still aspects, the silica substrate is selected from the group consisting of silica particles and silica gel.

In a further embodiment, the present invention provides a method for identifying an analyte that inhibits activity of CYPl A2, which comprises providing a mixture comprising CYP 1A2, [O- ethyl-14C]phenacetin, NADPH, optionally an NADPH regenerating system, and the analyte; incubating the mixture for a time sufficient for the CYP 1A2 activity to deethylate the [O-ethyI-14C]phenacetin; optionally removing the CYP 1A2 from the mixture; applying the mixture to a water- wettable polymer formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon to remove any remaining [O-ethyl-14c]phenacetin from the mixture; and, measuring [14C] radioactivity not bound to the sorbent (i.e., [14C] radioactivity in the mixture with the [O-ethyl-14C]phenacetin removed), wherein a decrease in the [14C] radioactivity in the mixture indicates that the analyte inhibits activity of the CYPlA2.

In further aspects of the above embodiment, the lipophilic monomer comprises a phenyl, phenylene, ether, or C2-C18 alkyl group. In a further still aspect, the lipophilic monomer is divinylbenzene.

In further aspects of the above embodiment, the hydrophilic monomer comprises a saturated, unsaturated, or aromatic heterocyclic group. In a further still aspect, the hydrophilic monomer is N-vinylpyrrolidone.

In further still aspects of the above embodiment, the water wettable polymer is poly(vinylbenzene-co-N-vinylpyrrolidone, preferably, a polymer wherein the poly(vinylbenzene-co-N- vinylpyrrolidone comprises more than 12 mole percent N-vinylpyrrolidone, more preferably, a polymer wherein the poly(vinylbenzene-co-N-vinylpyrrolidone comprises from about 15 mole percent to about 30 mole percent N-vinylpyrrolidone.

In a further embodiment, the present invention provides a method for identifying an analyte that inhibits activity of cytochrome CYP 1A2, which comprises providing a mixture comprising CYP 1A2, [O-ethyl-14c]phenacetin, NADPH, optionally an NADPH regenerating system, and the analyte; incubating the mixture for a time sufficient for the CYPl A2 activity to deethylate the [O-ethyl- 14C]phenacetin; optionally removing the CYP 1A2 from the mixture; applying the mixture to a water wettable polymer formed by copolymerizing divinylbenzene and N-vinylpyrrolidone at a ratio of divinylbenzene to N-vinylpyrrolidone such that the poly(vinylbenzene-co-N-vinylpyrrolidone formed is water-wettable and effective at retaining organic solutes thereon to remove the human liver microsomes and any remaining [O-ethyl-14C]phenacetin from the mixture; and measuring [14C] radioactivity not bound to the sorbent (i.e., [14C] radioactivity in the mixture with the [O-ethyl-14C]phenacetin removed), wherein a decrease in the [^C] radioactivity in the mixture indicates that the analyte inhibits activity of the CYPlA2.

In further aspects of the above embodiment, the poly(vinylbenzene-co-N- vinylpyrrolidone comprises more than 12 mole percent N-vinylpyrrolidone, more preferably, a polymer wherein the poly(vinylbenzene-co-N-vinylpyrrolidone comprises from about 15 mole percent to about 30 mole percent N-vinylpyrrolidone. In further still embodiments of the above, the sorbent or water wettable polymer is packed inside a solid phase extraction cartridge or column. In a particularly preferred embodiment of any one of the above, the method is performed in a multiwell plate format comprising a first multiwell plate for performing the incubation, a multicolumn plate in the same configuration as the multiwell plate for separating the [O-ethyl-14C]phenacetin from the [14c]acetaldehyde after the incubation, and a second multiwell plate for collecting the column void volume and washes from the multicolumn for determining the [14c]acetaldehyde therein.

During incubation of the above mixtures, the [O-ethyl-14c]phenacetin is deethylated to produce non-polar compound paracetamol and the polar compound [14c]acetaldehyde. The [14C]acetaldehyde can be further oxidized to the polar compound [14C]acetic acid. Therefore, in further still aspects of any one of the above embodiments or aspects, the [14C] radioactivity in the mixture after removing any remaining [O-ethyl-14c]phenacetin) can comprise [14C]acetaldehyde, [14c]acetic acid, or mixture thereof.

In further still aspects of any one of the above embodiments or aspects, the [14C] radioactivity in the mixture is compared to the amount [14C] radioactivity in the mixture from a control mixture comprising HLM, [O-ethyl-14c]phenacetin, and NADPH, and not the analyte.

The present invention further provides a method for identifying an analyte that inhibits activity of CYPl A2, which comprises providing a multiwell plate and a column plate having an array of solid phase extraction cartridges or columns having therein a sorbent which preferentially binds non- polar compounds; applying to each of the wells of the multiwell plate a mixture comprising CYP2A1, [O-ethyl-14C]phenacetin, and an analyte; contacting NADPH and optionally an NAPDH regenerating system to the mixture in each of the wells above and incubating for a time sufficient for the CYP 1A2 to deethylate the [O-ethyl-14C]phenacetin; optionally separating the CYP2A1 from the mixture in each of the wells of the multiwell plate; applying each mixture to a separate minicolumn of the column plate to remove any remaining [O-ethyl-14c]phenacetin from the mixture; and, measuring amount of [14c] radioactivity in the mixture with the [O-ethyl-14c]phenacetin removed wherein a decrease in the amount of the [14C] radioactivity indicates that the analyte inhibits activity of the CYPl A2.

In a further aspect of the above embodiment, the sorbent comprises a water- wettable polymer formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon. In further aspects of the above embodiment, the lipophilic monomer comprises a phenyl, phenylene, ether, or C2-C18 alkyl group. In a further still aspect, the lipophilic monomer is divinylbenzene. In further aspects of the above embodiments, the hydrophilic monomer comprises a saturated, unsaturated, or aromatic heterocyclic group. In a further still aspect, the hydrophilic monomer is N-vinylpyrrolidone. In further still aspects of the above embodiment, the water wettable polymer is poly(vinylbenzene-co-N-vinylpyrrolidone, preferably, a polymer wherein the poly(vinylbenzene-co-N- vinylpyrrolidone comprises more than 12 mole percent N-vinylpyrrolidone, more preferably, a polymer wherein the poly(vinylbenzene-co-N-vinylpyrrolidone comprises from about 15 mole percent to about 30 mole percent N-vinylpyrrolidone.

In a further embodiment, the present invention provides a method for identifying an analyte that inhibits activity of CYP1A2, which comprises providing a multiwell plate and a column plate having an array of solid phase extraction cartridges or columns having therein a water wettable polymer formed by copolymerizing divinylbenzene and N-vinylpyrrolidone at a ratio of divinylbenzene to N- vinylpyrrolidone such that the poly(vinylbenzene-co-N-vinylpyrrolidone formed is water-wettable and effective at retaining organic solutes thereon; applying to each of the wells of the multiwell plate a mixture comprising CYPl A2, [O-ethyl-14c]phenacetin, and an analyte; contacting NADPH and optionally an NAPDH regenerating system to the mixture in each of the wells and incubating for a time sufficient for the CYP 1A2 to deethylate the [O-ethyl-14c]phenacetin; optionally separating the CYP 1A2 from the mixture in each of the wells of the multiwell plate; applying each mixture to a separate minicolumn of the column plate to remove the [O-ethyl-14c]phenacetin from the mixture; and, measuring amount of [14C] radioactivity in the mixture with the [O-ethyl-14c]phenacetin removed wherein a decrease in the amount of the [1 ^C] radioactivity in the presence of the analyte indicates that the analyte inhibits activity of the CYP1A2.

