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Study of in vitro metabolism of m-nisoldipine in human liver microsomes and recombinant cytochrome P450 enzymes by liquid chromatography–mass spectrometry
Accepted Manuscript Title: Study of in vitro metabolism of m-nisoldipine in human liver microsomes and recombinant cytochrome P450 enzymes by liquid chromatography–mass spectrometry Author: Lin Yuan Peipei Jia Yupeng Sun Chengcheng Zhao Xuran Zhi Ning Sheng Lantong Zhang PII: DOI: Reference:
S0731-7085(14)00157-5 http://dx.doi.org/doi:10.1016/j.jpba.2014.03.030 PBA 9509
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
31-10-2013 17-3-2014 20-3-2014
Please cite this article as: L. Yuan, P. Jia, Y. Sun, C. Zhao, X. Zhi, N. Sheng, L. Zhang, Study of in vitro metabolism of m-nisoldipine in human liver microsomes and recombinant cytochrome P450 enzymes by liquid chromatographyndashmass spectrometry, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.03.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Study of in vitro metabolism of m-nisoldipine in human liver microsomes and recombinant
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cytochrome P450 enzymes by liquid chromatography–mass spectrometry
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Lin Yuan, Peipei Jia, Yupeng Sun, Chengcheng Zhao, Xuran Zhi, Ning Sheng, Lantong Zhang *
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Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University,
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Shijiazhuang, P. R. China
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∗ Corresponding author:
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Prof. Lantong Zhang, Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical
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University, Shijiazhuang, 050017, P. R. China
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E-mail address: [email protected]
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Tel: +86 311 86266419; fax: +86 311 86266419
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Abstract This is a report about the investigation of the metabolic fate of m-nisoldipine in human liver
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microsomes and the recombinant cytochrome P450 enzymes by using LC-MS/MS. A sensitive and
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reliable LC-MS/MS method was developed to obtain a rapid and complete characterization of new
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metabolites and the metabolism pathways. The analytes were separated on a reversed phase C18
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column with acetonitrile and 0.1% aqueous formic acid as the mobile phase. Tandem mass
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spectrometry with positive electrospray ionization was used to enable the structural characterization
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of the metabolites. A total of 10 metabolites were characterized with proposed structures in the
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incubation of human liver microsomes by comparing their retention times and spectral patterns with
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those of the parent drug. Dehydrogenation of the dihydropyridine core and reactions of side chains
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such as hydroxylation and hydrolysis of ester bonds were the major metabolic pathways. The
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specific cytochrome P450 (CYP) enzymes responsible for m-nisoldipine metabolites were identified
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using chemical inhibition and cDNA expressed CYP enzymes. The results indicated that CYP2C19
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and CYP3A4 might play major roles in the metabolism of m-nisoldipine in human liver
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microsomes.
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Keywords: m-Nisoldipine; Human liver microsomes; Recombinant cytochrome P450 enzymes;
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LC-MS/MS; Metabolism
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1. Introduction m-Nisoldipine is a new dihydropyridine calcium channel antagonist that was first synthesized
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in the School of Pharmacy at Hebei Medical University [1]. As the isomer of nisoldipine (the nitro
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is in the 2-position of the phenyl system), m-nisoldipine has a similar structure but displays
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significant stability when exposed to the light [2]. m-Nisoldipine is a vasodilator that could be used
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in the treatment of hypertension. Previous studies indicated that m-nisoldipine was mainly excreted
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as metabolites [3]. Therefore, the metabolism study of m-nisoldipine will play an important role in
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clarifying the in vivo process of m-nisoldipine.
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Liver is the major organ involved in the biotransformation of various endogenous compounds
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and drugs. The cytochrome P450 superfamily is a large and diverse group of enzymes that catalyze
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the oxidation of organic substances. CYPs are the major enzymes involved in drug metabolism and
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bioactivation, accounting for about 75% of the total number of different metabolic reactions [4].
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The liver microsome metabolic model is widely used in the research of drug metabolism for its own
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advantages [5,6]. Previous work showed that the main metabolites of dihydropyridine calcium
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channel antagonist were products of oxidation of the 1,4-dihydropyridine ring, hydrolysis of the
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ester group in the side chain of position 3 and position 5, and hydroxylation [7-8].
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Liquid chromatography-tandem mass spectrometry (LC-MS) has become a powerful and
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reliable analytical approach for metabolite identification because of its high sensitivity, low
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consumption and high speed of analysis [9-12]. This paper described the application of LC-MS for
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supporting the study of the metabolism of m-nisoldipine in human liver microsomes. The
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characterization of 10 metabolites was achieved by the LC-MS analysis and the possible metabolic
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pathway was proposed.
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2. Experimental
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2.1 Chemicals and Materials m-Nisoldipine (purity>99.5%) was provided by School of Pharmacy, Hebei Medical
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University (Shijiazhuang, China). Dehydrogenation product of m-nisoldipine (S1) was synthesized
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according to literature [2]. 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-2-carboxylic
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acid-2-hydroxy-methyl isobutyl (S2) and 2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-2-carboxylic
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acid-2-hydroxy-methyl isobutyl (S3) were obtained by microbial biotransformation for
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identification. Pooled human liver microsomes and microsomes containing c-DNA expressed P450s
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(CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4) were obtained from BD
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Biosciences
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dehydrogenase, sulfaphenazole, quinidine and diethyldithiocarbamate were purchased from Sigma
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Chemical (St. Louis, MO, USA). Furafylline, ticlopidine hydrochloride and ketoconazole were
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purchased from China Institute for Control of Pharmaceutical and Biological Products. Acetonitrile
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and formic acid were HPLC grade (Tedia, Fairfield, USA). Ultra pure water was used for all
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analysis. The other chemicals were all of analytical reagent.
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Glucose-6-phosphate,
β-NADP and
Glucose-6-phosphate
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(Woburn,
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2.2 Instruments
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The chromatographic separation system consisted of a quaternary pump (Agilent 1200), an
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online solvent degasser and an autosampler. 3200 QTRAPTM system from Applied Bios stems/MDS
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Sciex (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion trap mass
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spectrometer equipped with Turbo V sources and Turbo Ionspray interface. Instrument control and
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data acquisition were carried out with Applied Biosystems/MDS Sciex Analyst software
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(version1.4.2).
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HPLC analysis was performed at room temperature on a Diamonsil C18 column (5 µm, 250
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mm×4.6 mm). The mobile phase consisted of acetonitrile (A) and 0.1% aqueous formic acid (B).
