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WO2002004660A2 - Procede permettant de tester l'inhibition des isozymes cytochromes p450 a metabolisation de medicament - Google Patents

Procede permettant de tester l'inhibition des isozymes cytochromes p450 a metabolisation de medicament Download PDF

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Publication number
WO2002004660A2
WO2002004660A2 PCT/US2001/021909 US0121909W WO0204660A2 WO 2002004660 A2 WO2002004660 A2 WO 2002004660A2 US 0121909 W US0121909 W US 0121909W WO 0204660 A2 WO0204660 A2 WO 0204660A2
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WO
WIPO (PCT)
Prior art keywords
cyp450
isozymes
cocktail
sample
inhibition
Prior art date
Application number
PCT/US2001/021909
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English (en)
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WO2002004660A3 (fr
Inventor
Elizabeth A. Dierks
Heng-Keang Lim
Simon Edward Ball
Original Assignee
Wyeth
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wyeth filed Critical Wyeth
Priority to AU2001271985A priority Critical patent/AU2001271985A1/en
Publication of WO2002004660A2 publication Critical patent/WO2002004660A2/fr
Publication of WO2002004660A3 publication Critical patent/WO2002004660A3/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
    • C12Q1/007—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isoenzyme profiles
    • 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

  • This invention relates to a method for simultaneously testing the inhibition or
  • This invention also relates to a method for phenotyping cell
  • Cytochrome P450 (“CYP450") isozymes are the principal enzymes for the CYP450
  • Compounds that are potent inhibitoTs of one or more CYP450 isozymes have a potential for drug-drug interactions when administered to a
  • Potent drag-drug interactions e.g. ketoconazole
  • CYP450 isozymes are mixed function oxidases that possess a common
  • isozymes include CYP3A4, CYP2D6, , CYP2C9, CYPl A2, CYP2C19, CYP2A6, and CYP2C8. Potential drug candidates can be evaluated for their ability to inhibit
  • CYP450 isozymes by monitoring the effect of the drug candidate on the metabolism of selected compounds known as probe substrates. Compounds are considered
  • CYP450 probe substrates if a specific pathway of their metabolism is mediated by a single CYP450 isozyme. Specific, well-characterized CYP probe substrates are
  • CYP2D6 diclofenac (substrate for CYP2C9), ethoxyresorufin (substrate for CYP2D6), diclofenac (substrate for CYP2C9), ethoxyresorufin (substrate for CYP2D6), diclofenac (substrate for CYP2C9), ethoxyresorufin (substrate for CYP2D6), diclofenac (substrate for CYP2C9), ethoxyresorufin (substrate for CYP2D6), diclofenac (substrate for CYP2C9), ethoxyresorufin (substrate for CYP2D6), diclofenac (substrate for CYP2C9), ethoxyresorufin (substrate for CYP2D6), diclofenac (subs
  • CYP2A6 ' CYP2A6
  • paclitaxel also known as taxol
  • the metabolism of such compounds is typically studied in vitro using human tissue
  • Crespi et al. (6) and Moody et al. (7) require the use of expressed single CYP450 isozymes instead of human liver microsomes for at least some of the isozyme assays due to the poor specificity of the probe substrates used in both assays. Overestimation or underestimation of inhibition can occur when such
  • the products detected are the same for many of the isozymes in this assay and, as
  • sample preparation steps e.g. solid phase extraction
  • inability to assay multiple isozymes in a single sample (6-10).
  • CYP450 isozymes and are therefore less specific. This necessitates that separate
  • CYP450 probe substrates to monitor enzyme activity.
  • the method can be used to determine not only the inhibition of CYP450
  • the method can also be used for phenotyping cell or tissue samples or both based on the identification of the specific CYP450 isozyme(s) expressed by the cell or
  • Figure 1 provides the elution profiles obtained from using an LC-MS MS method of the metabolites (A) 7-hydroxycoumarin, (B) resorufin, (C) 4'- hydroxymephenytoin, (D) dextrorphan (internal standard), (E) r-hydroxybufuralol,
  • Figures 2(A)-(G) are graphs depicting the specificity of the probe substrates in
  • CYP450 isozymes that are E. c ⁇ ft-expressed include CYP3A4 ("3A4"), CYP2D6
  • the substrates are: (Fig. 2A) ethoxyresorufin, (Fig. 2B) coumarin, (Fig. 2C) paclitaxel (Fig. 2D) diclofenac, (Fig. 2E) S-mephenytoin,