In further aspects of the above embodiment, the poly(vinylbenzene-co-N- vinylpyrrolidone comprises more than 12 mole percent N-vinylpyrrolidone, more preferably, a polymer wherein the polyCvinylbenzene-co-N-vinylpyrrolidone comprises from about 15 mole percent to about 30 mole percent N-vinylpyrrolidone.

The present invention further provides a method for identifying an analyte that inhibits activity of cytochrome CYP 1A2, which comprises providing a multiwell plate and a column plate having an array of solid phase extraction cartridges or columns having therein a water-wettable polymer formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon; applying to each of the wells of the multiwell plate a mixture comprising CYP 1A2, [O-ethyl- 14C]phenacetin, and an analyte; contacting NADPH and optionally an NAPDH regenerating system to the mixture in each of the wells above and incubating for a time sufficient for the CYP 1A2 to deethylate the [O-ethyl-14c]phenacetin; optionally separating the CYP1A2 from the mixture in each of the wells of the multiwell plate; applying each mixture to a separate minicolumn of the column plate to remove any remaining [O-ethyl-14C]phenacetin from the mixture; and, measuring amount of the [^C] radioactivity in the mixture with the [O-ethyl-14C]phenacetin removed wherein a decrease in the amount of the [^C] radioactivity indicates that the analyte inhibits activity of the CYPl A2. In further aspects of the above embodiment, the lipophilic monomer comprises a phenyl, phenylene, ether, or C2-C18 alkyl group. In a further still aspect, the lipophilic monomer is divinylbenzene.

In further still aspects of any one of the above embodiments and aspects, each of the minicolumns of the column plate further comprises a porous retaining means for retaining the polymer therein. In a preferred embodiment, the wells of the multiwell plate and column plate each have a 96- well tissue culture plate format. In further still aspects of any one of the above embodiments or aspects, the [^C] radioactivity in the mixture after removing any remaining [O-ethyl-14C]phenacetin) can comprise [14c]acetaldehyde, [^C] acetic acid, or mixture thereof.

In further still aspects of any one of the above embodiments or aspects, the [^C] radioactivity in the mixture is compared to the amount [14c] radioactivity in the mixture from a control mixture comprising HLM, [O-ethyl-14c]phenacetin, and NADPH, and not the analyte.

In particular embodiments of any one of the above embodiments and aspects, the CYP2C8 is provided in microsomes. The microsomes can be produced from cells selected from the group consisting of mammalian and insect cells, wherein the cells include a vector (e.g., viral or plasmid vectors) expressing the CYP2C8 or the microsomes can be from kidney, liver, brain, muscle, or the like cells. Preferably, the microsomes are human liver microsomes (HLM). In particular embodiments of any one of the above embodiments and aspects which use HLM as the source for CYP2C8, the HLM are removed from the aqueous mixture by acidification and/or centrifugation.

In a further embodiment, the present invention provides a method for determining the activity of CYPl A2 in hepatocytes, which comprises providing a culture of the hepatocytes; incubating the hepatocytes in a medium comprising [O-ethyl-14C]phenacetin for a time sufficient for the CYP 1A2 to deethylate the [O-ethyl-14c]phenacetin; removing the medium from the culture of hepatocytes; applying the medium to a sorbent which preferentially binds non-polar compounds to remove any remaining [O-ethyl-14c]phenacetin from the medium; and measuring amount of the [1 ^C] radioactivity not bound to the sorbent (i.e., [14c] radioactivity in the mixture with the [O-ethyl-14C]phenacetin removed), which determines the relative activity of the CYP 1A2 in the hepatocytes.

In a further still embodiment, the present invention provides a method for identifying an analyte that induces CYPl A2 expression, which comprises providing a culture of hepatocytes; incubating the hepatocytes in a medium comprising the analyte; replacing the medium comprising the analyte with a second medium comprising [O-ethyl-14C]phenacetin and incubating the hepatocytes for a time sufficient for the CYP1A2 to deethylate the [O-ethyl-14c]phenacetin; removing the second medium from the culture of hepatocytes; applying the second medium to a sorbent, which preferentially binds non-polar compounds, to remove any remaining [O-ethyl-14c]phenacetin from the second medium; and measuring amount of [14C] radioactivity not bound to the sorbent (i.e., [14C] radioactivity in the second medium with the [O-ethyl-14C]phenacetin removed) wherein an increase in the amount of the [14c] radioactivity indicates that the analyte induces CYP 1A2 expression. Preferably, the hepatocytes are incubated in the medium comprising the analyte for between about 24 to 78 hours.

In a further embodiment, the present invention provides a method for identifying an analyte that inhibits CYP 1A2 activity, which comprises providing a culture of hepatocytes; incubating the hepatocytes in a medium comprising [O-ethyl-14c]phenacetin and the analyte for a time sufficient for the CYPl A2 to deethylate the [O-ethyl-14c]phenacetin; removing the medium from the culture of hepatocytes; applying the medium to a sorbent, which preferentially binds non-polar compounds, to remove any remaining [O-ethyl-14c]phenacetin from the medium; and measuring amount of the [^C] radioactivity not bound to the sorbent (i.e., [^C] radioactivity in the medium with the [O-ethyl- 14C]phenacetin removed) wherein a decrease in the amount of [14c]acetaldehyde indicates that the analyte inhibits the CYP 1A2 activity.

In a further aspect of the above embodiments, the culture of hepatocytes is provided in one or more wells of a multiwell plate and the sorbent is provided packed in one or more solid phase extraction cartridges or columns comprising a column plate. In a further aspect of the above embodiments, the sorbent comprises a water-wettable polymer formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon. In further aspects of the above embodiment, the lipophilic monomer comprises a phenyl, phenylene, ether, or C2-C18 alkyl group. In a further still aspect, the lipophilic monomer is divinylbenzene. In further aspects of the above embodiments, the hydrophilic monomer comprises a saturated, unsaturated, or aromatic heterocyclic group. In a further still aspect, the hydrophilic monomer is N-vinylpyrrolidone. In further still aspects of the above embodiment, the water wettable polymer is poly(vinylbenzene-co-N-vinylpyrrolidone, preferably, a polymer wherein the poly(vinylbenzene-co-N- vinylpyrrolidone comprises more than 12 mole percent N-vinylpyrrolidone, more preferably, a polymer wherein the poly(vinylbenzene-co-N-vinylpyrrolidone comprises from about 15 mole percent to about 30 mole percent N-vinylpyrrolidone.

In another aspect of the above embodiments, the sorbent comprises a non-polar group bonded to a silica substrate. In a further still aspect, the sorbent comprises one or more silanes selected from the group consisting phenyl silane, dimethylsilane, trimethylsilane, ethyl silane, butyl silane, hexyl silane, octyl silane, and octadecyl silane. In further still aspects, the silica substrate is selected from the group consisting of silica particles and silica gel.

In further still aspects of any one of the above embodiments or aspects, the [^C] radioactivity in the mixture after removing any remaining [O-ethyl-14c]phenacetin) can comprise [14c]acetaldehyde, [^C] acetic acid, or mixture thereof. In further still aspects of any one of the above embodiments or aspects, the [1 ^C] radioactivity in the medium is compared to the amount [l^C] radioactivity in the medium from a control culture of hepatocytes incubated with the [O-ethyl-14C]phenacetin and without the analyte.

As used herein, the term "analyte" refers to molecules, compounds, chemicals, compositions, drugs, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a cross-sectional view of an extraction cartridge or column 10.

