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University Hospital Tübingen, Department of Clinical Pharmacology, Tübingen, Germany (B.K., C.H.G.); Eberhard-Karls-University Tübingen, Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Tübingen, Germany (H.S., S.L.); and Merckle GmbH, Ulm, Germany (W.A.)
(Received October 19, 2006; accepted March 6, 2007)
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Abstract |
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, interleukin-1ß, and interleukin-6. Experimental models suggested that p38 inhibition can lead to an improvement or even remission of inflammatory diseases like arthritis (Berenbaum, 2004
; Peifer et al., 2006), Crohn's disease (Hommes et al., 2002), or asthma (Newton and Holden, 2003; Barnes, 2004). Consequently, p38 inhibitors have been considered as anti-TNF- therapeutics that could potentially replace established anti-TNF antibodies or TNF receptors. Many pharmaceutical companies initiated p38 discovery programs (Hynes and Leftheri, 2005; Natarajan and Doherty, 2005), but despite the fact that a few candidates have meanwhile reached phase II of clinical development, safety issues appear to represent the main hurdle for a successful development. SB203580 represents the prototype p38 inhibitor and has been frequently used as a benchmark for the evaluation of new compounds (Wagner and Laufer, 2006). SB203580 and most other p38 inhibitors bind to the enzyme's ATP-binding pocket, which is a highly conserved region of the so-called serine-threonine protein kinases. Thus, the selectivity profile, i.e., the extent of p38 inhibition versus inhibition of other protein kinases, represents a critical feature. In addition, many imidazoles are known to inhibit cytochrome P450 enzymes (P450), which have been associated with liver toxicity.
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). Pharmacokinetic or metabolism data, however, are essentially limited to in vitro tests on the inhibition of P450 isoforms. Detailed characteristics have been published for SB203580 (Ward et al., 2001) and a follow-up candidate, SB242235 (Ward et al., 2002a, b). Based on in vitro metabolism and pharmacokinetic studies in different animal species, both compounds were characterized as metabolically stable. Excretion data suggested renal elimination of the unchanged compound as the predominant mechanism of disposition. However, the fact that systemic exposure to both drugs increased in an over-proportional manner with ascending doses, together with the known affinity of 4-pyridinyl imidazoles to cytochrome P450 enzymes, suggests that metabolic elimination might contribute to their pharmacokinetics.
Several modifications of the diarylimidazoles resulted in an improvement of p38 MAP kinase selectivity and a reduction of P450 inhibition (Laufer et al., 2003). However, keeping in mind that minor modifications of the chemical structure could have a dramatic impact on the protein kinase selectivity, metabolism studies with protein kinase inhibitors are of great importance. ML3403 ({4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine) (Fig. 1) belongs to the second generation of diarylimidazole-type p38 MAP kinase inhibitors (Laufer et al., 2003). The potency toward p38 is approximately 2-fold compared with SB203580, and in BALB/c mice, the lipopolysaccharide/D-galactosamine-induced release of TNF- was completely inhibited when doses of 30 and 100 mg/kg body weight ML3403 were orally administered 1 h before the lipopolysaccharide/D-galactosamine challenge. Preliminary in vitro metabolism studies demonstrated a rapid oxidation of the sulfanyl group to the corresponding sulfoxide, denoted as ML3603 ({4-[5-(4-fluorophenyl)-2-methylsulfinyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine). The in vitro pharmacological profile of the metabolite was similar to that of the parent drug, but with a slightly reduced P450 inhibition potency (inhibition of CYP1A2, 2C9, 2C19, 2D6, and 3A4 at 10 µM by ML3403/ML3603: 68/52%, 76/60%, 66/54%, 33/23%, and 85/61%).
Besides ML3603, additional metabolites were detected. Detailed analyses confirmed that ML3403 and ML3603 were also subject to pyridine-N-oxidation and dealkylation. Based on the identification of metabolites, a biotransformation pathway has been proposed. Because these initial studies were carried out with liver microsomes from CD-1 mice, the relevance of the identified metabolites remained unknown. Therefore, to evaluate the importance of the metabolism of ML3403, in vitro metabolism studies with liver microsomes from different species, i.e., mouse, rat, primate, and human, were performed. In addition, the intrinsic clearance of ML3403 was determined in liver microsomes, and human recombinant P450 isoforms involved in the biotransformation were identified. In a pilot pharmacokinetic study, ML3403 was administered to male and female Wistar rats. Plasma samples were used for quantification of the parent drug and the main metabolite ML3603 as well as for detection of additional metabolites. It could be demonstrated that metabolites formed in Wistar rats in vivo are identical to those obtained in in vitro studies.