  • Figures 3(A)-(G) are graphs depicting a comparison of the inhibition of
  • CYP450 isozymes in the presence of a cocktail of substrates and the individual
  • the present invention is directed to a method for simultaneously testing the
  • method comprises incubating a sample comprising at least two CYP450 isozymes, a cocktail comprising probe substrates specific for the CYP450 isozymes, and a
  • the present invention is also directed to a method for simultaneously testing
  • CYP450 isozymes comprises incubating a sample comprising at least two CYP450
  • isozymes a cocktail comprising probe substrates specific for the CYP450 isozymes,
  • the present invention is also directed to a method for phenotyping a cell or a
  • tissue sample by identifying the profile of the multiple-drag metabolizing CYP450
  • the method comprises incubating an extract from the cell or tissue sample to be phenotyped with a cocktail comprising probe substrates specific for each of the CYP450 isozymes; preparing a standard curve of the metabolites produced by each isozyme; comparing the amounts of metabolites produced in the cell or tissue sample
  • An advantage of the present invention is that a CYP450 inhibition or
  • the present invention is that large numbers of cell or tissue samples can be rapidly
  • the present invention provides a novel method to test simultaneously for
  • the human drug-metabolizing CYP450 isozymes are preferably
  • the present invention advantageously provides a greatly increased throughput method with a single incubation and analysis for multiple different CYP450 isozymes.
  • This method utilizes an in vitro cocktail of two or more preferably seven, specific, well- characterized CYP450 probe substrates to monitor enzyme activity and inhibition.
  • the CYP450 isozymes may be expressed in bacteria, preferably E. coli,
  • insect cells mammalian cells, and yeast using standard techniques or can be
  • Sample analysis is done by mass spectrometry, preferably by fast gradient LC-MS/MS with selective reaction monitoring (“SRM”)
  • the method of the present invention rapidly predicts potent CYP450 inhibition
  • the present method can be used both to screen for potent CYP450
  • the present assay using a cocktail of probe substrates offers many advantages over prior methods for evaluation of cytochrome P450 activity and inhibition
  • substrates are midazolam, bufuralol, diclofenac, ethoxyresorafin, S-mephenytoin,
  • cytochrome P450 isozymes can be used in all assays. All of the substrates and
  • tandem mass spectrometry the preferred measurement technique, ensures that only
  • Samples are stable at 4° C for at least two days after preparation, which also is an advantage when generating large numbers of samples.
  • Sample volume can be decreased to
  • the cocktail of probe substrates of the present invention may also be used in the following
  • This method utilizes an in vitro cocktail of two or preferably seven,
  • CYP450 probe substrates to monitor the induction/activation of CYP450 isozymes. This is accomplished by incubating the
  • cocktail with human tissue preparations e.g., microsomes, cell cultures, or slices.
  • bacteria preferably E. coli, insect cells, mammalian cells, and
  • yeast using standard techniques or can be purchased from commercial sources.
  • Sample analysis is done by mass spectrometry, preferably by fast gradient LC-
  • the cocktail of probe substrates and the method of the present invention may be any one of the same type.
  • tissue preparations e.g., microsomes, cell cultures, or slices
  • Tris base dibasic potassium phosphate
  • Tranylcypromine and 8-methoxypsoralen were obtained from Aldrich Chemical
  • Ethoxyresorafin was from Molecular Probes, Inc.
  • Human liver microsomes were prepared by differential ultracentrifugation
  • Each sample to be incubated contained 0.5 mg/ml human liver microsomes, 10 mM
  • solvent A (0.1% formic acid in water)
  • solvent B (0.1% formic acid in
  • the positive ion APCI mass spectra were acquired by flow injection of 80 ⁇ l
  • the sample was desolvated at a capillary temperature of 170° C and
  • vaporizer temperature 500° C.
  • the sheath gas was set at 90 psi and the flow meter reading of auxiliary gas of 30.
  • the conversion dynode were set at 4.5 kV, 1700 kV, and 15 kV, respectively.
  • the mass scan used selected reaction monitoring ("SRM") for each metabolite at a scan
  • Figure 1 provides the elution profiles of the metabolites in Table 3 above using