Figure 2 shows a perspective view of a multicolumn microfϊltration/extraction plate 100.

Figure 3 shows the time-dependent formation of [14c]acetaldehyde from [O-ethyl- 14C]phenacetin in HLM.

Figure 4 shows the effect of furafylline on formation of [14C]acetaldehyde from [O- ethyl-14c]phenacetin in HLM.

Figure 5 shows the correlation between IC50 values from the assay of the present invention versus IC50 values from a conventional LC-MS/MS assay. Figure 6 shows a comparison between IC50 values of CYP inhibitors in radiometric assay (formation of [14c]-labeled reaction product) vs. conventional LC-MS/MS assay (formation of paracetamol).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a rapid and sensitive phenacetin deethylation assay for assessing cytochrome P-450 isoform 1A2 (CYP1A2) activity and for identifying modulators of CYP1A2 activity or expression. In particular, the present invention provides an assay for assessing the activity of CYP1A2 in mixtures comprising CYP1A2 or hepatocytes, the potential of an analyte to inhibit CYP1A2 activity in mixtures comprising CYP 1A2 or hepatocytes, and the potential of an analyte to induce CYP 1A2 expression in hepatocytes. The assays include both reversible inhibition assays and mechanism-based or time-dependent inhibition assays. Examples of mixtures comprising CYP 1A2 include microsomes from various tissues such as human liver microsomes (HLM); microsomes from mammalian or insect cells containing an expression vector which expresses recombinant CYP2C9; or hepatocytes, the potential of an analyte to inhibit CYP 1A2 activity in any of the above mixtures, and the potential of an analyte to induce CYP 1A2 expression in hepatocytes. Preferably, the CYP 1A2 is a human CYP1A2. The assay is based on detecting the release of [14c]acetaldehyde which occurs upon CYPlA2-mediated deethylation of phenacetin labeled in the O-ethyl group with 14c in the presence of the analyte wherein an increase or decrease in the release of [14C]acetaldehyde over time indicates that the analyte is a modulator of CYPl A2 activity. For example, a decrease in the release of [14C]acetaldehyde in HLM's in the presence of an analyte indicates that the analyte is an inhibitor of CYPl A2 activity whereas an increase in the release of [14C]acetaldehyde in hepatocytes after treatment of the hepatocytes with the an analyte indicates that the analyte is an inducer of CYPl A2 activity. The [14c]acetaldehyde water soluble product is separated from the [O-ethyl- 14c]phenacetin in a solid-phase extraction process using a sorbent a sorbent comprising a substrate which preferentially binds non-polar compounds such as phenacetin. All the steps of the assay, including incubations, product separation, and radioactivity counting are performed in a multiwell format, which can be automated.

Oxidation of [14C]acetaldehyde to [14c]acetic acid and [14C]CO2 can occur in the assays using hepatocytes. Oxidation of the [14C]acetaldehyde to [14C]acetic acid may also occur in the assays using HLM's. The [14C]acetic acid, like the [14C]acetaldehyde, is water soluble and separable from the [O-ethyl-14C]phenacetin using the solid phase extraction process disclosed herein. Because oxidation of [14c]acetaldehyde to [14c]acetic acid can occur in hepatocytes and HLM's, the [14C]acetaldehyde and [14C] acetic acid are both separable from the substrate, and the assay measures the amount of [14C]acetaldehyde produced by measuring water soluble radioactivity produced in the assay, the assays disclosed herein detect not only the [14c]acetaldehyde that is produced but also any [14C]acetic acid that might also be produced via oxidation of the [14c]acetaldehyde.

The embodiment for identifying analytes that induce or inhibit CYP 1A2 activity using hepatocytes in one aspect identifies analytes that inhibit or induce expression of the gene encoding CYP1A2, i.e., analytes which affect transcription of the gene encoding CYP1A2. The embodiment in another aspect identifies analytes that exert their inhibitory or inducing effect on CYP 1A2 activity by affecting posttranscriptional processing of mRNA encoding the CYP 1A2. The embodiment in a further aspect identifies analytes that exert their inhibitory or inducing effect on CYPlA2activity by affecting translation of the mRNA encoding the CYPl A2. The embodiment in a further still aspect identifies analytes that exert their inhibitory or inducing effect on CYP 1A2 activity by interacting directly or indirectly with the CYP 1A2.

The embodiment for assessing CYPlA2activity is useful for controlling the activity of commercial batches of hepatocytes or the quality of hepatocytes isolated in house, for instance, before using these hepatocytes to perform metabolic stability studies with new chemical entities. The embodiment for identifying CYP1A2 modulators is useful for assessing the CYPl A2inhibition or induction potential of drug candidates in order to exclude drug candidates that are potent inhibitors or inducers from further development. In either embodiment, the present invention is an improvement over assays of the prior art which rely on HPLC separation and mass spectrometry to assess the CYP 1A2 inhibition or induction potential of an analyte.

While the assays are described herein using HLM or hepatocytes, the assays can use purified recombinant CYP 1A2 or microsomes prepared from other tissues, for example, kidney, intestine, lung, or the like, or other subcellular fractions containing microsomes. The microsomes can be prepared from mammalian cells containing a plasmid or viral vector that expresses CYPl A2, preferably, a human CYPl A2. The microsomes can be from insect cells infected with recombinant baculovirus expressing CYP1A2 and a p450 reductase. The advantage of the cells expressing recombinant CYP1A2 is that CYP 1A2 is the only cytochrome P450 present in these microsomes and the specific activity is generally higher. The concentration range for assays using recombinant CYP 1A2 is from about 1 to 100 pmol/mL, preferred concentrations are between about 5 to 50 pmol/mL. For time-dependent assays, the enzyme should be 5- 10-fold higher (because of the final dilution in the second incubation).

To test an analyte for inhibition of CYPl A2 activity, a first container is provided which contains an aqueous mixture comprising the analyte to be tested for an inhibitory effect on CYP 1A2 activity, [O-ethyl-14c]phenacetin as the substrate probe, pooled HLM, and a buffer at a physiological pH. Typically, between about 10,000 to 1,000,000 dpm of [O-ethyl-14c]phenacetin is used, preferably, the labeled phenacetin is at about 100,000 dpm. The concentration of phenacetin is between about 1 to 100 μM, typically at about 10 μM. The pooled HLM are generally at about 0.05 to 1 mg/mL, typically, about 0.5 mg/mL. An example of a suitable buffer is 0.1 M potassium phosphate, pH 7.6). The final volume is preferably between about 100 μL to 200 μL. Preferably, a control containing an equivalent amount of the vehicle used for the analyte is provided.

Synthesis and purification of [O-ethyl-14C]phenacetin has been described in Rodrigues et al., Drug metab. Disp. 25: 10971100 (2005) and Kurumaya et al., Chem. Pham Bull. (Tokyo) 36: 2679-2681 (1988) and is shown in Example 1.

