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WO2007035335A2 - Dosage de l'isoforme 2c19 du cytochrome p450 - Google Patents

Dosage de l'isoforme 2c19 du cytochrome p450 Download PDF

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Publication number
WO2007035335A2
WO2007035335A2 PCT/US2006/035529 US2006035529W WO2007035335A2 WO 2007035335 A2 WO2007035335 A2 WO 2007035335A2 US 2006035529 W US2006035529 W US 2006035529W WO 2007035335 A2 WO2007035335 A2 WO 2007035335A2
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WO
WIPO (PCT)
Prior art keywords
tritium
mephenytoin
labeled
cyp2c19
mixture
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PCT/US2006/035529
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English (en)
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WO2007035335A3 (fr
Inventor
Ralph Laufer
Annalise Di Marco
Ashok Chaudhary
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Merck & Co., Inc.
Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A.
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Publication of WO2007035335A2 publication Critical patent/WO2007035335A2/fr
Publication of WO2007035335A3 publication Critical patent/WO2007035335A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

Definitions

  • the present invention relates to an assay for assessing the activity of Cytochrome P450 isoform 2Cl 9 (CYP2C19) and the potential of an analyte to modulate CYP2C19 activity, e.g., inhibitor of CYP2C19 activity or inducer of CYP2C19 expression.
  • CYP2C19 Cytochrome P450 isoform 2Cl 9
  • the assay determines CYP2C19 activity or expression by measuring CYP2C19-mediated hydroxylation of (S)-mephenytoin in reactions comprising CYP2C19, microsomes comprising CYP2C19, or hepatocytes using (S)-mephenytoin labeled with tritium in the 4 position of the phenyl ring as a substrate and a sorbent which preferentially binds non- polar compounds such as the tritium-labeled (S)-mephenytoin to separate the tritium-labeled (S)- mephenytoin from tritiated water formed during the hydroxylation.
  • the assay is useful for assessing CYP2C19 enzymatic activity and CYP2C19 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.
  • 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 toxification and detoxification as a result of increased formation of reactive metabolites (Lin and Lu, Clin. Pharmacokinet. 35: 361-390 (1998)).
  • CYP2C19 is a polymorphic enzyme that is absent in about 5% of the Caucasian population and up to 20% of the Asian population (Wrighton and Stevens, Crit. Rev. Toxicol. 22: 1-21 (1992). CYP2C19 is the major isoform responsible for the oxidation of a small number of drugs such as the (S)-enantiomer of mephenytoin and proton pump inhibitors such as omeprazole and lansoprazole.
  • CYP2C19 participates in metabolic clearance of various drugs, such as phenytoin, diazepam, imipramine, amitryptiline, and propranolol (Goldstein, Br. J. Clin. Pharmacol. 52: 349-355 (2001). Inhibition of CYP2C19 has been implicated in clinical drug interactions between fluvoxamine and proton pump inhibitors (Yasui-Furukori et ah, Journal of Clinical Pharmacology 44:1223-1229 (2004); Yasui-Furukori et ah, Br J Clin Pharmacol.
  • alprazolam Sudzuki et ah, Eur J Clin Pharmacol 58: 829-833 (2003)
  • mephenytoin mephenytoin, chloroguanide and diazepam
  • tricyclic antidepressants and phenytoin Shin et al, Drug Metab Disp 30:1102-1107 (2002)
  • carbamazepine/diazepam and phenytoin Lakehal et ah, Epilepsy Research 52 79-83 (2002)
  • clarithromycin and omeprazole Calabresi et ah, Pha ⁇ nacological Research 49: 493 ⁇ 199 (2004)).
  • the enzymatic reaction most commonly used to determine the enzymatic activity of CYP2C19 and the inhibitory potential of drugs on CYP2C19 activity is 4-hydroxylation of (S)- mephenytoin (Walsky and Obach, Drug Metab. Dispos. 32:647-660 (2004).
  • Current CYP2C19 assays using (S)-mephenytoin as the substrate are conducted in human liver microsomes (HLM) and require HPLC for product isolation and ultraviolet (UV) or mass spectrometric (MS) methods for detection and quantification of the product (Walsky and Obach, Drug Metab. Dispos. 32:647-660 (2004); Wedlund and Wilkinson, Methods Enzymology 272:105-114 (1996).