  • E. coli expression plasmids modified pCW containing human
  • NADPH-cytochrome P450 reductase and human CYP3 A4 were provided by the LINK industrial
  • CYP2C8 the substrate was metabolized exclusively or primarily by its specific isozyme. Multiple isozymes metabolize taxol, diclofenac, bufuralol, and
  • the substrate is a probe.
  • methoxypsoralen/CYP2A6 were incubated at a concentration of 10 ⁇ M with both the
  • probe substrates and individual substrates would yield similar results. Incubations were performed as described in Example 1 with 10 ⁇ M of the inhibitor, human liver microsomes, and either the cocktail of probe substrates or individual probe substrates
  • Figure 3 shows the results of incubations of the individual probe substrates and the cocktail of probe substrates with human liver microsomes in the presence of
  • ketoconazole is a potent inhibitor of CYP3A4 and
  • CYP2C8 quinidine is a potent inhibitor of only CYP2D6, sulfaphenazole of only
  • CYP2C9 tranylcypromine of CYP2C19 and CYP2A6, quercetm of CYPl A2 and
  • CYP2C8 furafylline of CYPl A2, and 8-methoxypsoralen of CYP2C19, CYP2C8, CYP2A6, and CYPl A2.
  • IC 50 values were determined for the inhibition of the CYP450 isozymes by the inhibitors ketoconazole, quinidine, sulfaphenazole,
  • Example 1 with the exception that 0, 0.1, 1, 10, or 50 ⁇ M of the inhibitor was
  • IC 50 values were determined by linear interpolation.
  • ketoconazole quinidine
  • sulfaphenazole tranylcypromine
  • quercetin quercetin
  • probe substrates and individual probe substrates predicted strong inhibition of
  • CYP3A4 (IC 50 of 0.1 ⁇ M for both) and CYP2C8 (IC 50 of 9 ⁇ M for the individual probe substrate incubation and 6 ⁇ M for the cocktail of probe substrates incubation).
  • probe substrates and individual probe substrates predicted an IC 50 value of 1 ⁇ M for
  • CYP2C19 (IC 50 of 7 ⁇ M for the individual probe substrate incubation and 6 ⁇ M for the cocktail of probe substrates incubation) and CYP2A6 (IC 50 of 0.6 ⁇ M for the
  • CYP450 isozymes CYP3 A4, CYP2D6, CYP2C9
  • CYPl A2, and CYP2C8 were either moderately or weakly inhibited (IC 50 values of 25
  • concentrations of the specific inhibitors demonstrate that the method of the present invention can be used both to screen for potent inhibition and to rapidly evaluate the
  • CYP2C 19 Significantly greater inhibition is seen of CYP2C 19 over CYP2C9 in both the cocktail of probe substrates incubation and the individual probe substrate incubation (see Table 7 in Example 4). This could be due to the use of different subsfrates or expressed enzymes versus human liver microsomes in the two studies.
  • the cocktail of seven probe substrates or a subset thereof could be used to quickly determine ⁇ values for inhibition of multiple CYP450 isozymes and to evaluate
  • CYP2C19 participates in tolbutamide hydroxylation by human liver
  • liver microsomes liver microsomes. Rapid Commun. Mass Spectrom. 14, 207-214.
  • microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
  • reductase generates a highly functional monooxygenase system in Escherichia coli. FEBSLett. 397, 210-214.
  • NADPH-cytochrome P-450 oxidoreductase affects drug metabolism. J.
  • P-450 validation of a pharmaceutical tool for drug metabolism research.
  • bufuralol the prototypic substrate of CYP2D6.
  • cytochrome P450 enzymes to study potential drug-drug interactions. Adv.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant de tester simultanément l'inhibition ou l'induction/activation, par un certain nombre de médicaments candidats potentiels, des isozymes cytochromes P450 (CYP450) à métabolisation de médicament, du type CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2C19, CYP2A6, et CYP2C8, en utilisant un mélange de substrats de sondes spécifiques pour les isozymes CYP450. L'invention concerne également un procédé qui permet de déterminer le phénotype d'un échantillon de cellule ou de tissu en déterminant le ou les isozymes CYP450 présents dans l'échantillon de cellule ou de tissu, sur la base du procédé décrit.
PCT/US2001/021909 2000-07-12 2001-07-11 Procede permettant de tester l'inhibition des isozymes cytochromes p450 a metabolisation de medicament WO2002004660A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001271985A AU2001271985A1 (en) 2000-07-12 2001-07-11 Method for testing for inhibition of drug-metabolizing cytochrome p450 isozymes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US21854700P 2000-07-12 2000-07-12
US60/218,547 2000-07-12