Following a preferred preincubation step of microsomes in buffer for several minutes at 37°C, about 1 mM NADPH with or without an NADPH regenerating system comprising about 5 mM glucose-6-phosphate, about 3 mM MgCl2, and about 1 unit/mL glucose-6-phosphate dehydrogenase is added to the aqueous mixture to form a reaction mixture which is then incubated at 37°C for a period of time sufficient to allow deethylation of the labeled phenacetin. In general, about 10 to 20 minutes is usually sufficient to detect CYP 1A2 activity. In some cases, a multiplicity of assays are performed for various lengths of time. The reaction mixture is then stopped by addition of an acid such as HCl at a concentration of about 0.1 N. Preferably, the HLM are removed from the aqueous mixture before transferring the reaction mixture to an extraction cartridge or column for separating [14c]acetaldehyde (and any [14C]acetic acid produced from oxidation of the [14c]acetaldehyde) from the [O-ethyl-

14C]phenacetin. The HLM can be removed from the aqueous layer by filtration, centrifugation, or the like. In a preferred embodiment, the HLM are removed by centrifugation. Because the acidification of the reaction causes the proteins in the HLM to precipitate, the proteins of the HLM can be removed using low speed centrifugation. The aqueous mixture with the HLM removed or the reaction mixture containing the

HLM is transferred to an extraction cartridge or column containing a sorbent which preferentially binds non-polar compounds such as phenacetin and any remaining [O-ethyl-14c]phenacetin. The aqueous void volume or flow-through from the column is collected in a second container. The sorbent in the column is washed with water and the washes transferred to the second container. Scintillation fluid is added to the second container containing the aqueous void volume and washes and the radioactivity of the [14C]acetaldehyde released from the [O-ethyl-l⁴C]phenacetin by CYP1A2 (and any [J4c]acetic acid produced from oxidation of the [14C]acetaldehyde) is measured. Alternatively, the void volume or flow- through and washes are transferred to a scintillation vial and mixed with scintillation fluid for measuring the 14c radioactivity in a scintillation counter. The absence of 14c radioactivity or reduced amounts of 14c radioactivity compared to the amounts of [14C]acetaldehyde in the positive controls indicate that the analyte is an inhibitor of CYPl A2 activity.

The CYP 1A2 activity of a preparation of hepatocytes from liver tissue is determined as follows. Primary cultures of hepatocytes, which can comprise hepatocytes freshly isolated from liver tissue or which had been isolated previously, frozen for storage, and thawed for the assay, are provided. The hepatocytes are maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air or oxygen in a culture medium or aqueous mixture suitable for culturing hepatocytes (See for example, Dich and Grunnet in Methods in Molecular Biology, Vol. 5: Animal Cell Culture (Pollard, and Walker, eds) pp. 161-176, Humana Press, Clifton, New Jersey. (1989). The assay can be performed using either cells in suspension or cultured cells attached to cell culture plates. For suspension assays, typically, the hepatocytes are incubated at a concentration of about 1 x 10$ cells/mL to 1 x Iθ6 cells/mL, preferably 1 x 106 cells/mL. Thus, each culture well contains about 1 x Iθ6 cells, 1 mL of hepatocyte culture medium (HCM) (Dich and Grunnet, ibid.), and [O-ethyl-14C]phenacetin. Typically, between about 100,000 to 1,000,000 dpm of [O-ethyl-14C]phenacetin is used. The concentration of phenacetin is between about 1 to 200 μM, typically at about 10 μM. For assays in plated cells, the hepatocytes are plated onto tissue culture plates (preferably, the culture plates are collagen-coated 24- or 96-well tissue culture plates) and maintained at 37°C in a humidified atmosphere of 5% CO2 in a culture medium suitable for culturing fresh hepatocytes, e.g., HCM. Preferably, the medium is supplemented with ITS (insulin-transferrin-selenium mixture). Typically, the hepatocytes are plated at a density of about 150,000 to 200,000 cells/cm2. Following the incubation, the incubation medium is removed from the cells, for instance by centrifugation, and transferred to an extraction cartridge or column containing a sorbent which preferentially binds non-polar compounds such as phenacetin. The void volume or flow-through from the column is collected in a second container. The sorbent in the column is washed several times with water and the washes transferred to the second container. Scintillation fluid is added to the second container and the radioactivity of the [14C]acetaldehyde released from the [O-ethyl-14c] phenacetin by CYP1A2 (and any [14C]acetic acid produced from oxidation of the [14C]acetaldehyde) is measured. Alternatively, the void volume or flow-through and washes are transferred to a scintillation vial and mixed with scintillation fluid for measuring the 14c radioactivity in a scintillation counter. The amounts of 14c radioactivity produced determines the relative CYP 1A2 activity of the hepatocytes. The assay for determining the ability of an analyte to inhibit CYP 1A2 activity in hepatocytes is as follows. Primary cultures of hepatocytes, which can comprise hepatocytes freshly isolated from liver tissue or which had been isolated previously, frozen for storage, and thawed for the assay, are provided. The assay can be performed using either cells in suspension or cultured cells attached to cell culture plates. For suspension assays, the hepatocytes are maintained at 37°C in a humidified atmosphere of 5% CO2 in a culture medium suitable for culturing hepatocytes as above.

Typically, the hepatocytes are incubated at a concentration of about 1 x Iθ6 cells/mL. For non- suspension assays, the hepatocytes are plated to collagen-coated plates and maintained at 37°C in a humidified atmosphere of 5% CO2 in a culture medium suitable for culturing hepatocytes, e.g., HCM. Thus, each culture well contains about 2 x 10^ cells, 0.2 mL of HCM, the analyte being tested for inhibitory effect on CYP1A2 activity, and [O-ethyl-14c]phenacetin. Typically, between about 100,000 to 1,000,000 dpm of [O-ethyl-14c]phenacetin is used. The concentration of phenacetin is between about 1 to 200 μM, typically at about 10 μM. Preferably, controls that include the vehicle for the analyte or a CYP 1A2 inhibitor such as furafylline provided. Following the incubation, the incubation medium is removed from the cells and transferred to an extraction cartridge or column containing a sorbent which preferentially binds non-polar compounds such as phenacetin. The aqueous void volume or flow-through from the column is collected in a second container. The sorbent in the column is washed several times with water and the washes transferred to the second container. Scintillation fluid is added to the second container and the radioactivity of the [14C]acetaldehyde released from the [O-ethyl-l⁴C]phenacetin by CYP1A2 (and any [14c]acetic acid produced from oxidation of the [14c]acetaldehyde) is measured. Alternatively, the void volume or flow-through and washes are transferred to a scintillation vial and mixed with scintillation fluid for measuring the 14c radioactivity in a scintillation counter. The absence of 14c radioactivity or reduced amounts of 14c radioactivity compared to the amounts of 14c radioactivity in the control comprising the vehicle only indicates that the analyte is an inhibitor of CYP1A2 activity.

The assay for determining the ability of an analyte to induce CYP 1A2 activity is as follows. Primary cultures of hepatocytes, which can comprise hepatocytes freshly isolated from liver tissue or which had been isolated previously, frozen for storage, and thawed for the assay, are provided. The hepatocytes are plated onto tissue culture plates (preferably, the culture plates are collagen-coated 24- or 96-well tissue culture plates) and maintained at 37°C in a humidified atmosphere of 5% CO2 in a culture medium suitable for culturing fresh hepatocytes, e.g., HCM. Preferably, the medium is supplemented with ITS. Typically, the hepatocytes are plated at a density of about 150,000 to 200,000 cells/cm2. Twenty-four to 78 hours later, the culture medium is removed and fresh medium and the analyte to be tested for induction potential are added to the hepatocytes. Preferably, controls are provided which comprise either the vehicle for the analyte or a known inducer such as methylcholanthrene and omeprazol. After incubating the hepatocytes as above for time sufficient for induction of CYP1A2, usually between about 24 to 78 hours, CYP1A2 enzyme activity is determined.