  • the present invention provides a rapid and sensitive radiometric assay for assessing the activity of cytochrome P-450 (CYP) 2C19 and the potential of an analyte to inhibit CYP2C19 activity or induce CYP2C19 expression. All the steps of the assay, including incubations, product separation, and radioactivity counting are preferably performed in a multiwell format, which can be automated.
  • CYP cytochrome P-450
  • the present invention provides a method for identifying an analyte that inhibits activity of CYP2C19, which comprises providing a mixture comprising CYP2C19, (S)-mephenytoin labeled with tritium in the 4 position of the phenyl ring, NADPH, optionally an NADPH regenerating system, and the analyte; incubating the mixture for a time sufficient for the CYP2C19 activity to hydroxylate the tritium-labeled (S)-mephenytoin at the 4 position of the phenyl ring; removing the CYP2C19 from the mixture; applying the mixture to a sorbent which preferentially binds non-polar compounds such as (S)-me ⁇ henytoin to remove any remaining tritium-labeled (S)- mephenytoin from the mixture; and, measuring amount of tritium in the mixture with the tritium-labeled (S)-mephenytoin removed wherein
  • 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 non-polar group is a C2 to Ci 8 alkyl group.
  • the non-polar group is a trifunctional octadecyl.
  • 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 activated charcoal as the substrate.
  • the present invention provides a method for identifying an analyte that inhibits activity of cytochrome CYP2C19, which comprises providing a mixture comprising CYP2C19, (S)-mephenytoin labeled with tritium in the 4 position of the phenyl ring, NADPH, optionally an NADPH regenerating system, and the analyte; incubating the aqueous mixture for a time sufficient for the CYP2C19 activity to hydroxy late the tritium-labeled (S)-mephenytoin at the 4 position of the phenyl ring; optionally removing the CYP2C19 from the mixture; applying the mixture to a sorbent comprising a non-polar group bonded to a silica substrate to remove the human liver microsomes and tritium-labeled (S)-mephenytoin from the mixture; and measuring amount of the
  • the non-polar group is a trifunctional octadecyl.
  • the present invention provides a method for identifying an analyte that inhibits activity of cytochrome CYP2C19, which comprises providing a mixture comprising CYP2C19, (S)-mephenytoin labeled with tritium in the 4 position of the phenyl ring, NADPH, optionally an NADPH regenerating system, and the analyte; incubating the aqueous mixture for a time sufficient for the CYP2C19 activity to hydroxylate the tritium-labeled (S)-mephenytoin at the 4 position of the phenyl ring; optionally removing the CYP2C19 from the mixture; applying the mixture to a sorbent comprising silica bonded to a trifunctional octadecyl to remove the human liver microsomes and tritium-labeled (S)- mephenytoin from the mixture; and measuring amount of the tritium not bound to the sorbent (i.e
  • the CYP2C19 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 CYP2C19 or the microsomes can be from kidney, liver, brain, muscle, or the like cells.
  • the microsomes are human liver microsomes (HLM).
  • the microsomes are removed from the aqueous mixture by acidification and/or centrifugation.
  • the sorbent 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 tritium-labeled (S)-mephenytoin from the tritium after the incubation, and a second multiwell plate for collecting the column void volume and washes from the multicolumn for determining the tritium therein.
  • the tritium in the mixture is compared to the amount of tritium in the mixture from a control mixture comprising CYP2C19, tritium-labeled (S)-mephenytoin, and NADPH, and not the analyte.
  • the present invention further provides a method for identifying an analyte that inhibits activity of CYP2C19, 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 such as (S)-mephenytoin; applying to each of the wells of the multiwell plate a mixture comprising CYP2C19, (S)-mephenytoin labeled with tritium at the 4 position of the phenyl ring, 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 CYP2C19 to hydroxylate the tritium-labeled (S)- mephenytoin at the 4 position of the phenyl ring; optionally separating the CYP2C19 from the mixture in each of the wells of the multiwell plate; applying each mixture to
  • 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 non-polar group is a C2 to Ci 8 alkyl group. In a preferred aspect, the non-polar group is a trifunctional octadecyl.
  • 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 activated charcoal.
