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WO2006114275A1 - Dosage du cytochrome p450 1a2 - Google Patents

Dosage du cytochrome p450 1a2 Download PDF

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Publication number
WO2006114275A1
WO2006114275A1 PCT/EP2006/003812 EP2006003812W WO2006114275A1 WO 2006114275 A1 WO2006114275 A1 WO 2006114275A1 EP 2006003812 W EP2006003812 W EP 2006003812W WO 2006114275 A1 WO2006114275 A1 WO 2006114275A1
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Prior art keywords
phenacetin
mixture
ethyl
analyte
cyp
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PCT/EP2006/003812
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English (en)
Inventor
Annalise Di Marco
Ralph Laufer
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Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa
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Publication of WO2006114275A1 publication Critical patent/WO2006114275A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances

Definitions

  • 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.
  • cytochrome P450 cytochrome P450
  • CYP cytochrome P450
  • 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)).
  • CYPs are also strongly inducible by xenobiotics, up to 50 to 100 fold.
  • drug therapy there are two major concerns with respect to CYP induction.
  • induction may cause a reduction in therapeutic efficacy by decreasing systemic exposure as a result of increased drug metabolism.
  • induction may create an undesirable imbalance between toxif ⁇ cation 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)).
  • HMM human liver microsomes
  • 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. 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).
  • 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
  • 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.
  • 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.
  • 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 1
  • 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.
  • the lipophilic monomer comprises a phenyl, phenylene, ether, or C2-C18 alkyl group.
  • the lipophilic monomer is divinylbenzene.
  • the hydrophilic monomer comprises a saturated, unsaturated, or aromatic heterocyclic group.
  • the hydrophilic monomer is N-vinylpyrrolidone.
  • the water wettable polymer is poly(vinylbenzene-co-N-vinylpyr ⁇ olidone, 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.
  • the sorbent comprises a non-polar group bonded to a silica substrate.
  • 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.
  • the silica substrate is selected from the group consisting of silica particles and silica gel.
  • 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
  • the lipophilic monomer comprises a phenyl, phenylene, ether, or C2-C18 alkyl group.
  • the lipophilic monomer is divinylbenzene.
  • the hydrophilic monomer comprises a saturated, unsaturated, or aromatic heterocyclic group.
  • the hydrophilic monomer is N-vinylpyrrolidone.
  • 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.
  • 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
  • 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.
  • the sorbent or water wettable polymer is packed inside a solid phase extraction cartridge or column.
  • 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.
  • 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.
  • 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 [14
  • 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.
  • the lipophilic monomer comprises a phenyl, phenylene, ether, or C2-C18 alkyl group.
  • the lipophilic monomer is divinylbenzene.
  • the hydrophilic monomer comprises a saturated, unsaturated, or aromatic heterocyclic group.
  • the hydrophilic monomer is N-vinylpyrrolidone.
  • 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.
  • the sorbent comprises a non-polar group bonded to a silica substrate.
  • 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.
  • the silica substrate is selected from the group consisting of silica particles and silica gel.
  • 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-ethy
  • 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
  • the hydrophilic monomer comprises a saturated, unsaturated, or aromatic heterocyclic group.
  • the hydrophilic monomer is N-vinylpyrrolidone.
  • 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.
  • each of the minicolumns of the column plate further comprises a porous retaining means for retaining the polymer therein.
  • the wells of the multiwell plate and column plate each have a 96- well tissue culture plate format.
  • the [ ⁇ C] radioactivity in the mixture after removing any remaining [O-ethyl-14C]phenacetin) can comprise [14c]acetaldehyde, [ ⁇ C] acetic acid, or mixture thereof.
  • 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.
  • 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.
  • the microsomes are human liver microsomes (HLM).
  • HLM human liver microsomes
  • the HLM are removed from the aqueous mixture by acidification and/or centrifugation.
  • 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.
  • 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]phenacet
  • 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 inhibit
  • 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.
  • 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.
  • the lipophilic monomer comprises a phenyl, phenylene, ether, or C2-C18 alkyl group.
  • the lipophilic monomer is divinylbenzene.
  • the hydrophilic monomer comprises a saturated, unsaturated, or aromatic heterocyclic group.
  • the hydrophilic monomer is N-vinylpyrrolidone.
  • 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.
  • the sorbent comprises a non-polar group bonded to a silica substrate.
  • 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.
  • the silica substrate is selected from the group consisting of silica particles and silica gel.
  • the [ ⁇ C] radioactivity in the mixture after removing any remaining [O-ethyl-14c]phenacetin) can comprise [14c]acetaldehyde, [ ⁇ C] acetic acid, or mixture thereof.
  • 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.
  • analyte refers to molecules, compounds, chemicals, compositions, drugs, and the like.
  • Figure 1 shows a cross-sectional view of an extraction cartridge or column 10.
  • Figure 2 shows a perspective view of a multicolumn microf ⁇ ltration/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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the enzyme should be 5- 10-fold higher (because of the final dilution in the second incubation).
  • a first container 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.
  • 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.
  • the labeled phenacetin is at about 100,000 dpm.
  • the concentration of phenacetin is between about 1 to 100 ⁇ M, typically at about 10 ⁇ M.
  • 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 ⁇ L to 200 ⁇ L.
  • a control containing an equivalent amount of the vehicle used for the analyte is provided.
  • aqueous mixture 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.
  • an acid such as HCl
  • 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-
  • 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.