The hepatocytes are incubated in an incubation medium containing a balanced salt solution containing a buffer at physiological pH, for example, pH 7.4. An example of a balanced salt solution is Hank's balanced salt solution and an example of a suitable buffer is 10 mM HEPES. Then a mixture containing [O-ethyl-14C]phenacetin is added and the hepatocytes incubated as above for a suitable time to assess CYP 1A2 activity, about an hour is usually sufficient. Typically, between about 100,000 to 1,000,000 dpm/mL of [O-ethyl-14c]phenacetin is used, preferably, the [O-ethyl- 14C]phenacetin is at about 1,000,000 dpm/mL. The concentration of phenacetin is between about 1 to 300 μM, typically at about 10 μM. Optionally, parallel incubations are performed, which contain a

CYP 1A2 inhibitor such as furafylline, to ascertain that detected enzyme activity is specifically mediated by CYPlA2.

Following the incubation, the incubation medium is removed from the cells and transferred to an extraction cartridge or column containing a sorbent which preferentially binds non-polar compounds such as phenacetin. The aqueous void volume or flow-through from the column is collected in a second container. The sorbent in the column is washed with water and the washes transferred to the second container. Scintillation fluid is added to the second container and the radioactivity of the [14c]acetaldehyde released from the [O-ethyl-14c]phenacetin by CYP1A2 (and any [14c]acetic acid produced from oxidation of the [14C]acetaldehyde) is measured. Alternatively, the void volume or flow- through and washes are transferred to a scintillation vial and mixed with scintillation fluid for measuring 14c radioactivity in a scintillation counter. The presence of 14c radioactivity or increased amounts of 14c radioactivity compared to the amounts of 14c radioactivity in the control with the vehicle only indicates that the analyte is an inducer of CYPl A2 activity.

As discussed below and shown in Example 2, in a preferred aspect of the present invention, the assay is performed in a multiwell format, preferably, a 96-well format. The multiwell format enables a plurality of analytes to be tested simultaneously. In the multiwell format, each reaction is conducted in the well of a multiwell plate (first container). The separation of [14c]acetaldehyde from [O-ethyl-14C]phenacetin at the conclusion of the reaction and following the optional step of removing the HLM is performed by applying each reaction to a separate column of a microfiltration/extraction column plate comprising a plurality of miniature columns, each containing the sorbent disclosed herein. Preferably, the columns of the microfiltration/extraction column plate are arranged in the same format as the format for the multiwell plate. The void volume and washes are collected in a second multiwell plate in the same format as the microfiltration/extraction column plate, mixed with scintillation fluid, and counted in a scintillation counter adapted for counting samples in a multiwell format. The sorbent preferentially binds non-polar compounds such as phenacetin, i.e., the sorbent can adsorb or bind the labeled phenacetin but not the labeled acetaldehyde produced by the deethylation or acetic acid produced by further oxidation of acetaldehyde. Sorbents which preferentially bind non-polar compounds such as phenacetin include, but are not limited to, sorbents comprising a hydrophobic or lipophilic polymer such as polystrene-divinylbenzene or poly(divinyl-benzene- vinylpyrrolidone), water-wettable polymers comprising lipophilic and hydrophilic monomers in a ratio that enables the sorbent to bind the labeled phenacetin but not labeled acetaldehyde ,and silicon-based sorbents such as the C2-C18 silanes.

The sorbent comprising a water-wettable polymer is formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon. The lipophilic monomer can comprise a lipophilic moiety such as phenyl, phenylene, and C2-Ci8-alkyl groups. Particularly useful lipophilic monomers include divinylbenzene and styrene. The hydrophilic monomer can comprise a hydrophilic moiety such as a saturated, unsaturated, or aromatic heterocyclic groups, for example, a pyrrolidonyl group or a pyridyl group. Alternatively, the hydrophilic group can be an ether group. Particularly useful monomers include N-vinylpyrrolidone, 2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, and ethylene oxide. In one embodiment of the water-wettable polymer, the polymer is a poly(divinylbenzene-co-N- vinylpyrrolidone) copolymer comprising greater than about 12 mole percent N-vinylpyrrolidone, preferably, from about 15 mole percent to about 30 mole percent N-vinylpyrrolidone. Examples of preferred water wettable polymers are disclosed in WO9738774 and U.S. Patent No. 6,726,842, both to Bouvier et al. A preferred sorbent is the OASIS HLB sorbent,, which comprises a balanced ratio of N- vinylpyrrolidone and divinylbenzene monomers, and is commercially available from Waters Corporation (Newcastle, DE).

Sorbents comprising a silicon-based substrate or matrix include a non-polar group bonded to a silica substrate. The sorbent can comprise one or more silanes well known in the art for extracting non-polar compounds. Such sorbents include, but are not limited to, phenyl silane, butyldimethyl silane, dimethylsilane, trimethylsilane, ethyl silane, butyl silane, hexyl silane, octyl silane, or octadecyl silane. The silanes can be monofunctional or trifunctional. The silica substrate or matrix includes, but is not limited to, solid or porous silica or ceramic particles or microparticles or silica gel. In a preferred embodiment of the method, the sorbent is provided as particles, beads, or the like are packed within an open-ended container to form a solid phase extraction cartridge or column. In particular embodiments of the method, the sorbent is packed into the solid phase extraction cartridge or column enmeshed in a porous membrane. In other embodiments, the solid phase extraction cartridge or column further includes a porous retaining means, such as a filter element, or frit at or near one or both ends of the solid phase extraction cartridge or column adjacent to the sorbent. The porous retaining means is to retain the sorbent within the solid phase extraction cartridge or column. In a further embodiment, the sorbent is disposed between a pair of porous retaining means, the first porous retaining means to retain the sorbent within the solid phase extraction cartridge or column and the second retaining means also aids in retaining the sorbent within the column and to prevent solid materials such as HLM from mixing with the sorbent. The filter or frit can be, for example, fritted glass, or a porous polymer such as high density polyethylene, TEFLON (E.I du Pont de Nemours and Company, DE), or polycarbonate.

Figure 1 shows a cross-sectional view of an example of a solid phase extraction cartridge or column 10 which is suitable for practicing the method of the present invention. The column 10 comprises an elongated body 12 having wall 14, which defines an axial hollow portion 16, an inlet 18 at the distal end 20 of the column 10 for receiving an aqueous mixture, and outlet 22 at the proximal end 24 of the column 10 for exit of the aqueous mixture. As further shown in Figure 1, adjacent to the proximal end 24 is a porous retaining means 26 which has surface 28. The porous retaining means 26 is positioned adjacent to the proximal end 24 in column 10 so that surface 28 is perpendicular to wall 14 of column 10. Disposed on surface 28 of the porous retaining means 26 is sorbent 30. Optionally, as shown, a second porous retaining means 32 can be positioned adjacent to or near the distal end 20 and the sorbent 30 disposed therebetween. The column 10 enables the aqueous mixture to enter the container through the inlet 18, contact the sorbent 30 within the column 10, and exit the column 10 through the outlet 22. Preferably, the sorbent 30 is packed in the column 10 as small particles such as beads having a diameter preferably between about 30 to 60 μm.