  • the present invention provides a method for identifying an analyte that inhibits activity of CYP2C19, which comprises providing a multiwell plate and a column plate having an array of solid phase extraction cartridges or columns comprising a sorbent comprising non-polar group bound to a silica substrate; applying to each of the wells of the multiwell plate a mixture comprising CYP2C19, (S)-mephenytoin labeled with tritium at the 4 position of the phenyl ring, 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 CYP2C19 to hydroxylate the tritium-labeled (S)- mephenytoin at the 4 position of the phenyl ring; optionally separating the CYP2C19 from the mixture in each of the wells of the multi
  • the CYP2C19 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 CYP2C19 or the microsomes can be from kidney, liver, brain, muscle, or the like cells.
  • the microsomes are HLMs.
  • the non-polar group is trifunctional octadecyl.
  • the tritium in the mixture is compared to the amount of tritium in the mixture from a control mixture comprising HLM, tritium-labeled (S)-mephenytoin, and NADPH, and not the analyte.
  • the present invention further provides a method for identifying an analyte that irreversibly inhibits activity of cytochrome 2Cl 9 (CYP2C19), which comprises providing a mixture comprising CYP2C19, NADPH regenerating system, and the analyte; incubating the mixture for different times; diluting the mixture and then adding to the diluted mixture (S)-mephenytoin labeled with tritium in the 4 position of the phenyl ring and NADPH; incubating the diluted mixture for a time sufficient for the CYP2C19 to hydroxylate tritium-labeled (S)-me ⁇ henytoin; removing the CYP2C19 from the mixture; applying the mixture to a sorbent which preferentially binds non-polar compounds to remove the tritium- labeled (S)-mephenytoin from the mixture; and measuring amount of the tritium in the mixture of step (d) with the tritium-labeled (S)-me
  • 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 non-polar group is a C2 to Cl 8 alkyl group. In a preferred aspect, the non-polar group is a trifunctional octadecyl.
  • 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 activated charcoal.
  • the CYP2C19 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 CYP2C19 or the microsomes can be from kidney, liver, brain, muscle, or the like cells.
  • the microsomes are HLMs.
  • the present invention provides a method for determining the activity of CYP2C19 in hepatocytes, which comprises providing a culture of the hepatocytes; incubating the hepatocytes in a medium comprising (S)-mephenytoin labeled with tritium at the 4 position of the phenyl ring for a time sufficient for the CYP2C19 to hydroxylate the tritium-labeled (S)-mephenytoin at the 4 position of the phenyl ring; removing the medium from the culture of hepatocytes; (d) applying the medium to a sorbent which preferentially binds non-polar compounds to remove any remaining tritium- labeled (S)-mephenytoin from the medium; and measuring amount of tritium in the medium with the [tritium-labeled (S)-mephenytoin removed, which determines the relative activity of the CYP2C19 in the hepatocytes.
  • the present invention provides a method for identifying an analyte that induces CYP2C19 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 (S)-mephenytoin labeled with tritium at the 4 position of the phenyl ring and incubating the hepatocytes for a time sufficient for the CYP2C19 to hydroxylate the tritium-labeled (S)-mephenytoin at the 4 position of the phenyl ring; 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 tritium-labeled (S)-mephenytoin from the second medium; and measuring amount of tritium in the second medium with the tritium-labele
  • the present invention provides a method for identifying an analyte that inhibits CYP2C19 activity, which comprises providing a culture of hepatocytes; incubating the hepatocytes in a medium comprising (S)-mephenytoin labeled with tritium at the 4 position of the phenyl ring and the analyte for a time sufficient for the CYP2C19 to hydroxylate the tritium-labeled (S)- mephenytoin at the 4 position of the phenyl ring; removing the medium from the culture of hepatocytes; applying the medium to a sorbent, which preferentially binds non-polar compounds, to remove any remaining tritium-labeled (S)-mephenytoin from the medium; and measuring amount of tritium in the medium with the tritium-labeled (S)-mephenytoin removed wherein a decrease in the amount of the radioactivity compared to a control culture of hepat
  • 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 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 non-polar group is a C2 to Ci 8 alkyl group. In a preferred aspect, the non-polar group is a trifunctional octadecyl.
  • 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 monoiner 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 activated charcoal.
  • the tritium in the mixture is compared to the amount of tritium in the mixture from a control mixture comprising HLM, tritium-labeled (S)-mephenytoin, and NADPH, and not the analyte.