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The gradient program started at 40% A, changed to 95% A within 20 min, held at 95% A for 5 min,
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and finally went back to 40% A in 1 min. This was followed by an eight minutes equilibrium period
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with initial conditions prior to injection of the next sample. The injection volume was 10 μL and the
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flow rate was 0.8 mL/min.
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The mass spectral analysis was performed in a positive electrospray ionization mode. The
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following parameters were optimized in order to achieve the highest sensitivity: turbo spray voltage
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(IS) 5500V, turbo spray temperature 650°C, nebulizer gas (gas 1) 60 arbitrary units, heater gas (gas
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2) 65 arbitrary units, curtain gas 25 arbitrary units, interface heater on. Nitrogen was used in all
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cases. m-Nisoldipine and its metabolites were identified by EMS-IDA-EPI, MRM-IDA-EPI and
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PREC-IDA-EPI mode.
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Typical incubation mixture [13] (1 mL final volume) was carried out in a 0.1 M K2HPO4 buffer
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(pH 7.4) containing human liver microsomes (1.0 mg/mL), 3.3 mM MgCl2, 1.3 mM β-NADP, 3.3
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mM glucose-6-phosphate, 1.0 U/mL glucose-6-phosphate dehydrogenase and 100 μM
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m-nisoldipine methanol solution (1% of final volume). The mixture was pre-incubated for 5 min at
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37°C and then initiated by adding NADPH generating system. After incubation at 37°C for 30 min,
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1 mL ice-cold acetonitrile were added to stop the reaction. The mixture was extracted with ether:
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n-hexane (50:50, v/v) 5 mL by vortex-mixing for 2 min. After centrifugation at 10,000×g for 10 min,
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the organic phase was collected and evaporated under nitrogen gas. The residues were reconstituted
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in 200 μL methanol and an aliquot (10 μL) was injected into the chromatographic system for
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analysis. Blank sample was incubated without m-nisoldipine while control sample was incubated
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without NADPH generating system followed the same treatment.
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2.4 Chemical inhibition
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The inhibition study was performed with three concentrations of the inhibitors, human liver
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microsomes (1 mg/mL) and m-nisoldipine (100 μM). The chemical inhibitors used were as follows:
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furafylline for CYP1A2, sulfaphenazole for CYP2C9, ticlopidine hydrochloride for CYP2C19,
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quinidine for CYP2D6, diethyldithiocarbamate for CYP2E1 and ketoconazole for CYP3A4 [14].
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The inhibitors were pre-incubated for 30 min with human liver microsomes and NADPH system at
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37°C, then the m-nisoldipine was added to initiate the reaction.
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The incubation of microsomes containing individual CYP enzyme (100 pmol P450/mL) was
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2.5 Metabolism by recombinant cytochrome P450 enzymes
conducted the same as microsomal incubation.
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3. Results and discussion
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3.1 Characterization of metabolites in human liver microsomes and the recombinant cytochrome
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P450 enzymes
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Under the condition of electrospray ionization, m-nisoldipine (C20H24N2O6, MW 388 Da) can
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easily form the [M+H]+ ion at m/z 389 in positive ion mode. Compared with the control and the
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blank samples, 10 major metabolites (M1–M10, numbered according to retention time) were
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detected (Fig.1). Each metabolite was identified on the basis of chromatographic behavior and
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characteristic mass spectrometric fragmentation features observed in the LC-MS spectrums. The
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extracted ion chromatograms of metabolites showed that the peaks at identified m/z ratios did not
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have any interference observed in either blank or control samples. The predominant fragmentation
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patterns of m-nisoldipine and S1 were illustrated in Fig.2. The information of the four known
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compounds facilitated the characterization of the 10 metabolites. The retention times and
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characteristic fragments used to identify the metabolites were summarized in Table 1.
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3.1.1 Structural characterization of 10 metabolites in human liver microsomes
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For M1 eluting at 7.72 min, the [M+H]+ ion at m/z 331 was 56 Da less than that of S1. It
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showed a series of product ions at m/z 299,285,168 and shared the same fragmentation pattern of S1.
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That indicated that M1 was a metabolite generated by dehydrogenation and hydrolysis of the methyl
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ester group in the side chain at position 3 of the m-nisoldipine. The MS2 spectrum of M2 ([M+H]+
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at m/z 391) eluting at 8.02 min was compared with that of S2 because of the same fragment ions at
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m/z 301 and 284. Other product ions at m/z 373 [M+H–H2O]+ and 347 [M+H–CO2]+ were found. In
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addition, with a mass of 14 Da lower than that of S2, M2 should be the product of hydroxylation in
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the side chain at position 3 and hydrolysis of the methyl ester group at position 5.
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For M3 ([M+H]+ at m/z 419) eluting at 8.20 min, the same product ions as S1 (m/z
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347,331,299 and 285) were detected, as well as another product ion at m/z 401 [M+H–H2O]+. The
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[M+H]+ ion of M3 was 32 Da more than that of S1, which can be assigned to two hydroxyl groups
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to the molecule. Based on the structure of product ions and other dihydropyridine calcium channel
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blockers, the 6-hydroxymethyl metabolite was observed [15]. Thus, M3 was suggested as a
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dehydrogenation and hydroxylation product of m-nisoldipine, and the localization of the hydroxyl
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group should be at position 6 and in the side chain at position 3.
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For M4 ([M+H]+ at m/z 421), eluting at 8.45 min, it was suggested to be a 1,4-dihydropyridine
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analogue to m-nisoldipine with two hydroxyl groups at position 6 and in the side chain at position 3.
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The product ions were 2 Da more than those of M3, and M4 shared the same fragmentation pattern
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of m-nisoldipine (m/z 315,298,283). M5 eluting at 10.87 min showed the [M+H]+ ion at m/z 389,
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which was 2 Da less than that of M2. And the characteristic product ions at m/z 331 and 285 were
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detected. Hence dehydrogenation, hydroxylation in the side chain at position 3 and hydrolysis of the
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methyl ester group at position 5 were suggested for M5.