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Materials and Methods |
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NADP+ sodium salt, Trizma base and Trizma HCl, magnesium chloride hexahydrate, glucose 6-phosphate sodium salt, and glucose-6-phosphate dehydrogenase from yeast were purchased from Sigma-Aldrich Chemie GmbH (Muenchen, Germany); human plasma of different donors was kindly provided by Blutbank/UKT (University Hospital Tübingen, Germany). Water was used from an in-house bi-distillation device (Department of Pharmaceutical Biology, University of Tübingen, Germany).
In Vitro Metabolism Studies. Liver microsomes of CD-1 mice, rat (RLMs), cynomolgus monkey, and human (HLMs) prepared by the Department of Drug Research, Merckle GmbH (Ulm, Germany), were used. These liver microsomes were characterized in protein and cytochrome P-450 content.
Baculovirus cDNA-expressed isoenzymes (Supersomes) of CYP1A2 with P450 reductase, CYP2C8 with P450 reductase/cytochrome b5, CYP2C9*1 (Arg144) with P450 reductase/cytochrome b5, CYP2C19 with P450 reductase/cytochrome b5, CYP2D6*1 with P450 reductase, CYP3A4 with P450 reductase/cytochrome b5, flavin monooxygenase isoform 3 (FMO3), Supermix, P450 reductase/cytochrome b5 insect cell control, insect cell control, and pooled male and female RLMs and HLMs were purchased from BD Gentest (Woburn, MA).
All incubations (final total volume 500 µl) were made in the presence of an NADPH-regenerating system consisting of 5 mM glucose 6-phosphate, 1 IU/ml glucose-6-phosphate dehydrogenase, and 1 mM NADP+. ML3403 (2 µM), NADPH regenerating system, and 1.6 mM MgCl2 x 6H2O in 0.1 M Tris buffer (pH 7.4) were preincubated for 10 min in a shaking water bath at 37°C, 50 rpm, and the reaction was started by addition of microsomes (0.5 mg protein/ml or 25 pmol P450/mg). To follow the time course of metabolism, at different time points (0, 10, 30, 60, and 90 min), 50-µl aliquots were withdrawn and transferred into an ice-cooled vial containing 150 µl of H2O, 150 µl of ACN, and 50 µl of internal standard solution (ML3435 and ML3596 in concentrations of 100 ng/ml each). The samples were vortexed for 15 s and centrifuged (19,800 relative centrifugal force/4°C/10 min). The supernatant was directly used for analysis. All incubations were conducted in triplicates; average mean values of these incubations are shown in the figures. In all incubations a limit of 1% organic solvent was not to be exceeded (Chauret et al., 1998; Busby et al., 1999).
Clearance Predictions by Substrate Depletion Approach (in Vitro t
Method). For microsomal incubations of different species, the substrate depletion approach was used to calculate the in vitro intrinsic clearance (Obach et al., 1997). ML3403 was incubated for 90 min with the same protein content in each of the different species' liver microsomes. Five aliquots were drawn at time points 0, 10, 30, 60, and 90 min as described above. ML3403 concentrations were then determined by the online solid-phase extraction (SPE)-LC-MS/MS method and normalized to the amount of ML3403 at t = 0 min. The logarithm of the percentage remaining versus time was fitted by least-squares linear regression of at least four time points. If substrate decline demonstrated nonlinearity, only those initial time points where log-linearity was observed were used to determine the depletion rate constant. The in vitro t for the microsomes was used to calculate CLint for microsomal incubations:
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Liver weights and the standard value of 45 mg of microsomal protein content per gram of liver of mouse, rat, monkey, and human were taken from Davies and Morris (1993) and Houston (1994).