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WO2002004660A2 true WO2002004660A2 (fr) 2002-01-17
WO2002004660A3 WO2002004660A3 (fr) 2003-02-27

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1164200A3 (fr) * 2000-05-31 2004-02-18 Pfizer Products Inc. Méthode et dispositif pour la préparation d'échantillons de test d'interactions médicament-médicament
RU2316327C1 (ru) * 2006-10-05 2008-02-10 Институт органической и физической химии им. А.Е. Арбузова Казанского научного центра РАН (ИОФХ им. А.Е. Арбузова КазНЦ РАН) Ксимедон в качестве индуктора активности микросомальных оксидаз печени человека
US7632656B2 (en) * 2003-03-04 2009-12-15 Cellseed Inc. High performance liquid chromatography with an aqueous mobile phase for analysis of drug and its metabolite
CN105136962A (zh) * 2015-09-30 2015-12-09 成都华西海圻医药科技有限公司 一种肝微粒体中1-羟基咪达唑仑浓度的uplc/ms/ms检测方法
CN105241992A (zh) * 2015-09-30 2016-01-13 成都华西海圻医药科技有限公司 一种肝微粒体中4’-羟基双氯芬酸浓度的uplc/ms/ms检测方法
CN105277635A (zh) * 2015-09-30 2016-01-27 成都华西海圻医药科技有限公司 一种肝微粒体中右啡烷浓度的uplc/ms/ms检测方法
CN109596752A (zh) * 2019-02-01 2019-04-09 重庆市农业科学院 一种高效液相色谱串联质谱法检测蚯蚓体内cyp1a2和cyp3a4酶活力的方法
CN110713921A (zh) * 2018-07-13 2020-01-21 复旦大学 一套体外动态的药物代谢孵育系统
CN113720944A (zh) * 2021-09-30 2021-11-30 珠海润都制药股份有限公司 一种氢溴酸右美沙芬硫酸奎尼丁胶囊含量的检测方法
WO2024223797A1 (fr) 2023-04-28 2024-10-31 Institut National de la Santé et de la Recherche Médicale Utilisation d'inhibiteurs de cyp3a4 pour le traitement d'infections par le virus de l'hépatite d (vhd)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE186747T1 (de) * 1993-05-25 1999-12-15 American Cyanamid Co Cytochrome p-450 reduktase screening für ergosterol biosynthese-inhibitoren
US6060253A (en) * 1997-01-23 2000-05-09 The United States Of America As Represented By The Department Of Health And Human Services Agents that bind to and inhibit human cytochrome P450 2D6
GB9810016D0 (en) * 1998-05-08 1998-07-08 Smithkline Beecham Plc Compounds
ATE256120T1 (de) * 1998-07-16 2003-12-15 Gentest Corp Reagentien für cyp2d fluoreszenztest
DE69907962T2 (de) * 1998-12-14 2004-05-19 Vertex Pharmaceuticals (San Diego) Llc, San Diego Optische molekularsensoren für cytochrome p-450 aktivität

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1164200A3 (fr) * 2000-05-31 2004-02-18 Pfizer Products Inc. Méthode et dispositif pour la préparation d'échantillons de test d'interactions médicament-médicament
US7632656B2 (en) * 2003-03-04 2009-12-15 Cellseed Inc. High performance liquid chromatography with an aqueous mobile phase for analysis of drug and its metabolite
RU2316327C1 (ru) * 2006-10-05 2008-02-10 Институт органической и физической химии им. А.Е. Арбузова Казанского научного центра РАН (ИОФХ им. А.Е. Арбузова КазНЦ РАН) Ксимедон в качестве индуктора активности микросомальных оксидаз печени человека
CN105136962A (zh) * 2015-09-30 2015-12-09 成都华西海圻医药科技有限公司 一种肝微粒体中1-羟基咪达唑仑浓度的uplc/ms/ms检测方法
CN105241992A (zh) * 2015-09-30 2016-01-13 成都华西海圻医药科技有限公司 一种肝微粒体中4’-羟基双氯芬酸浓度的uplc/ms/ms检测方法
CN105277635A (zh) * 2015-09-30 2016-01-27 成都华西海圻医药科技有限公司 一种肝微粒体中右啡烷浓度的uplc/ms/ms检测方法
CN110713921A (zh) * 2018-07-13 2020-01-21 复旦大学 一套体外动态的药物代谢孵育系统
CN110713921B (zh) * 2018-07-13 2022-12-20 复旦大学 一套体外动态的药物代谢孵育系统
CN109596752A (zh) * 2019-02-01 2019-04-09 重庆市农业科学院 一种高效液相色谱串联质谱法检测蚯蚓体内cyp1a2和cyp3a4酶活力的方法
CN113720944A (zh) * 2021-09-30 2021-11-30 珠海润都制药股份有限公司 一种氢溴酸右美沙芬硫酸奎尼丁胶囊含量的检测方法
WO2024223797A1 (fr) 2023-04-28 2024-10-31 Institut National de la Santé et de la Recherche Médicale Utilisation d'inhibiteurs de cyp3a4 pour le traitement d'infections par le virus de l'hépatite d (vhd)

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WO2002004660A3 (fr) 2003-02-27

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