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M6 ([M+H]+ at m/z 403), M7 ([M+H]+ at m/z 405) and M9 ([M+H]+ at m/z 387), eluting at
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11.92, 11.99 and 19.83 min respectively, showed identical product ion spectrums compared to S3,
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S2 and S1. And with the retention time comparison of the standards under the same HPLC
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conditions, M6, M7 and M9 could all be identified with no doubt. Although M7 was also detected
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in the control sample, the concentration was far below. This is because that m-nisoldipine could be
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oxidated not only by the enzyme in liver microsomes, but also by the air of oxygen [16]. The three
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metabolites were also confirmed and detected in rat plasma, and were considered as major
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metabolites of m-nisoldipine in our previous work [17].
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It was suggested the hydrolysis of the methyl ester group at position 5 happened for M8. The
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[M+H]+ ion at m/z 375 was decreased by 14 Da when compared with that of m-nisoldipine, and the
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characteristic product ions at m/z 357,301,283 also appeared. M10 showed the [M+H]+ ion at m/z
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429. With a mass of 42 Da higher than that of S1, it may be an acetylated metabolite. The product
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ions at m/z 373 and 347 were detected, as well as the similar ions of S1 (m/z 299, 285). For steric
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reasons, acetylation at position 6 was more likely.
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3.1.2 Metabolites detected in the recombinant cytochrome P450 enzymes The incubations of m-nisoldipine with the recombinant P450s respectively were analyzed just
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the same as the human liver microsomal incubation. 6 metabolites (M2, M5, M6, M7, M8 and M9)
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were detected. M9 seemed to be the major metabolite because it could be found in all P450s in our
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experiment with a concentration much higher than others. In the incubation samples of CYP1A2, 2D6 and 3A4, M6, M7 and M9 were found. While only
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M8 and M9 were detected in both CYP2C9 and CYP2E1. CYP2C19 transformed m-nisoldipine
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into 6 metabolites mentioned above. That indicated CYP2C19 may play an important role in the
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metabolism of m-nisoldipine.
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In liver microsomes, drugs can be transformed by different enzymes so that more than one
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reaction may happen. However, the individual CYP enzyme shows specificity in catalyzing. So the
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metabolites which are products of reactions of different enzymes found in human liver microsomes
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may not be detected in individual CYP enzyme.
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3.1.3 Metabolic pathway of m-nisoldipine
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According to the results above, 10 metabolites of m-nisoldipine were detected and
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characterized in human liver microsomes. And the possible metabolic pathways of m-nisoldipine in
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human liver microsomes were proposed as shown in Fig.3. That indicated dehydrogenation of the
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dihydropyridine core and hydroxylation of the side chain at position 3 were major metabolic
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pathways of m-nisoldipine.
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3.2 Identification of the recombinant cytochrome P450 enzymes involved in the metabolism of
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m-nisoldipine
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According to the metabolic pathways mentioned above, m-nisoldipine mainly transformed into
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M7 and M9, and then both of them could convert into M6. That indicated the three structural
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confirmed compounds to be the major metabolites in human liver microsomes. To investigate the
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role that recombinant cytochrome P450 enzyme played in m-nisoldipine transformation, chemical
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inhibition experiment and the incubation with individual CYP450 enzyme were conducted.
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Although the authentic metabolite standard was not available in the present study, we could
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compare the relative production of each metabolite by using the area under the curve of controls
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versus treated groups.
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3.2.1 Chemical inhibition experiment
Table 2 showed the effects of selective inhibitors of CYPs on the three metabolites formation
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of m-nisoldipine in human liver microsomes. M7 was inhibited strongly by ticlopidine
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hydrochloride (inhibitor of CYP2C19), and to a lesser degree inhibited by ketoconazole (inhibitor
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of CYP3A4). Also furafylline and quinidine (inhibitor of CYP1A2 and CYP2D6) affected the
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formation in a minor extent. For M9, its production dropped obviously with the concentration of
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ketoconazole increasing. Other inhibitors prevented the formation slightly. M6 was inhibited mainly
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by ticlopidine hydrochloride and ketoconazole.
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3.2.2 Incubation of m-nisoldipine with the recombinant CYP450s
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From the chemical inhibition experiment, we considered CYP2C19 and CYP3A4 might be
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primarily responsible for the formation of the major metabolites. For further determination,
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m-nisoldipine (100 μM) was incubated with individual recombinant CYP450 enzyme (100 pmol
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P450/mL). Fig.4A showed a significant biodegradation of m-nisoldipine in CYP450 enzymes. The
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concentration of m-nisoldipine declined over 80% of the initial concentration in CYP2C19, and
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nearly 70% in CYP3A4. Also, approximately 20-40% m-nisoldipine was metabolized in CYP1A2,
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CYP2C9, CYP2D6 and CYP2E1. As shown in Fig.4B, at the same enzyme concentration (100 pmol P450/mL), CYP2C19 was
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the major enzyme for the formation of M7. CYP3A4 also catalysed M7 formation, but the level was
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20% of that formed by CYP2C19. Only a small amount of M7 could be detected in the incubations
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of CYP1A2 and CYP2D6, and it was hardly found in CYP2C9 or CYP2E1. In contrast, CYP3A4
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showed a high catalytic efficiency in M9 formation as shown in Fig.4C. The level of M9 formation
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was nearly 2 or 3 times of that formed in other CYP450 enzymes.
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In Fig.4D, CYP2C19 and CYP3A4 showed higher efficiency than other CYP450 enzymes
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clearly in formation of the M6. Moreover, the formation level of M6 in CYP3A4 was approximately
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3 times of that in CYP2C19. That meant a strong ability of CYP3A4 to catalyze both
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dehydrogenation and hydroxylation reactions. M7 formed in CYP3A4 might be more likely
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transformed into M6 because of the high efficiency of CYP3A4 in catalyzing dehydrogenation
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reaction. No M6 detected in CYP2C9 or CYP2E1 just the same as M7 shown in Fig.4B, this
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convinced us that neither CYP2C9 nor CYP2E1 could catalyze the hydroxylation reaction of
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m-nisoldipine in the incubation.
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3.3 Method assessment
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In this experiment, a LC-MS method was used to characterize the metabolites of m-nisoldipine
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according to former research [18-20]. The possible metabolites were identified by comparing the
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retention times and spectral patterns with the original drug. Peaks at identified m/z ratios did not
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have any interference observed in either blank or control samples.