In a preferred embodiment, a multiplicity of the columns 10 are arranged to provide a format which is particularly suitable for high throughput screening. For example, a multicolumn microfiltration/extraction column plate comprising a multiplicity of wells adapted to provide solid phase extraction cartridges or columns (preferably, miniature solid phase extraction cartridges or columns, i.e., minicolumns). A preferred multicolumn microfiltration/extraction column plate format has the minicolumns arranged in a format that corresponds to the format used for multiwell tissue culture plates. For example, the minicolumns of the microfiltration/extraction column plate can be arranged in a 6-well, 12-well, 24-well, 48-well, 96-well, or 384-well format. In a preferred embodiment, the multicolumn microfiltration/extraction column plate has the minicolumns arranged in a 96-well format. As an example, Figure 2 shows a multicolumn microfiltration/extraction plate 100 comprising a multiplicity of minicolumns 102 with opening 104 for receiving an aqueous mixture and outlet 106 for exit of the aqueous mixture wherein each of the minicolumns 102 comprises an internal arrangement similar to that shown for column 10 of Figure 2 arrayed in a 96-minicolumn format. Movement of the aqueous mixture through the column and into a collecting plate containing wells arranged in a 96-well format can be achieved by centrifugation or by vacuum. Multi-column microfilitration/extraction column plates and methods and apparatus for using the plates have been disclosed in a number of U.S. Patents, for example, U.S. Patent No. 6,506,343 to Bodner et al, U.S. Patent No. 6,491,873 to Roberts and Woelk, and U.S. Patent No. 6338802 to Bodner et al, and U.S. Published Patent Application No. 20030143124 to Roberts and Grenz. In addition to reversible inhibition of CYP, irreversible or quasi-irreversible inactivation by certain analytes or their CYP-generated metabolites can occur. This type of inhibition, termed mechanism-based or time-dependent inhibition (MBI), is characterized by a progressive time-dependent decrease in enzyme activity in the presence of inhibitor. Three types of mechanism-based (time- dependent) inactivation of CYP have been reported: (i) inhibitor covalently binds to enzyme apoprotein; (ii) inhibitor covalently binds to prosthetic heme; (iii) inhibitor tightly (quasi-irreversibly) binds to heme or apoprotein. Most human hepatic drug-metabolizing CYPs, including CYP3A4/5, CYP2C9, CYP 1A2, CYP2D6, CYP2C19, CYP2A6, CYP2B6 and CYP2E1 are subject to mechanism-based inhibition (MBI) (Zhang and Wong, Curr. Drug Metab. 6: 241-257 (2005); Venkatakrishnan et al., Curr. Drug Metab. 4: 423-459 (2003); Zhou et al, Curr. Drug Metab. 5: 415-442 (2004); Zhou et al, Clin. Pharmacokinet. 44: 279-304 (2005)).

In contrast to reversible CYP inhibition, whose effects are not always manifest in vivo, MBI almost invariably leads to clinically relevant drug-drug interactions. Indeed, it is currently thought that MBI might be one of the major causes for clinical drug-drug interactions, which has been potentially overlooked in the past. Since MBI leads to a time-dependent loss of active enzyme, the clinical effects of a time- dependent CYP inhibitor on the pharmacokinetics of a drug that is metabolized by the same CYP is as follows:

• MBI causes non-stationary PK upon multiple dosing

• The extent of drug-drug interaction is time-dependent in onset and offset • High concentrations of inhibitor in intestinal lumen will cause significant effects on substrates whose oral bioavailability is limited by intestinal metabolism.

Therefore, the present invention also provides mechanism-based or time-dependent assays in addition to the reversible or quasi-reversible assays described above. To assess the potential of a compound to act as a time-dependent CYP inhibitor, the analyte is preincubated with CYP 1A2 in the presence of an NADPH regenerating system for a series of different lengths of time (typically from 0 minutes to 60 minutes). In general, CYPl A2 is provided at an amount about 5 to 10 times greater than the amount used in the reversible inhibition assays. Control incubations are performed in the absence of inhibitor to monitor for losses in enzyme activity due to thermal instability. At the end of the preincubation, the change in the amount of enzymatically active CYP relative to the time 0 preincubation time control is determined. This is achieved by performing a second incubation in which the preincubation is diluted about 10-fold and substrate is added. Enzyme activity is determined by measuring the amount of product formed during a specified time interval. Typical substrates used for time-dependent CYP inhibition assays are the same as those used for reversible inhibition assays above. For example, the K_m for CYP 1A2 with phenacetin is about 50 μM and the preferred concentration of testosterone is between about 250 to 500 uM. Example 5 provides an example of a time dependent assay using HLM.

In order to minimize any reversible CYP inhibition effect caused by the test analyte in the second incubation, the preincubation mixture is diluted several-fold (typically 5-20 times), the CYP substrate is added at a concentration several times (typically 5-10 times) higher than the concentration required for half-maximal activity (to minimize competitive inhibition by test compound), and the incubation time is short (typically 10 min). If an analyte acts as a time-dependent inhibitor, preincubation with CYP will cause a loss of enzyme activity with pseudo-first order kinetics. For each inhibitor concentration, the percentage of remaining enzyme activity (relative to a control without inhibitor) will change with time according to the equation:

% of remaining enzyme activity = 100 x e ^{('k x x)} _eαUation 1

where k is the observed pseudo-first order inactivation rate constant, which is related to the inhibitor concentration during preincubation according to the following relationship:

k = "inact

K ⁰⁵ " +1" equation 2

where I is the inhibitor concentration, k_mact is the maximal inactivation rate constant, Ko.5 is the inhibitor concentration at 50% k_mact, and n is the Hill coefficient. To determine k_mact and Ko.5, the curve of k versus I is fitted to equation 2 using non-linear regression analysis.

The following example is intended to promote a further understanding of the present invention.

EXAMPLE l Synthesis and purification of [O-ethyl-14c]phenacetin was made as follows according to the procedure of Kurumaya et ah, Chem. Pham Bull. (Tokyo) 36: 2679-2681 (1988).

In a 25 mL of flask, N-acetyl-p-aminophenol (30.2 mg, 0.2 mmol) and potassium carbonate (28 mg, 0.2 mmol) were dissolved in 5 mL of dry acetone. Then [l-14c]-iodoethane (32 mg, 0.2 mmol, 10 mCi) was added and the reaction mixture was refluxed for 60 hours. Afterwards, the reaction mixture was filtered and the filtrate evaporated to give a white solid. The white solid was partitioned between chloroform and saturated sodium bicarbonate. The aqueous layer was extracted with chloroform (2x 10 mL). The combined organic layers were washed with water, dried over anhydrous magnesium sulfate, and evaporated to give N-(4-([l-14C] Ethoxy) phenylacetamide ([0-ethyl- 14C]phenacetin). The residue was then purified by preparatory HPLC (ZORBAX RX C8 (ZORBAX is a trademark of DuPont), mobile phase: 20% ACN (acetonitrile) with 0.1%TFA (trifluoroacetate) to give the [O-ethyl-14c]phenacetin (26.8 mg, 7.5 mCi)

EXAMPLE 2

This example illustrates the usefulness of the assay of the present invention to identify inhibitors of CYP 1 A2 activity.

The assay was carried out in 96-well microtiter plates containing radiolabeled substrates (70,000 dpm [O-ethyl-14C]phenacetin (10 μM), pooled HLM (0.5 mg/mL), 1 mM NADPH with NADPH regenerating system (5 mM glucose-6-phosphate, 3 mM MgC12₅ 1 U/mL glucose-6-phosphate dehydrogenase) and 0.1 M potassium phosphate, pH 7.6, in a final volume of 100 μL. The assays were conducted for various lengths of time at 37°C in the presence or absence of test compounds. Reactions were stopped by addition of HCl to a final concentration of 0.1 N. Plates were centrifuged in a microplate rotor, and supernatants were loaded on a preconditioned 10 mg OASIS 96-well HLB plate (Waters Corporation (Newcastle, DE)). Vacuum was applied and the flow- through was collected in the collection plate. Then, 80 μL of water was added, vacuum was applied again and the wash was collected into the same plate. This step was then repeated. Pooled eluates were transferred into scintillation vials and counted.