  • 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-l 4 C]phenacetin by CYP1A2 (and any [J4c]acetic acid produced from oxidation of the [14C]acetaldehyde) is measured.
  • 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.
  • the hepatocytes are incubated at a concentration of about 1 x 10$ cells/mL to 1 x I ⁇ 6 cells/mL, preferably 1 x 106 cells/mL.
  • each culture well contains about 1 x I ⁇ 6 cells, 1 mL of hepatocyte culture medium (HCM) (Dich and Grunnet, ibid.), and [O-ethyl-14C]phenacetin.
  • HCM hepatocyte culture medium
  • [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 ⁇ M, typically at about 10 ⁇ M.
  • 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.
  • a culture medium suitable for culturing fresh hepatocytes e.g., HCM.
  • the medium is supplemented with ITS (insulin-transferrin-selenium mixture).
  • the hepatocytes are plated at a density of about 150,000 to 200,000 cells/cm2.
  • 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.
  • 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.
  • the hepatocytes are maintained at 37°C in a humidified atmosphere of 5% CO2 in a culture medium suitable for culturing hepatocytes as above.
  • the hepatocytes are incubated at a concentration of about 1 x I ⁇ 6 cells/mL.
  • 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.
  • HCM a culture medium suitable for culturing hepatocytes
  • 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.
  • the concentration of phenacetin is between about 1 to 200 ⁇ M, typically at about 10 ⁇ M.
  • controls that include the vehicle for the analyte or a CYP 1A2 inhibitor such as furafylline provided.
  • 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-l 4 C]phenacetin by CYP1A2 (and any [14c]acetic acid produced from oxidation of the [14c]acetaldehyde) is measured.
  • 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.
  • the medium is supplemented with ITS.
  • 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.
  • 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.
  • a balanced salt solution is Hank's balanced salt solution and an example of a suitable buffer is 10 mM HEPES.
  • 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.
  • 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.
  • 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.
  • CYP 1A2 inhibitor such as furafylline, to ascertain that detected enzyme activity is specifically mediated by CYPlA2.
  • 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.
  • 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 assay is performed in a multiwell format, preferably, a 96-well format.
  • the multiwell format enables a plurality of analytes to be tested simultaneously.
  • 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.
  • 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.
  • 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 ace
  • 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.
  • the hydrophilic group can be an ether group.
  • Particularly useful monomers include N-vinylpyrrolidone, 2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, and ethylene oxide.
  • 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.
  • 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.
  • the sorbent is packed into the solid phase extraction cartridge or column enmeshed in a porous membrane.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • 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.
  • the sorbent 30 is packed in the column 10 as small particles such as beads having a diameter preferably between about 30 to 60 ⁇ m.
  • a multiplicity of the columns 10 are arranged to provide a format which is particularly suitable for high throughput screening.
  • 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.
  • 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.
  • the multicolumn microfiltration/extraction column plate has the minicolumns arranged in a 96-well format.
  • 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 Stahl.
  • irreversible or quasi-irreversible inactivation by certain analytes or their CYP-generated metabolites can occur.
  • mechanism-based or time-dependent inhibition is characterized by a progressive time-dependent decrease in enzyme activity in the presence of inhibitor.
  • 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.
  • 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)).
  • MBI mechanism-based inhibition
  • MBI 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:
  • the present invention also provides mechanism-based or time-dependent assays in addition to the reversible or quasi-reversible assays described above.
  • 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).
  • 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.
  • 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 ⁇ M 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.
  • 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).
  • 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).
  • 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:
  • k is the observed pseudo-first order inactivation rate constant, which is related to the inhibitor concentration during preincubation according to the following relationship:
  • N-acetyl-p-aminophenol 30.2 mg, 0.2 mmol
  • potassium carbonate 28 mg, 0.2 mmol
  • [l-14c]-iodoethane 32 mg, 0.2 mmol, 10 mCi
  • the white solid was partitioned between chloroform and saturated sodium bicarbonate. The aqueous layer was extracted with chloroform (2x 10 mL).
  • 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 ⁇ M), pooled HLM (0.5 mg/mL), 1 mM NADPH with NADPH regenerating system (5 mM glucose-6-phosphate, 3 mM MgC12 5 1 U/mL glucose-6-phosphate dehydrogenase) and 0.1 M potassium phosphate, pH 7.6, in a final volume of 100 ⁇ L.
  • 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.
  • 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 ⁇ M and Vmax of 460 pmol/min/mg protein were calculated.
  • 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.
  • 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.
  • HCM hepatocyte culture medium
  • 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.
  • HCM hepatocyte culture medium
  • ITS+ Cold Research, Waltham, MA
  • 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 ⁇ M 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 ⁇ L 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.
  • HBSS Hank's balanced salt solution
  • 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 ⁇ L HLM (3.3 mg/ml of protein, preferred final concentration 2 mg/mL; range 0.1 to 5 mg/mL), 1 ⁇ L of test analyte (dissolved in 35 % DMSO, 65% Methanol), 9 ⁇ L of assay buffer (0.1 M potassium phosphate, pH 7.6). Preincubations are started by adding 10 ⁇ L 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).
  • NADPH NADPH
  • 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 ⁇ L of assay buffer containing [O-ethyl- 14C]phenacetin (about 800,000 dpm), 250 to 500 ⁇ M 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 ⁇ L of IN HCl.
  • Plates are centrifuged at room temperature at 2800 rpm for 15 minutes. About 300 ⁇ L of supernatant are loaded on a preconditioned 30 mg OASIS plate. The flow-through is collected and aliquots of 120 ⁇ L are transferred into 96 well scintillation counting plates (Packard). 180 ⁇ L of MICROSCINT 40 scintillation fluid is added and plates are sealed, shaken, and counted in a Packard TOPCOUNT scintillation counter.