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In order to get as much information as possible, three identification methods were used in our
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experiment. The EMS-IDA-EPI method provides most comprehensive information while it’s
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needed to get a completely separation of the metabolites. That usually takes a long time and there is
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also a difficult task for identification. Under the MRM-IDA-EPI mode, the possible structures of
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metabolites were auto-generated base on the original drug. And it is not necessary to get all the
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metabolites completely separated because the extract ion chromatograms give us the exact
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information without any interference. PREC-IDA-EPI method is good at monitoring a certain kind
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of compounds which can produce the given ion. That provides a supplement for early work so that
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as many metabolites as possible could be detected. Among the three methods, MRM-IDA-EPI may
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be considered as the most useful and effective method.
4. Conclusions
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m-Nisoldipine was mainly transformed in three primary metabolic pathways, including
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dehydrogenation of the dihydropyridine core, hydroxylation in the side chain and hydrolysis of ester
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bonds. Dehydrogenation and hydroxylation were considered as the major pathways in the
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metabolism of m-nisoldipine because of the large amounts of M6, M7 and M9 found in both human
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liver microsomes and recombinant CYP450 enzymes.
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Individual CYP450 enzyme incubation test showed that CYP2C19 had the ability to catalyze
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dehydrogenation, hydroxylation and hydrolysis reactions. CYP3A4 also played an important role in
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dehydrogenation and hydroxylation reactions, for its catalyzing efficiency was much higher than
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that of other CYP450 enzymes. Both CYP2C19 and CYP3A4 could be considered as the major
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enzymes in the biotransformation of m-nisoldipine in human liver microsomes. This study provided
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evidences in the clinical application and the combination of medicine therapy.
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In the present study, the metabolism in human liver microsomes of m-nisoldipine had been
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investigated. Further research is needed in patients to confirm the clinical significance of the
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metabolic pathways identified in this study so we can systematically clarify the metabolic
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mechanism of m-nisoldipine, and finally know the in vivo process of the drug more clearly.
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Acknowledgements
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This project was supported by the National Natural Science Foundation of China（No.
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81273475）. Furthermore, we also would like to thank Mr. Yumin Du, School of Pharmacy, Hebei
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Medical University, for his support in the study.
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[14] D.J. Newton, R.W. Wang, A.Y.H. Lu, Cytochrome P450 inhibitors: evaluation of specificities
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in the in vitro metabolism of therapeutic agents by human liver microsomes, Drug Metab. Dispos.
mass
spectrometry,
Rapid
Commun.
Mass
Spectrom.
d
tandem
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ionization
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14
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23(1995)154-158.
306
[15] M. Hu, K. Krausz, J. Chen, X. Ge, J.Q. Li, H.L. Gelboin, F.J. Gonzalez, Identification of
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CYP1A2 as the main isoform for the phase I hydroxylated metabolism of genistein and a prodrug
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converting enzyme of methylated isoflavones, Drug Metab. Dispos. 31(2003)924-931.
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[16] A.B. Baranda, R.M. Alonso, R.M. Jimenez, W. Weinmann, Instability of calcium channel
310
antagonists during sample preparation for LC-MS-MS analysis of serum samples, Forensic Sci. Int.
311
156(2006)23-34.
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[17] D.Z. Kong, S.L. Li, X.W. Zhang, Simultaneous determination of m-nisoldipine and its three
313
metabolites in rat plasma by liquid chromatography-mass spectrometry, J. Chromatogr. B.
314
878(2010)2989-2996.
315
[18] A. Deroussent, M. Ré, H. Hoellinger, T. Cresteil, Metabolism of sanguinarine in human and in
316
rat: Characterization of oxidative metabolites produced by human CYP1A1 and CYP1A2 and rat
317
liver microsomes using liquid chromatography–tandem mass spectrometry, J. Pharmaceut Biomed.
318
52(2010)391–397.
319
[19] L. Tang, L. Ye, C. Lv, Z.J. Zheng, Y. Gong, Z.Q. Liu, Involvement of CYP3A4/5 and CYP2D6
320
in the metabolism of aconitine using human liver microsomes and recombinant CYP450 enzymes,
321
Toxicol Lett. 202(2011)47–54.
322
[20] K. Choi, H. Joo, J. L. Campbell Jr., R. A. Clewell, M.E. Andersen, H.J. Clewell III, In vitro
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metabolism of di(2-ethylhexyl) phthalate (DEHP) by various tissues and cytochrome P450s of
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human and rat, Toxicol In Vitro. 26(2012)315–322.
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Figure Captions
326
Fig.1 LC–MS/MS chromatograms of m-nisoldipine incubations with human liver microsomes
327
Fig.2 Explanation of product ion spectra of m-nisoldipine and its synthetic derivatives S1–S3 used
328
as standards
329
Fig.3 Structures of m-nisoldipine metabolites and suggested scheme of biotransformation in human
330
liver microsomes
331
Fig.4 Transformation of m-nisoldipine and formation of the metabolites (%) in incubations of
332
c-DNA expressed recombinant CYP450 enzymes. (A) Transformation of m-nisoldipine; (B)
333
Formation of M7; (C) Formation of M9; (D) Formation of M6. The enzyme activities were an
334
average of two measurements
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*Graphical Abstract
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*Highlights (for review)
Highlights ► A simple and reliable LC-MS/MS method was developed to characterize 10 metabolites. ► The main metabolic rules of m-nisoldipine in human liver microsomes were summarize.