When the radiolab led substrate [O-ethyl-14c]phenacetin, dissolved in microsomal assay buffer, was applied to 96-well OASIS plates, over 99.7% of radioactivity was retained on the extraction plate, and could be recovered by elution with methanol. In contrast, about 80% of the [14C]acetaldehyde, the product of CYPlA2-mediated oxidation of [O-ethyl-14c]phenacetin, was recovered in the combined void volume and aqueous washing of OASIS extraction plates.

When [0-ethyl-14c]phenacetin was incubated with HLM in the presence of an NADPH regenerating system, the radiolabeled reaction product was produced in a time-dependent manner (Figure 4). The reaction products were not retained by the OASIS polymeric reversed-phase sorbent, similarly to standards [14C]acetaldehyde and [14c]acetic acid (and unlike the substrate [O-ethyl- 14c]phenacetin). Product formation was dependent on NADPH indicating that the reaction is mediated by cytochrome P450. The specific CYP 1A2 inhibitor furafylline potently inhibited product formation, with an IC50 of about 1.5 μM (Figure 5), indicating that the reaction is mediated mainly by CYPl A2. Product formation increased linearly with the concentration of microsomes up to a protein concentration of 1 mg/mL.

Signal to noise ratio is defined as the ratio between product counts obtained in the presence vs. absence of NADPH. The specific conversion rate is expressed as percent of total radiolabeled substrate converted into product per unit time and per mg of microsomal protein. Signal to noise ratio was 10 when assays were performed for 20 min, using 0.5 mg/mL of HLM. Specific conversion rate was about 3%/min/mg. From competition experiments using radiolabeled and unlabelled phenacetin, a Km of 60 μM and Vmax of 460 pmol/min/mg protein were calculated. These values are in good agreement with Km and Vmax values for formation of the unlabeled product paracetamol (quantified by HPLC coupled to triple quadrupole mass spectrometric analysis), 99 μM and 380 pmol/min/mg, respectively.

Experiments with specific CYP inhibitors confirmed that the reaction is mediated by CYP1A2. Thus, the reaction was not significantly inhibited by coumarin (inhibits CYP2A6), sulfaphenazole (inhibits CYP2C9), quinidine (inhibits CYP2D6), diethyldithiocarbamate (inhibits CYP2E1), and ketoconazole (inhibits CYP3A4). To compare IC50 values of several known CYP 1A2 inhibitors obtained in the new radiometric assay with those obtained in the conventional HPLC-mass spectrometric assay, reactions were performed in the absence or presence of 10 different concentrations of known inhibitors.

Formation of the [14c]acetaldehyde product and of the unlabeled paracetamol product were determined in the same reaction mixture. Paracetamol was quantified by HPLC coupled to triple quadrupole mass spectrometric analysis. As shown in Figure 6, IC50 values for the two products were almost identical, with a correlation coefficient r2 = 0.937 from linear regression analysis (Figure 5). These results demonstrate that the present assay provides a reliable measurement of the potency (IC50) of CYPl A2 inhibitors. EXAMPLE 3

This example illustrates the use of the present invention to determine and quantity the enzymatic activity and the effect of CYPl A2 inhibitors in intact hepatocytes.

Human hepatocytes are prepared from fresh liver samples. Hepatocytes are isolated and cryopreserved in liquid nitrogen according to established protocols (See for example, Hengstler et al., Drug Metab. Rev. 32: 81-118 (2000); Ferrini et al, Methods MoI. Biol. 107: 341-52 (1998)). Cells are thawed and incubated for one hour at 37°C in a shaking water bath under a humidified atmosphere of 5% CO2 , 95% oxygen, in 12-well culture plates. Each culture well contains about one million cells, 1 mL of hepatocyte culture medium (HCM) (Dich and Grunnet, in Methods in Molecular Biology, Vol. 5: Animal Cell Culture (Pollard and Walker, eds) pp. 161-176, Humana Press, Clifton, New Jersey. (1989)), and about 370,000 dpm of [O-ethyl-l⁴C]phenacetin. When assaying an analyte for inhibition of CYPl A2 activity, the analyte is added to the above reaction. In this example, the inhibitor 10 μM furafylline is added to parallel incubations. Control incubations are also performed comprising a known inhibitor such as 10 μM furafylline or the vehicle for the analyte. After one hour, aliquots of the incubation medium are loaded onto individual wells of preconditioned 30 mg OASIS plates, which are washed two times with 200 μL of water. For each well, the flow-through is combined with the water washes and counted in a beta-counter after addition of scintillation fluid.

EXAMPLE 4 This example illustrates the use of the present invention to determine and quantify the effect of CYPl A2 inducers in hepatocytes.

Cryopreserved human hepatocytes from two different donors are obtained from Tissue Transformation Technologies (Edison, NJ). Cells (ca. 320,000) are plated in 24-well collagen-coated culture plates and maintained at 37°C in a humidified atmosphere of 5% CO2 , 95% air, in hepatocyte culture medium (HCM) (Dich and Grunnet, ibid.) supplemented with ITS+ (Collaborative Research, Waltham, MA). Twenty-four hours later, the culture medium for each well of cells is removed, fresh HCM with ITS is added, and cells are treated with either vehicle (control), methylcholanthrene (positive control), or analyte being tested for ability to induce CYP1A2 activity for 48 hours. CYP1A2 enzyme activity is then determined as follows. For each well, the medium is removed and the cells are incubated in 0.5 mL of Hank's balanced salt solution (HBSS) containing 10 mM Hepes, pH 7.4, and ca. 200,000 dpm of [O-ethyl- 14C]phenacetin for 1 hour at 37°C. For each, parallel incubations are also performed in the presence of 10 μM furafylline to ascertain that enzyme activity is specifically mediated by CYP1A2. The incubation medium is then loaded onto individual wells of preconditioned 30 mg OASIS plates, which are washed two times with 200 μL of water. For each well, the flow-through is combined with the water washes and counted in a beta-counter after addition of scintillation fluid.

EXAMPLE 5 This example shows an example of how to perform a time-dependent CYP 1A2 assay using HLM.

The preincubation step is performed as follows. Preincubation mixtures containing about 30 μL HLM (3.3 mg/ml of protein, preferred final concentration 2 mg/mL; range 0.1 to 5 mg/mL), 1 μL of test analyte (dissolved in 35 % DMSO, 65% Methanol), 9 μL of assay buffer (0.1 M potassium phosphate, pH 7.6). Preincubations are started by adding 10 μL of NADPH regenerating system (5 mM NADPH, 25 mM Glucose-6-phosphate, 17 mM MgCl2, 5 U/mL Glucose-6-phosphate dehydrogenase, in assay buffer). Preincubations are started at different times in reverse order (longest preincubation was started first, shortest preincubation was started last). Mixtures are preincubated in a shaking water bath for 0-30 minutes at 37°C. Determination of remaining activity is as follows. The second incubation is started by about 10-fold dilution of the preincubation mixtures with 450 μL of assay buffer containing [O-ethyl- 14C]phenacetin (about 800,000 dpm), 250 to 500 μM unlabelled phenacetin and 1 mM NADPH. Incubations are performed in a shaking water bath for 10 min at 37°C. Reactions are stopped by addition of about 50 μL of IN HCl. Plates are centrifuged at room temperature at 2800 rpm for 15 minutes. About 300 μL of supernatant are loaded on a preconditioned 30 mg OASIS plate. The flow-through is collected and aliquots of 120 μL are transferred into 96 well scintillation counting plates (Packard). 180 μL of MICROSCINT 40 scintillation fluid is added and plates are sealed, shaken, and counted in a Packard TOPCOUNT scintillation counter.