  • the tritium-labeled (S)-mephenytoin is hydroxylated at the 4 position of the phenyl ring to produce non-polar compound 4-hydroxy-(S)- mephenytoin and the polar compound tritium-labeled water.
  • the 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 labeled diclofenac from the tritiated water after the incubation, and a second multiwell plate for collecting the column void volume and washes from the multicolumn for determining the tritium therein.
  • the present invention further provides a method for making (S)-mephenytoin labeled with tritium at solely the 4 position of the phenyl ring comprising the steps of iodinating the (S)- mephenytoin for a time sufficient to produce a [phenyl-4-iodo](S)-mephenytoin; and mixing the [phenyl- 4-iodo](S)-mephenytoin with tritium gas for a time sufficient to produce the (S)-mephenytoin labeled with tritium solely at the 4 position of the phenyl ring.
  • the present invention further provides a method for making (S)-mephenytoin labeled with tritium at the 3,4, and 5 position of the phenyl ring comprising the steps of iodinating the (S)- mephenytoin for a time sufficient to produce a [phenyl-3,4,5-triiodo](S)-mephenytoin; and mixing the [phenyl-3,4,5-triiodo](S)-mephenytoin with tritium gas for a time sufficient to produce the (S)- mephenytoin labeled with tritium solely at the 3, 4, and 5 positions of the phenyl ring.
  • the (S)-mephenytoin is reacted with N- iodosuccinimaide in the presence of an acid.
  • the terms "(S)-mephenytoin labeled in the 4 position of the phenyl ring", “(S)-mephenytoin labeled in the 4 position", and "tritium-labeled (S)-mephenytoin” refer to (S)- mephenytoin labeled at the 4 position of the phenyl ring only or labeled at the 4 position and at either the 3 or 5 positions of the phenyl ring, or both the 3 and 5 positions of the phenyl ring.
  • Figure 1 shows a cross-sectional view of an extraction cartridge or column 10.
  • Figure 2 shows a perspective view of a multicolumn microfiltration/extraction plate 100.
  • Figure 3 shows the formation of tritiated water from [phenyl-3,4,5 ⁇ 3H3](S)-mephenytoin in HLM as a function of time and microsome concentration.
  • Figure 4A shows the kinetics of (S)-mephenytoin 4-hydroxylation measured using LC- MS/MS.
  • Figure 4B shows the kinetics of (S)-mephenytoin 4-hydroxylation in HLM measured using [phenyl-4-3H](S)-mephenytoin as the substrate.
  • Figure 4C shows the kinetics of (S)-mephenytoin 4-hydroxylation in HLM measured using [phenyl-3,4,5-3H3](S)-mephenytoin as the substrate.
  • Figure 5 A shows the effect of CYP2C19 inhibitor ticlopidine on tritiated water formation from [phenyl-3,4,5-3H3](S)-mephenytoin in HLM.
  • Figure 5B shows the effect of CYP2C19 inhibitor progesterone on tritiated water formation from [phenyl-3, 4,5-3 H3](S)-mephenytoin i n HLM.
  • Figure 5C shows the effect of CYP2C19 inhibitor omeprazole on tritiated water formation from [phenyl-3,4,5-3H3](S)-mephenytoin in HLM.
  • Figure 5D shows the effect of CYP2C 19 inhibitor tranylcypromine on tritiated water formation from [phenyl-3,4,5-3H3](S)-mephenytoin in HLM.
  • Figure 5E shows the effect of CYP2C19 inhibitor miconazole on tritiated water fo ⁇ nation from [phenyl-3,4,5-3H3](S)-mephenytoin in HLM.
  • Figure 5F shows the effect of CYP2C19 inhibitor ketoconazole on tritiated water formation from [phenyl-3 ,4,5-3H3](S)-mephenytoin in HLM.
  • Figure 6 shows there is a correlation between IC50 values of 15 compounds obtained in the assays of the present invention vs. conventional LC-MS/MS assays DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides a rapid and sensitive (S)-mephenytoin hydroxylation assay for assessing cytochrome P-450 isoform 2C19 (CYP2C19) activity and for identifying modulators of CYP2C19 activity or expression.