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Abstract

L'invention concerne un dosage radiométrique rapide et sensible permettant d'évaluer l'activité du cytochrome P-450 (CYP) 1A2 et le pouvoir d'un analyte d'inhiber l'activité du CYP3A1A2 ou d'induire l'expression du CYP1A2. Il est préférable d'effectuer toutes les étapes du dosage, y compris les incubations, la séparation de produits, et le comptage de la radioactivité, en format à puits multiples, ce qui peut être automatiser.
PCT/EP2006/003812 2005-04-26 2006-04-21 Dosage du cytochrome p450 1a2 WO2006114275A1 (fr)

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JP2014095696A (ja) * 2012-10-12 2014-05-22 Toyama Chem Co Ltd 反応性代謝物の検出方法
EP2881470A1 (fr) * 2013-12-05 2015-06-10 Les Laboratoires Servier Microsomes irreversiblement inhibés pour des CYP450 leurs utilisations dans le phénotypage enzymatique des voies métaboliques

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014095696A (ja) * 2012-10-12 2014-05-22 Toyama Chem Co Ltd 反応性代謝物の検出方法
EP2881470A1 (fr) * 2013-12-05 2015-06-10 Les Laboratoires Servier Microsomes irreversiblement inhibés pour des CYP450 leurs utilisations dans le phénotypage enzymatique des voies métaboliques
WO2015082694A1 (fr) * 2013-12-05 2015-06-11 Les Laboratoires Servier Microsomes irreversiblement inhibes pour des cyp450 leurs utilisations dans le phenotypage enzymatique des voies metaboliques

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