While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

Claims

WHAT IS CLAMED:

1. A method for identifying an analyte that inhibits activity of cytochrome 1A2 (CYP1A2), which comprises: (a) providing a mixture comprising CYPl A2, [O-ethyl-14C]phenacetin, NADPH, and the analyte;

(b) incubating the mixture for a time sufficient for the CYP 1A2 to deethylate the [O- ethyl- 14C]phenacetin;

(c) removing the CYP 1A2 from the mixture; (d) applying the reaction mixture to a sorbent, which preferentially binds non-polar compounds, to remove [O-ethyl-14c]phenacetin from the mixture; and

(e) measuring [14C] radioactivity in the mixture of step (d) with the [O-ethyl-

14C]phenacetin removed, wherein a decrease in the [^C] radioactivity in the mixture indicates that the analyte inhibits activity of the CYP1A2.

2. The method of claim 2 wherein the sorbent is selected from the group consisting of water- wettable polymers formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon, and silica substrates comprising a non-polar group bonded to the silica substrate.

3. The method of Claim 2 wherein the sorbent is poly(vinylbenzene-co-N- vinylpyrrolidone.

4. The method of Claim 1 wherein the aqueous mixture further comprises an NADPH regenerating system.

5. The method of Claim 1 wherein the tritium in the mixture in step (e) is compared to the amount of tritium in the mixture from a control mixture comprising CYPl A2, [O-ethyl- 14C]phenacetin, and NADPH, and not the analyte.

6. The method of Claim 1 wherein the CYP1A2 is provided in microsomes.

7. The method of claim 6 wherein the microsomes are human liver microsomes.

8. The method of Claim 6 wherein the microsomes are produced from cells selected from the group consisting of mammalian and insect cells, wherein the cells include a vector expressing the CYP 1A2.

9. The method of Claim 1 wherein the aqueous mixture further comprises an

NADPH regenerating system.

10. The method of claim 1 wherein the [^C] radioactivity comprises [14c]acetaldehyde, [14C]acetic acid, or mixture thereof.

11. The method of Claim 1 wherein the [1 ^C] radioactivity in the mixture in step (e) is compared to the amount [^C] radioactivity in the mixture from a control mixture comprising HLM, [O-ethyl-14c]phenacetin, and NADPH, and not the analyte.

12. A method for determining the activity of cytochrome 1A2 (CYP 1A2) in hepatocytes, which comprises:

(a) providing a culture of the hepatocytes;

(b) incubating the hepatocytes in a medium comprising [O-ethyl-14c]phenacetin for a time sufficient for the CYP 1A2 to deethylate the [O-ethyl-14C]phenacetin; (c) removing the medium from the culture of hepatocytes;

(d) applying the medium to a sorbent which preferentially binds non-polar compounds to remove the [O-ethyl-14C]phenacetin from the medium; and

(e) measuring [14C] radioactivity in the medium of step (d) with the [O-ethyl- 14C]phenacetin removed, wherein the amount of [14C] radioactivity in the medium determines the activity of the CYP 1 A2 in the hepatocytes.

13. The method of claim 14 wherein the sorbent is selected from the group consisting of water-wettable polymers formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon and silica substrates comprising a non-polar group bonded to the silica substrate.

14. A method for identifying an analyte that inhibits cytochrome 1A2 (CYP1A2) activity, which comprises: (a) providing a culture of hepatocytes; (b) incubating the hepatocytes in a medium comprising [O-ethyl-14c]phenacetin and the analyte for a time sufficient for the CYP 1A2 to deethylate the [0-ethyl-14c]phenacetin;

(c) removing the medium from the culture of hepatocytes;

(d) applying the medium to a sorbent, which preferentially binds non-polar compounds, to remove the [O-ethyl-14C]phenacetin from the medium; and

(e) measuring amount of [14C] radioactivity in the medium of step (d) with the [O- ethyl-14C]phenacetin removed wherein a decrease in the amount of [14c] radioactivity indicates that the analyte inhibits the CYP 1A2 activity.

15. The method of claim 14 wherein the sorbent is selected from the group consisting of water-wettable polymers formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon and silica substrates comprising a non-polar group bonded to the silica substrate.

16. A method for identifying an analyte that induces cytochrome 1 A2 (CYPl A2) expression, which comprises:

(a) providing a culture of hepatocytes;

(b) incubating the hepatocytes in a medium comprising the analyte; (c) replacing the medium comprising the analyte with a second medium comprising

[O-ethyl-14c]phenacetin and incubating the hepatocytes for a time sufficient for the CYPl A2 to deethylate the [O-ethyl-14c]phenacetin;

(d) removing the second medium from the culture of hepatocytes;

(e) applying the second medium to a sorbent, which preferentially binds non-polar compounds, to remove the [O-ethyl-14c]phenacetin from the second medium; and

(f) measuring amount of [ 14c] radioactivity in the second medium of step (e) with the [O-ethyl-14C]phenacetin removed wherein an increase in the amount of the [^C] radioactivity indicates that the analyte induces CYPl A2 expression.

17. The method of claim 16 wherein the sorbent is selected from the group consisting of water-wettable polymers formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon and silica substrates comprising a non-polar group bonded to the silica substrate.

18. A method for identifying an analyte that inhibits activity of CYPl A2, which comprises:

(a) providing a multiwell plate and a column plate having an array of solid phase extraction cartridges or columns having therein a sorbent which preferentially binds non-polar compounds;

(b) applying to each of the wells of the multiwell plate a mixture comprising CYP 1A2, [O-ethyl-14c]phenacetin, and an analyte;

(c) contacting NADPH and optionally an NAPDH regenerating system to the mixture in each of the wells above and incubating for a time sufficient for the CYP1A2 to deethylate the [O-ethyl-l⁴C]phenacetin;

(d) separating the CYP 1A2 from the mixture in each of the wells of the multiwell plate; (e) applying each mixture to a separate minicolumn of the column plate to remove any remaining [O-ethyl-14C]phenacetin from the mixture; and,

(f) measuring amount of [^C] radioactivity in the mixture with the [O-ethyl- 14c]phenacetin removed wherein a decrease in the amount of the [^C] radioactivity indicates that the analyte inhibits activity of the CYP 1A2.

19. The method of claim 18 wherein the sorbent is selected from the group consisting of water-wettable polymers formed by copolymerizing at least one hydrophilic monomer and at least one lipophilic monomer in a ratio sufficient for the polymer to be water-wettable and effective at retaining organic solutes thereon and silica substrates comprising a non-polar group bonded to the silica substrate.

20. A method for identifying an analyte that irreversibly inhibits activity of cytochrome 1A2 (CYP1A2), which comprises:

(a) providing a mixture comprising CYPl A2, NADPH regenerating system, and the analyte;

(b) incubating the mixture for different times;

(c) diluting the mixture and then adding to the diluted mixture [O-ethyl- 14c]phenacetin and NADPH;

(d) incubating the diluted mixture for a time sufficient for the CYP 1A2 to deethylate the [O-ethyl-14C]phenacetin;

(e) removing the CYP 1A2 from the mixture;

(f) applying the mixture to a sorbent which preferentially binds non-polar compounds to remove the [O-ethyl-14c]phenacetin from the mixture; and (g) measuring amount of radioactivity in the mixture of step (d) with the [O-ethyl- 14C]phenacetin removed, wherein a decrease in the amount of the radioactivity indicates that the analyte irreversibly inhibits activity of the CYP1A2.