  • the present invention provides an assay for assessing the activity of CYP2C19 in mixtures containing CYP2C19.
  • the assays include both reversible inhibition assays and mechanism-based or time-dependent inhibition assays.
  • microsomes from various tissues such as human liver microsomes (HLM); microsomes from mammalian or insect cells containing an expression vector which expresses recombinant CYP2C19; or hepatocytes, the potential of an analyte to inhibit CYP2C19 activity in any of the above mixtures, and the potential of an analyte to induce CYP2C19 expression in hepatocytes.
  • HLM human liver microsomes
  • hepatocytes the potential of an analyte to inhibit CYP2C19 activity in any of the above mixtures, and the potential of an analyte to induce CYP2C19 expression in hepatocytes.
  • the CYP2C19 is a human CYP2C19.
  • the assay is based on detecting the release of tritium in the form of [3H]-H2 ⁇ , which occurs upon CYP2C19-mediated hydroxylation of (S)-mephenytoin labeled with tritium in the 4 position of the phenyl ring in the presence of the analyte wherein an increase or decrease in the release of tritium from the tritium-labeled (S)-mephenytoin over time indicates that the analyte is a modulator of CYP2C 19 activity.
  • a decrease in the release of tritium in any of the above mixtures in the presence of an analyte indicates that the analyte is an inhibitor of CYP2C19 activity whereas an increase in the release of tritium in hepatocytes after treatment of the hepatocytes with the an analyte indicates that the analyte is an inducer of CYP2C19 activity.
  • the tritium-labeled water is separated from the tritium- labeled (S)-mephenytoin in a solid-phase extraction process using a sorbent a sorbent comprising a substrate which preferentially binds non-polar compounds such as (S)-mephenytoin. All the steps of the assay, including incubations, product separation, and radioactivity counting are performed in a multiwell format, which can be automated.
  • Scheme 1 shows CYP2C19 hydroxylation of (S)-mephenytoin at the 4 position of the phenyl ring to produce [ ⁇ henyl-4-hydroxy]-(S)-mephenytoin (or 4-hydroxy-(S)-mephenytoin).
  • the embodiment for identifying analytes that induce or inhibit CYP2C19 activity using hepatocytes in one aspect identifies analytes that inhibit or induce expression of the gene encoding CYP2C19, i.e., analytes which affect transcription of the gene encoding CYP2C19.
  • the embodiment in another aspect identifies analytes that exert their inhibitory or inducing effect on CYP2C19 activity by affecting posttranscriptional processing of mRNA encoding the CYP2C19.
  • the embodiment in a further aspect identifies analytes that exert their inhibitory or inducing effect on CYP2C19 activity by affecting translation of the mRNA encoding the CYP2C19.
  • the embodiment in a further still aspect identifies analytes that exert their inhibitory or inducing effect on CYP2C19 activity by interacting directly or indirectly with the CYP2C19.
  • the embodiment for assessing CYP2C19 activity 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 CYP2C19 modulators is useful for assessing the CYP2C19inhibition 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 CYP2C19 inhibition or induction potential of an analyte.
  • the assays can use purified recombinant CYP2C19 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 CYP2C19, preferably, a human CYP2C19.
  • the microsomes can be from insect cells infected with recombinant baculoviras expressing CYP2C19 and a p450 reductase.
  • the advantage of the cells expressing recombinant CYP2C19 is that CYP2C19 is the only cytochrome P450 present in these microsomes and the specific activity is generally higher.
  • the concentration range for assays using recombinant CYP2C19 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).
  • (S)-mephenytoin labeled with tritium solely at the 4 position of the phenyl ring can be prepared using the iodination method as shown in Example 1 or, for example, using 4- bromopropiophenone to produce 4-bromo-5-phenyl-5-ethylhydantoin, which is then tritiated at the 4 position of the phenyl ring and methylated at the N3 position of the hydantoin ring to produce the (S)- mephenytoin labeled with tritium solely at the 4 position as shown in Kupfer et al., J. Pharmacol. Exp. Thera. 218: 193-199 (1981)).
  • (S)-mephenytoin labeled with tritium can be prepared using the iodination method as shown in Example 1.
  • a first container which contains an aqueous mixture comprising the analyte to be tested for an inhibitory effect on CYP2C19 activity, tritium-labeled (S)-mephenytoin as the substrate probe, unlabelled (S)- mephenytoin to provide an adequate concentration of substrate, pooled HLM, and a buffer at a physiological pH.