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► Chemical inhibition and incubation with individual CYP450 enzyme were
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Table(s)
Metabolites
Precursor ion(m/z)
tR (min)
M1
331
7.72
M2
391
8.02
M3
419
8.20
M4
421
8.45
M5
389
10.87
M6
403
11.92
M7
405
11.99
M8
375
M
Table 1. Chromatographic and mass spectrometric of metabolites of m-nisoldipine
M9
387
19.83
M10
429
ed
14.38
Dehydrogenation, 3-hydrolysis 299,285,168
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Hydroxylation, 5-demethylation 373,347,301,284 Dehydrogenation, dioxygen(position 3 and position 6) 401,347,331,299,285 Dioxygen(position 3 and position 6) 315,298,283,67 Dehydrogenation, hydroxylation(position 3), 3-hydrolysis 331,285 Dehydrogenation, hydroxylation(position 3) 331,299,285,168 Hydroxylation (position 3) 387,355,333,315,301,298,283 5- Demethylation 357,301,283 Dehydrogenation 331,299,285,168 Dehydrogenation,6- acetylation 373,317,299,285
Ac
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24.41
Proposed metabolic change Observed product ions(m/z)
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Table 2. Effects of CYP inhibitors on the metabolism of m-nisoldipine in human liver microsomes Relative percentage of control M6
M7
M9
0
100
100
100
Furafylline (CYP1A2)
20 50 100
98.2 95.3 93.1
95.3 89.7 86.6
97.3 94.8 90.5
Sulfaphenazole (CYP2C9)
20 50 100
95.7 93.1 90.6
92.8 90.2 89.3
95.1 92.7 88.1
Ticlopidine hydrochloride (CYP2C19)
20 50 100
90.4 81.3 70.9
73.5 51.9 40.1
98.3 92.3 90.2
Quinidine (CYP2D6)
5 10 20
97.5 95.1 92.7
96.7 83.1 80.9
96.4 91.7 90.1
Diethydithiocarbamate (CYP2E1)
10 20 50
96.3 94.1 90.1
90.3 88.4 87.1
98.1 92.3 83.5
89.7 75.4 68.3
92.8 87.2 75.6
82.8 61.3 45.5
1 2 5
Ac
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Ketoconazole (CYP3A4)
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Control
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Inhibitor concentration (μM)
Inhibitor
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Fig.1
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Fig.2
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Fig.3
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S0731-7085(14)00157-5 http://dx.doi.org/doi:10.1016/j.jpba.2014.03.030 PBA 9509
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
31-10-2013 17-3-2014 20-3-2014
Please cite this article as: L. Yuan, P. Jia, Y. Sun, C. Zhao, X. Zhi, N. Sheng, L. Zhang, Study of in vitro metabolism of m-nisoldipine in human liver microsomes and recombinant cytochrome P450 enzymes by liquid chromatographyndashmass spectrometry, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.03.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Study of in vitro metabolism of m-nisoldipine in human liver microsomes and recombinant
2
cytochrome P450 enzymes by liquid chromatography–mass spectrometry
3
Lin Yuan, Peipei Jia, Yupeng Sun, Chengcheng Zhao, Xuran Zhi, Ning Sheng, Lantong Zhang *
4
Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University,
5
Shijiazhuang, P. R. China
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∗ Corresponding author:
8
Prof. Lantong Zhang, Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical
9
University, Shijiazhuang, 050017, P. R. China
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E-mail address: [email protected]
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Tel: +86 311 86266419; fax: +86 311 86266419
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Abstract This is a report about the investigation of the metabolic fate of m-nisoldipine in human liver
14
microsomes and the recombinant cytochrome P450 enzymes by using LC-MS/MS. A sensitive and
15
reliable LC-MS/MS method was developed to obtain a rapid and complete characterization of new
16
metabolites and the metabolism pathways. The analytes were separated on a reversed phase C18
17
column with acetonitrile and 0.1% aqueous formic acid as the mobile phase. Tandem mass
18
spectrometry with positive electrospray ionization was used to enable the structural characterization
19
of the metabolites. A total of 10 metabolites were characterized with proposed structures in the
20
incubation of human liver microsomes by comparing their retention times and spectral patterns with
21
those of the parent drug. Dehydrogenation of the dihydropyridine core and reactions of side chains
22
such as hydroxylation and hydrolysis of ester bonds were the major metabolic pathways. The
23
specific cytochrome P450 (CYP) enzymes responsible for m-nisoldipine metabolites were identified
24
using chemical inhibition and cDNA expressed CYP enzymes. The results indicated that CYP2C19
25
and CYP3A4 might play major roles in the metabolism of m-nisoldipine in human liver
26
microsomes.
27
Keywords: m-Nisoldipine; Human liver microsomes; Recombinant cytochrome P450 enzymes;
28
LC-MS/MS; Metabolism
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1. Introduction m-Nisoldipine is a new dihydropyridine calcium channel antagonist that was first synthesized
31
in the School of Pharmacy at Hebei Medical University [1]. As the isomer of nisoldipine (the nitro
32
is in the 2-position of the phenyl system), m-nisoldipine has a similar structure but displays
33
significant stability when exposed to the light [2]. m-Nisoldipine is a vasodilator that could be used
34
in the treatment of hypertension. Previous studies indicated that m-nisoldipine was mainly excreted
35
as metabolites [3]. Therefore, the metabolism study of m-nisoldipine will play an important role in
36
clarifying the in vivo process of m-nisoldipine.
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Liver is the major organ involved in the biotransformation of various endogenous compounds
38
and drugs. The cytochrome P450 superfamily is a large and diverse group of enzymes that catalyze
39
the oxidation of organic substances. CYPs are the major enzymes involved in drug metabolism and
40
bioactivation, accounting for about 75% of the total number of different metabolic reactions [4].
41
The liver microsome metabolic model is widely used in the research of drug metabolism for its own
42
advantages [5,6]. Previous work showed that the main metabolites of dihydropyridine calcium
43
channel antagonist were products of oxidation of the 1,4-dihydropyridine ring, hydrolysis of the
44
ester group in the side chain of position 3 and position 5, and hydroxylation [7-8].
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Liquid chromatography-tandem mass spectrometry (LC-MS) has become a powerful and
46
reliable analytical approach for metabolite identification because of its high sensitivity, low
47
consumption and high speed of analysis [9-12]. This paper described the application of LC-MS for
48
supporting the study of the metabolism of m-nisoldipine in human liver microsomes. The
49
characterization of 10 metabolites was achieved by the LC-MS analysis and the possible metabolic
50
pathway was proposed.
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52
2. Experimental
53
2.1 Chemicals and Materials m-Nisoldipine (purity>99.5%) was provided by School of Pharmacy, Hebei Medical
55
University (Shijiazhuang, China). Dehydrogenation product of m-nisoldipine (S1) was synthesized
56
according to literature [2]. 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-2-carboxylic
57
acid-2-hydroxy-methyl isobutyl (S2) and 2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-2-carboxylic
58
acid-2-hydroxy-methyl isobutyl (S3) were obtained by microbial biotransformation for
59
identification. Pooled human liver microsomes and microsomes containing c-DNA expressed P450s
60
(CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4) were obtained from BD
61
Biosciences
62
dehydrogenase, sulfaphenazole, quinidine and diethyldithiocarbamate were purchased from Sigma
63
Chemical (St. Louis, MO, USA). Furafylline, ticlopidine hydrochloride and ketoconazole were
64
purchased from China Institute for Control of Pharmaceutical and Biological Products. Acetonitrile
65
and formic acid were HPLC grade (Tedia, Fairfield, USA). Ultra pure water was used for all
66
analysis. The other chemicals were all of analytical reagent.