  • tritium-labeled (S)- mephenytoin typically, the tritium-labeled (S)-mephenytoin is at about 500,000 dpm.
  • concentration of unlabeled (S)-mephenytoin is between about 1 to 100 ⁇ M, typically about 20 ⁇ M.
  • the pooled HLM are generally at about 0.05 to 2 mg/mL, typically, about 1 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.
  • controls that include the vehicle for the analyte or a CYP2C19 inhibitor such as tranylcypromine are provided.
  • aqueous mixture Following a preferred preincubation step of microsomes in buffer for several minutes at 31° C, about 1 mM NADPH with or without an NADPH regenerating system comprising about 5 mM glucose-6- ⁇ hosphate, about 3 mM MgCt ⁇ , 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 hydroxylation of the tritium-labeled (S)-mephenytoin. In general, about 30 minutes is usually sufficient to detect CYP2C19 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 at a concentration of about 0.1 N.
  • the HLM are removed from the aqueous mixture before transferring the reaction mixture to an extraction cartridge or column for separating tritium- labeled water from the tritium-labeled (S)-mephenytoin.
  • 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 (S)-mephenytoin.
  • 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 tritium-labeled water produced from the hydroxylation of the tritium-labeled (S)-mephenytoin labeled with tritium at the 4 position of the phenyl ring by CYP2C19 is measured.
  • the void volume or flow-through and washes are transferred to a scintillation vial and mixed with scintillation fluid for measuring the tritium-labeled water in a scintillation counter.
  • the absence of tritium-labeled water or reduced amounts of tritium- labeled water compared to the amounts of tritium-labeled water in the vehicle controls indicate that the analyte is an inhibitor of CYP2C19 activity.
  • the CYP2C19 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% ah- 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 I x IO ⁇ cells/mL to 1 x I ⁇ 6 cells/mL, preferably 1 x 106 cells/mL.
  • each culture well contains about 1 x 105 cells, 0.1 mL of hepatocyte culture medium (HCM) (Dich and Grunnet, ibid.), and tritium-labeled (S)-mephenytoin.
  • HCM hepatocyte culture medium
  • S tritium-labeled
  • S tritium-labeled
  • the concentration of unlabeled (S)-mephenytoin is between about 1 to 100 ⁇ M, typically about 20 ⁇ 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 0 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.
  • 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 (S)-mephenytoin.
  • 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 tritium-labeled water produced by the hydroxy lation of the tritium-labeled (S)- mephenytoin labeled with tritium at the 4 position of the phenyl ring by CYP2C19 is measured.
  • the void volume or flow-through and washes are transferred to a scintillation vial and mixed with scintillation fluid for measuring the tritium-labeled water in a scintillation counter.
  • the amounts of tritium-labeled water produced determines the relative CYP2C19 activity of the hepatocytes.
  • the assay for determining the ability of an analyte to inhibit CYP2C19 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 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.
  • a culture medium suitable for culturing hepatocytes e.g., HCM.
  • each culture well contains about 1 x 10 5 cells, 0.1 mL of HCM, the analyte being tested for inhibitory effect on CYP2C 19 activity, unlabelled (S)-mephenytoin, and tritium-labeled (S)-mephenytoin.
  • S tritium-labeled
  • the concentration of the unlabeled (S)-mephenytoin is between about 1 to 100 ⁇ M, typically at about 20 ⁇ M.
  • controls that include the vehicle for the analyte or a CYP2C19 inhibitor such as tranylcypromine are 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 (S)-me ⁇ henytoin.
  • 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 tritium-labeled water produced from the hydroxylation of the tritium-labeled (S)-mephenytoin labeled with tritium at the 4 position of the phenyl ring by CYP2C19 is measured.
  • the void volume or flow-through and washes are transferred to a scintillation vial and mixed with scintillation fluid for measuring the tritium-labeled water in a scintillation counter.
  • the assay for determining the ability of an analyte to induce CYP2C19 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.
  • 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 Rifampicin.
  • a known inducer such as Rifampicin.
  • the hepatocytes are incubated in an incubation medium containing a balanced salt solution and 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 unlabelled (S)-mephenytoin and tritium-labeled (S)-mephenytoin is added and the hepatocytes incubated as above for a suitable time to assess CYP2C19 activity, about an hour is usually sufficient.
  • tritium-labeled (S)-mephenytoin typically, the tritium-labeled (S)-mephenytoin is at about 500,000 dpm/mL.
  • concentration of unlabeled (S)-mephenytoin is between about 1 to 100 ⁇ M, typically at about 20 ⁇ M.
  • 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 (S)-mephenytoin.
  • 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 tritium- labeled water produced by the hydroxylation of the tritium-labeled (S)-mephenytoin labeled wit tritium at the 4 position of the phenyl ring by CYP2C19 is measured.
  • the void volume or flow- through and washes are transferred to a scintillation vial and mixed with scintillation fluid for measuring the tritium-labeled water in a scintillation counter.
  • the presence of tritium-labeled water or increased amounts of tritium-labeled water compared to the amounts of tritium-labeled water in the control with the vehicle only indicates that the analyte is an inducer of CYP2C19 activity.
  • the tritium-labeled (S)-mephenytoin is hydroxylated at the 4 position of the phenyl ring to produce the non-polar compound 4-hydroxy-(S)- mephenytoin and the polar compound tritium-labeled water.
  • the (S)-mephenytoin is labeled with tritium solely at the 4 position of the phenyl ring, hydroxylation produces unlabeled 4-hydroxy-(S)- mephenytoin and some labeled 4-hydroxy-(S)-mephenytoin.
  • the tritium-labeled 4-hydroxy-(S)- mephenytoin is likely the result of NIH shift wherein the tritium at position 4 on some of the molecules migrates to an adjacent carbon.
  • NIH shift wherein the tritium at position 4 on some of the molecules migrates to an adjacent carbon.
  • hydroxylation produces 4-hydroxy-(S)-mephenytoin and tritium-labeled water.
  • the assay is performed in a multiwell format, preferably, a 96-well fo ⁇ nat.
  • 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 tritium-labeled water produced from the hydroxylation of the tritium-labeled (S)-mephenytoin labeled with tritium at the 4 position of the phenyl ring 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 (S)-mephenytoin, i.e., the sorbent can adsorb or bind the tritium-labeled (S)-mephenytoin but not the tritium-labeled water produced by the hydroxylation of the tritium-labeled (S)-mephenytoin at the 4 position of the phenyl ring.
  • Sorbents which preferentially bind non-polar compounds such as (S)-mephenytoin 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 tritium-labeled (S)- mephenytoin but not tritium-labeled water , silicon-based sorbents such as the C2-C18 silanes, and activated charcoal.
  • 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
  • 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 a silicon-based matrix or silanes comprising C2-C18 alkyl groups, that is ethyl, octyl, monofunctional octadecyl, or trifunctional octadecyl alkyl groups.
  • the alkyl group is a trifunctional octadecyl functional alkyl.
  • Sorbents comprising a water-wettable polymer are 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-Cig-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).
  • 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 fo ⁇ nat.
  • 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.
  • 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.
  • 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 CYP2C19in the presence of an NADPH regenerating system for a series of different lengths of time (typically from 0 minutes to 60 minutes).
  • CYP2C19 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 CYP2C19 with (S)-mephenytoin is about 50 ⁇ M and the preferred concentration of (S)-mephenytoin is between about 200 to 600 ⁇ M.
  • Example 3 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:
  • the required fractions were collected and passed through SEP-PAK C- 18, followed by eluting with 10 mL ethanol to yield 5 mg of (5S)-5-ethyl-3-methyl ⁇ 5-(4'iodophenyl) imidazolidine-2,4-dione or [phenyl-4'-iodo ](S)-mephenytoin (4) in 30% isolated yield.
  • LC/MS 345 (M+H)+,
  • Radiometric CYP2C19 assays using tritiated (S)-mephenytoin were as follows. To remove polar impurities, an aliquot of [phenyl-3,4,5-3H3](S)-mephenytoin was dried under vacuum, reconstituted in water and loaded on 100-mg Cl 8 VERSAPLATE extraction plate (Varian Inc., Palo Alto, CA,). After washing with 9 ml of water, [3'4'5'3H3](S)-mephenytoin was eluted with 2 mL of methanol, dried, and reconstituted in ethanol. The purified tritium-labeled (S)-mephytoin was kept at - 80 0 C.