68
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MA).
Glucose-6-phosphate,
β-NADP and
Glucose-6-phosphate
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M
(Woburn,
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2.2 Instruments
69
The chromatographic separation system consisted of a quaternary pump (Agilent 1200), an
70
online solvent degasser and an autosampler. 3200 QTRAPTM system from Applied Bios stems/MDS
71
Sciex (Applied Biosystems, Foster City, CA, USA), a hybrid triple quadrupole linear ion trap mass
72
spectrometer equipped with Turbo V sources and Turbo Ionspray interface. Instrument control and
73
data acquisition were carried out with Applied Biosystems/MDS Sciex Analyst software
74
(version1.4.2).
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HPLC analysis was performed at room temperature on a Diamonsil C18 column (5 µm, 250
76
mm×4.6 mm). The mobile phase consisted of acetonitrile (A) and 0.1% aqueous formic acid (B).
77
The gradient program started at 40% A, changed to 95% A within 20 min, held at 95% A for 5 min,
78
and finally went back to 40% A in 1 min. This was followed by an eight minutes equilibrium period
79
with initial conditions prior to injection of the next sample. The injection volume was 10 μL and the
80
flow rate was 0.8 mL/min.
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The mass spectral analysis was performed in a positive electrospray ionization mode. The
82
following parameters were optimized in order to achieve the highest sensitivity: turbo spray voltage
83
(IS) 5500V, turbo spray temperature 650°C, nebulizer gas (gas 1) 60 arbitrary units, heater gas (gas
84
2) 65 arbitrary units, curtain gas 25 arbitrary units, interface heater on. Nitrogen was used in all
85
cases. m-Nisoldipine and its metabolites were identified by EMS-IDA-EPI, MRM-IDA-EPI and
86
PREC-IDA-EPI mode.
d Ac ce p
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Typical incubation mixture [13] (1 mL final volume) was carried out in a 0.1 M K2HPO4 buffer
90
(pH 7.4) containing human liver microsomes (1.0 mg/mL), 3.3 mM MgCl2, 1.3 mM β-NADP, 3.3
91
mM glucose-6-phosphate, 1.0 U/mL glucose-6-phosphate dehydrogenase and 100 μM
92
m-nisoldipine methanol solution (1% of final volume). The mixture was pre-incubated for 5 min at
93
37°C and then initiated by adding NADPH generating system. After incubation at 37°C for 30 min,
94
1 mL ice-cold acetonitrile were added to stop the reaction. The mixture was extracted with ether:
95
n-hexane (50:50, v/v) 5 mL by vortex-mixing for 2 min. After centrifugation at 10,000×g for 10 min,
96
the organic phase was collected and evaporated under nitrogen gas. The residues were reconstituted
97
in 200 μL methanol and an aliquot (10 μL) was injected into the chromatographic system for
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98
analysis. Blank sample was incubated without m-nisoldipine while control sample was incubated
99
without NADPH generating system followed the same treatment.
100
2.4 Chemical inhibition
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The inhibition study was performed with three concentrations of the inhibitors, human liver
103
microsomes (1 mg/mL) and m-nisoldipine (100 μM). The chemical inhibitors used were as follows:
104
furafylline for CYP1A2, sulfaphenazole for CYP2C9, ticlopidine hydrochloride for CYP2C19,
105
quinidine for CYP2D6, diethyldithiocarbamate for CYP2E1 and ketoconazole for CYP3A4 [14].
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The inhibitors were pre-incubated for 30 min with human liver microsomes and NADPH system at
107
37°C, then the m-nisoldipine was added to initiate the reaction.
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108
111 112
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The incubation of microsomes containing individual CYP enzyme (100 pmol P450/mL) was
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2.5 Metabolism by recombinant cytochrome P450 enzymes
conducted the same as microsomal incubation.
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3. Results and discussion
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3.1 Characterization of metabolites in human liver microsomes and the recombinant cytochrome
115
P450 enzymes
116
Under the condition of electrospray ionization, m-nisoldipine (C20H24N2O6, MW 388 Da) can
117
easily form the [M+H]+ ion at m/z 389 in positive ion mode. Compared with the control and the
118
blank samples, 10 major metabolites (M1–M10, numbered according to retention time) were
119
detected (Fig.1). Each metabolite was identified on the basis of chromatographic behavior and
120
characteristic mass spectrometric fragmentation features observed in the LC-MS spectrums. The
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extracted ion chromatograms of metabolites showed that the peaks at identified m/z ratios did not
122
have any interference observed in either blank or control samples. The predominant fragmentation
123
patterns of m-nisoldipine and S1 were illustrated in Fig.2. The information of the four known
124
compounds facilitated the characterization of the 10 metabolites. The retention times and
125
characteristic fragments used to identify the metabolites were summarized in Table 1.
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3.1.1 Structural characterization of 10 metabolites in human liver microsomes
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For M1 eluting at 7.72 min, the [M+H]+ ion at m/z 331 was 56 Da less than that of S1. It
129
showed a series of product ions at m/z 299,285,168 and shared the same fragmentation pattern of S1.
130
That indicated that M1 was a metabolite generated by dehydrogenation and hydrolysis of the methyl
131
ester group in the side chain at position 3 of the m-nisoldipine. The MS2 spectrum of M2 ([M+H]+
132
at m/z 391) eluting at 8.02 min was compared with that of S2 because of the same fragment ions at
133
m/z 301 and 284. Other product ions at m/z 373 [M+H–H2O]+ and 347 [M+H–CO2]+ were found. In
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addition, with a mass of 14 Da lower than that of S2, M2 should be the product of hydroxylation in
135
the side chain at position 3 and hydrolysis of the methyl ester group at position 5.
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136
For M3 ([M+H]+ at m/z 419) eluting at 8.20 min, the same product ions as S1 (m/z
137
347,331,299 and 285) were detected, as well as another product ion at m/z 401 [M+H–H2O]+. The
138
[M+H]+ ion of M3 was 32 Da more than that of S1, which can be assigned to two hydroxyl groups
139
to the molecule. Based on the structure of product ions and other dihydropyridine calcium channel
140
blockers, the 6-hydroxymethyl metabolite was observed [15]. Thus, M3 was suggested as a
141
dehydrogenation and hydroxylation product of m-nisoldipine, and the localization of the hydroxyl
142
group should be at position 6 and in the side chain at position 3.
143
For M4 ([M+H]+ at m/z 421), eluting at 8.45 min, it was suggested to be a 1,4-dihydropyridine
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analogue to m-nisoldipine with two hydroxyl groups at position 6 and in the side chain at position 3.
145
The product ions were 2 Da more than those of M3, and M4 shared the same fragmentation pattern
146
of m-nisoldipine (m/z 315,298,283). M5 eluting at 10.87 min showed the [M+H]+ ion at m/z 389,
147
which was 2 Da less than that of M2. And the characteristic product ions at m/z 331 and 285 were
148
detected. Hence dehydrogenation, hydroxylation in the side chain at position 3 and hydrolysis of the
149
methyl ester group at position 5 were suggested for M5.
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M6 ([M+H]+ at m/z 403), M7 ([M+H]+ at m/z 405) and M9 ([M+H]+ at m/z 387), eluting at
151
11.92, 11.99 and 19.83 min respectively, showed identical product ion spectrums compared to S3,
152
S2 and S1. And with the retention time comparison of the standards under the same HPLC
153
conditions, M6, M7 and M9 could all be identified with no doubt. Although M7 was also detected
154
in the control sample, the concentration was far below. This is because that m-nisoldipine could be
155
oxidated not only by the enzyme in liver microsomes, but also by the air of oxygen [16]. The three
156
metabolites were also confirmed and detected in rat plasma, and were considered as major
157
metabolites of m-nisoldipine in our previous work [17].
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It was suggested the hydrolysis of the methyl ester group at position 5 happened for M8. The
159
[M+H]+ ion at m/z 375 was decreased by 14 Da when compared with that of m-nisoldipine, and the
160
characteristic product ions at m/z 357,301,283 also appeared. M10 showed the [M+H]+ ion at m/z
161
429. With a mass of 42 Da higher than that of S1, it may be an acetylated metabolite. The product
162
ions at m/z 373 and 347 were detected, as well as the similar ions of S1 (m/z 299, 285). For steric
163
reasons, acetylation at position 6 was more likely.
164 165 166
3.1.2 Metabolites detected in the recombinant cytochrome P450 enzymes The incubations of m-nisoldipine with the recombinant P450s respectively were analyzed just
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167
the same as the human liver microsomal incubation. 6 metabolites (M2, M5, M6, M7, M8 and M9)
168
were detected. M9 seemed to be the major metabolite because it could be found in all P450s in our
169
experiment with a concentration much higher than others. In the incubation samples of CYP1A2, 2D6 and 3A4, M6, M7 and M9 were found. While only
171
M8 and M9 were detected in both CYP2C9 and CYP2E1. CYP2C19 transformed m-nisoldipine
172
into 6 metabolites mentioned above. That indicated CYP2C19 may play an important role in the
173
metabolism of m-nisoldipine.
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In liver microsomes, drugs can be transformed by different enzymes so that more than one
175
reaction may happen. However, the individual CYP enzyme shows specificity in catalyzing. So the
176
metabolites which are products of reactions of different enzymes found in human liver microsomes
177
may not be detected in individual CYP enzyme.
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3.1.3 Metabolic pathway of m-nisoldipine
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According to the results above, 10 metabolites of m-nisoldipine were detected and
181
characterized in human liver microsomes. And the possible metabolic pathways of m-nisoldipine in
182
human liver microsomes were proposed as shown in Fig.3. That indicated dehydrogenation of the
183
dihydropyridine core and hydroxylation of the side chain at position 3 were major metabolic
184
pathways of m-nisoldipine.
185
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186
3.2 Identification of the recombinant cytochrome P450 enzymes involved in the metabolism of
187
m-nisoldipine
188
According to the metabolic pathways mentioned above, m-nisoldipine mainly transformed into
189
M7 and M9, and then both of them could convert into M6. That indicated the three structural
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Page 9 of 24
confirmed compounds to be the major metabolites in human liver microsomes. To investigate the
191
role that recombinant cytochrome P450 enzyme played in m-nisoldipine transformation, chemical
192
inhibition experiment and the incubation with individual CYP450 enzyme were conducted.
193
Although the authentic metabolite standard was not available in the present study, we could
194
compare the relative production of each metabolite by using the area under the curve of controls
195
versus treated groups.
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3.2.1 Chemical inhibition experiment
Table 2 showed the effects of selective inhibitors of CYPs on the three metabolites formation
199
of m-nisoldipine in human liver microsomes. M7 was inhibited strongly by ticlopidine
200
hydrochloride (inhibitor of CYP2C19), and to a lesser degree inhibited by ketoconazole (inhibitor
201
of CYP3A4). Also furafylline and quinidine (inhibitor of CYP1A2 and CYP2D6) affected the
202
formation in a minor extent. For M9, its production dropped obviously with the concentration of
203
ketoconazole increasing. Other inhibitors prevented the formation slightly. M6 was inhibited mainly
204
by ticlopidine hydrochloride and ketoconazole.
206
M
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3.2.2 Incubation of m-nisoldipine with the recombinant CYP450s
207
From the chemical inhibition experiment, we considered CYP2C19 and CYP3A4 might be
208
primarily responsible for the formation of the major metabolites. For further determination,
209
m-nisoldipine (100 μM) was incubated with individual recombinant CYP450 enzyme (100 pmol
210
P450/mL). Fig.4A showed a significant biodegradation of m-nisoldipine in CYP450 enzymes. The
211
concentration of m-nisoldipine declined over 80% of the initial concentration in CYP2C19, and
212
nearly 70% in CYP3A4. Also, approximately 20-40% m-nisoldipine was metabolized in CYP1A2,
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Page 10 of 24
213
CYP2C9, CYP2D6 and CYP2E1. As shown in Fig.4B, at the same enzyme concentration (100 pmol P450/mL), CYP2C19 was
215
the major enzyme for the formation of M7. CYP3A4 also catalysed M7 formation, but the level was
216
20% of that formed by CYP2C19. Only a small amount of M7 could be detected in the incubations
217
of CYP1A2 and CYP2D6, and it was hardly found in CYP2C9 or CYP2E1. In contrast, CYP3A4
218
showed a high catalytic efficiency in M9 formation as shown in Fig.4C. The level of M9 formation
219
was nearly 2 or 3 times of that formed in other CYP450 enzymes.