  • the sorbent comprises a Cl 8 silane.
  • Quantification of 4-hydroxymephenytoin was as follows. Aliquots of the assay reaction mixture and of metabolite standard curves were analyzed by HPLC using an Agilent HPl 100 liquid chromatograph (Agilent Technologies) equipped with a CTC Analytics PAL Autosampler (HTS PAL; CTC Analytics AG, Switzerland). Flufenamic acid was used as internal standard.
  • the eluate was diverted to waste for the first minute, and then to a Sciex API-3000 triple quadrupole mass spectrometer (available, for example, from Perkin Elmer, Boston, MA) with a Turbo Ionspray ionization source operated in the negative ion mode. 4-hydroxymephenytoin was detected and identified using the transition m/z 232.90 ⁇ 189.85. Metabolite concentrations were determined by weighted linear least-squares regression analysis, using Analyst Quantitation Wizard software version 1.2 (Applied Biosystems, Foster City, CA). Curve fitting was as follows. Curve fitting to the Hill equation or to a four-parameter logistic inhibition model (Rodbard and Frazier, Meth. Enzymol. 37: 3-22 (1975)) was performed by nonlinear regression using XLFIT 4.0 (ID Business Solutions, Inc., Guildford, UK ; Emeryville, CA).
  • tritium-labeled water from 500 to 50,000 dpm was not retained on the SPE resin under the same conditions. Recovery of tritium-labeled water eluted in the combined void volume and aqueous washes was quantitative.
  • tritium-labeled water from [phenyl-3,4,5-3H3](S)-mephenytoin in HLM.
  • [phenyl-3,4,5-3H3](S)-mephenytoin was incubated with HLM in the presence of NADPH, tritium- labeled water was formed in a time-dependent manner. Product formation increased linearly with the concentration of microsomes up to a protein concentration of 1.0 mg/mL (See Figure 3). Formation of tritium-labeled water was dependent on NADPH, indicating that the reaction was mediated by cytochrome P450.
  • Signal to noise ratio is defined as the ratio between product counts obtained in the presence vs. absence of NADPH.
  • the fractional conversion rate is expressed as percent of total radiolabeled substrate converted into tritiated water per unit time and per mg of microsomal protein. Signal to noise ratios were about 18 when assays were performed for 30 minutes using 1 mg/mL of HLM. Fractional conversion rate was about 0.12%/min/mg.
  • tritium-labeled (S)-mephenytoin is used as an isotopic tracer
  • the formation rate of tritium-labeled water (v*) is representative of that of unlabeled product, namely water derived from 4-hydroxylation of the tritium-labeled (S)-mephenytoin (which is formed stoichiometrically with the formation of 4-hydroxy- (S)-mephenytoin).
  • the dependence of v* on substrate concentration (S) can be used to obtain information about the dependence on substrate concentration of the unlabeled product, even if the latter is not measured directly.
  • IC50 values differed less than 3-fold in every case. Most importantly, not a single compound out of the 15 tested would have been misclassified as either a strong or weak inhibitor based on the results of the radiometric assay. Linear regression analysis, excluding the 2 compounds with IC50 values
  • This example shows an example of how to perfo ⁇ n a time-dependent CYP2C19 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). 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 0 C.
  • HLM 3.3 mg/ml of protein, preferred final concentration 2 mg/m
  • 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 tritium- labeled (S)-mephenytoin at the 4 position of the phenyl ring (about 800,000 dpm), 300 to 600 ⁇ M unlabelled (S)-mephenytoin and 1 niM 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.

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Abstract

La présente invention concerne un dosage radiométrique rapide et précis qui permet d'évaluer l'activité du cytochrome P-450 (CYP) 2C19 et le potentiel d'un analyte à inhiber l'activité de CYP2C19 ou à induire l'expression de CYP2C19. Toutes les étapes du dosage, y compris les incubations, la séparation des produits et le comptage de radioactivité sont préférentiellement menées en multipuits, ce qui permet d'automatiser la méthode.
PCT/US2006/035529 2005-09-16 2006-09-13 Dosage de l'isoforme 2c19 du cytochrome p450 WO2007035335A2 (fr)

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