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In Fig.4D, CYP2C19 and CYP3A4 showed higher efficiency than other CYP450 enzymes
221
clearly in formation of the M6. Moreover, the formation level of M6 in CYP3A4 was approximately
222
3 times of that in CYP2C19. That meant a strong ability of CYP3A4 to catalyze both
223
dehydrogenation and hydroxylation reactions. M7 formed in CYP3A4 might be more likely
224
transformed into M6 because of the high efficiency of CYP3A4 in catalyzing dehydrogenation
225
reaction. No M6 detected in CYP2C9 or CYP2E1 just the same as M7 shown in Fig.4B, this
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convinced us that neither CYP2C9 nor CYP2E1 could catalyze the hydroxylation reaction of
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m-nisoldipine in the incubation.
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3.3 Method assessment
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In this experiment, a LC-MS method was used to characterize the metabolites of m-nisoldipine
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according to former research [18-20]. The possible metabolites were identified by comparing the
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retention times and spectral patterns with the original drug. Peaks at identified m/z ratios did not
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have any interference observed in either blank or control samples.
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In order to get as much information as possible, three identification methods were used in our
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experiment. The EMS-IDA-EPI method provides most comprehensive information while it’s
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needed to get a completely separation of the metabolites. That usually takes a long time and there is
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also a difficult task for identification. Under the MRM-IDA-EPI mode, the possible structures of
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metabolites were auto-generated base on the original drug. And it is not necessary to get all the
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metabolites completely separated because the extract ion chromatograms give us the exact
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information without any interference. PREC-IDA-EPI method is good at monitoring a certain kind
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of compounds which can produce the given ion. That provides a supplement for early work so that
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as many metabolites as possible could be detected. Among the three methods, MRM-IDA-EPI may
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be considered as the most useful and effective method.
4. Conclusions
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m-Nisoldipine was mainly transformed in three primary metabolic pathways, including
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dehydrogenation of the dihydropyridine core, hydroxylation in the side chain and hydrolysis of ester
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bonds. Dehydrogenation and hydroxylation were considered as the major pathways in the
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metabolism of m-nisoldipine because of the large amounts of M6, M7 and M9 found in both human
250
liver microsomes and recombinant CYP450 enzymes.
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Individual CYP450 enzyme incubation test showed that CYP2C19 had the ability to catalyze
252
dehydrogenation, hydroxylation and hydrolysis reactions. CYP3A4 also played an important role in
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dehydrogenation and hydroxylation reactions, for its catalyzing efficiency was much higher than
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that of other CYP450 enzymes. Both CYP2C19 and CYP3A4 could be considered as the major
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enzymes in the biotransformation of m-nisoldipine in human liver microsomes. This study provided
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evidences in the clinical application and the combination of medicine therapy.
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In the present study, the metabolism in human liver microsomes of m-nisoldipine had been
258
investigated. Further research is needed in patients to confirm the clinical significance of the
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metabolic pathways identified in this study so we can systematically clarify the metabolic
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mechanism of m-nisoldipine, and finally know the in vivo process of the drug more clearly.
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Acknowledgements
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This project was supported by the National Natural Science Foundation of China（No.
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81273475）. Furthermore, we also would like to thank Mr. Yumin Du, School of Pharmacy, Hebei
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Medical University, for his support in the study.
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Figure Captions
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Fig.1 LC–MS/MS chromatograms of m-nisoldipine incubations with human liver microsomes
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Fig.2 Explanation of product ion spectra of m-nisoldipine and its synthetic derivatives S1–S3 used
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as standards
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Fig.3 Structures of m-nisoldipine metabolites and suggested scheme of biotransformation in human
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liver microsomes
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Fig.4 Transformation of m-nisoldipine and formation of the metabolites (%) in incubations of
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c-DNA expressed recombinant CYP450 enzymes. (A) Transformation of m-nisoldipine; (B)
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Formation of M7; (C) Formation of M9; (D) Formation of M6. The enzyme activities were an
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average of two measurements
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*Graphical Abstract
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*Highlights (for review)
Highlights ► A simple and reliable LC-MS/MS method was developed to characterize 10 metabolites. ► The main metabolic rules of m-nisoldipine in human liver microsomes were summarize.
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► Chemical inhibition and incubation with individual CYP450 enzyme were
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Table(s)
Metabolites
Precursor ion(m/z)
tR (min)
M1
331
7.72
M2
391
8.02
M3
419
8.20
M4
421
8.45
M5
389
10.87
M6
403
11.92
M7
405
11.99
M8
375
M
Table 1. Chromatographic and mass spectrometric of metabolites of m-nisoldipine
M9
387
19.83
M10
429
ed
14.38
Dehydrogenation, 3-hydrolysis 299,285,168
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Hydroxylation, 5-demethylation 373,347,301,284 Dehydrogenation, dioxygen(position 3 and position 6) 401,347,331,299,285 Dioxygen(position 3 and position 6) 315,298,283,67 Dehydrogenation, hydroxylation(position 3), 3-hydrolysis 331,285 Dehydrogenation, hydroxylation(position 3) 331,299,285,168 Hydroxylation (position 3) 387,355,333,315,301,298,283 5- Demethylation 357,301,283 Dehydrogenation 331,299,285,168 Dehydrogenation,6- acetylation 373,317,299,285
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Proposed metabolic change Observed product ions(m/z)
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Table 2. Effects of CYP inhibitors on the metabolism of m-nisoldipine in human liver microsomes Relative percentage of control M6
M7
M9
0
100
100
100
Furafylline (CYP1A2)
20 50 100
98.2 95.3 93.1
95.3 89.7 86.6
97.3 94.8 90.5
Sulfaphenazole (CYP2C9)
20 50 100
95.7 93.1 90.6
92.8 90.2 89.3
95.1 92.7 88.1
Ticlopidine hydrochloride (CYP2C19)
20 50 100
90.4 81.3 70.9
73.5 51.9 40.1
98.3 92.3 90.2
Quinidine (CYP2D6)
5 10 20
97.5 95.1 92.7
96.7 83.1 80.9
96.4 91.7 90.1
Diethydithiocarbamate (CYP2E1)
10 20 50
96.3 94.1 90.1
90.3 88.4 87.1
98.1 92.3 83.5
89.7 75.4 68.3
92.8 87.2 75.6
82.8 61.3 45.5
1 2 5
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Ketoconazole (CYP3A4)
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Control
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Inhibitor concentration (μM)
Inhibitor
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Fig.4
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