In vivo clearance, CLp in vivo, was calculated from in vitro intrinsic clearance, CLint microsomes, using the well stirred and parallel-tube models (Pang and Rowland, 1977), where Q is the hepatic blood flow (Davies and Morris, 1993) and fu represents the free fraction in blood (here considered as 1). Well stirred model:
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Parallel-tube model:
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Determination of Kinetic Parameters for the Sulfoxidation of ML3403 to ML3603. km and Vmax were determined in pooled female and male human liver microsomes, using baculovirus cDNA-expressed isoenzymes (Supersomes) of CYP1A2 with P450 reductase, CYP2C19 with P450 reductase/cytochrome b5, CYP2D6*1 with P450 reductase, and CYP3A4 with P450 reductase/cytochrome b5. The substrate concentrations ranged from 0.1 µM to 50 µM. The protein content and time linearity were proven in the preceding analysis. The protein content determined was 0.03 mg/ml for HLMs and the cytochrome P450 content was 0.5 pmol/ml for cDNA-expressed isoenzymes. Incubations were conducted in a total volume of 250 µl for an incubation time of 10 min, and were performed in duplicates for the liver microsomes or triplicates for the rP450. Data reported are averages ± standard errors of the determinations. Estimation of the enzyme kinetic parameters was made by fitting to the Michaelis-Menten equation by best-fit nonlinear regression using the Enzyme Kinetics Module 1.1/Sigmaplot 8.02a software (Systat Software Inc., Point Richmond CA). Goodness of fit was assessed by calculation of the correlation coefficient (R2) and Akaike information criterion and visual examination of the data.
The microsomal and the CYP2D6 metabolite appearance data were best described using the Substrate Inhibition Kinetic model (Tracy and Hummel, 2004).
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All other Supersomes metabolite appearance data were modeled with the typical Michaelis-Menten equation
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The "total normalized rate" (TNR) of each recombinant isoenzyme is calculated based on the method described by Rodrigues (1999).
Pharmacokinetic Studies in Wistar Rats. The animal experimental part of this study was performed at Charles River Wiga Deutschland GmbH (Kisslegg, Germany). ML3403 was administered to eight female and male Wistar rats with a single oral gavage (p.o.) dose of 30 mg/kg body weight. The drug formulation was prepared by grinding 60 mg of ML3403 in a mortar; a small amount of 1% methylcellulose was added until a smooth paste was obtained. The paste was transferred into a graduated flask and made up with 1% methylcellulose to a volume of 10 ml. A dose of 5 ml/kg was administered with continuous stirring of the suspension.
For blood sampling, groups were allocated to schedules A or B. In schedule A, five samples were taken at time points 0 h (predose), 0.5 h, 2.0 h, 6.0 h, and 11.0 h after dosing in animals 1 to 4. In schedule B, five samples were taken at time points 0 h, 1.0 h, 4.0 h, 9.0 h, and 24.0 h after dosing in animals 5 to 8.
Blood samples of approximately 300 µl were drawn from the vena jugularis through an indwelling catheter into lithium-heparinized Eppendorf tubes. The catheter tube was removed 1 to 2 cm out of the subcutaneous spinal pouch and rinsed with a 0.2- to 0.3-ml heparin-glucose solution. The blood samples were then withdrawn with a 1-ml syringe and a prepared pipette tip. The catheter tube was rinsed again and pushed back into the subcutaneous spinal pouch. Blood samples were centrifuged for 10 min at 3000 rpm at 10°C, and plasma (approximately 150 µl) was transferred into Eppendorf tubes and stored at -80°C. Pharmacokinetic data were evaluated according to a noncompartmental pharmacokinetic model using the software WinNonlin 4.0.1 (Pharsight Corp., Mountain View, CA).
Online LC-SPE-MS/MS Analysis of Plasma Samples. For quantitative analysis of ML3403 and ML3603 in plasma samples, a fast and simple method was developed. The method is based on an online SPE in the first dimension to remove polar matrix elements coupled to a reversed-phase chromatography in the second dimension with a sensitive triple quadrupole mass spectrometric (MS) detection in the selective reaction monitoring (SRM) mode. With this method ML3403 and ML3603 is capable of being analyzed with two internal standards in 5 min without time-consuming offline extraction steps.
Plasma samples were thawed at room temperature. An aliquot of 50 µl was drawn and mixed with 50 µl of internal standard solution containing 100 ng/ml ML3435 and ML3596 each. For protein precipitation, 300 µl of ACN was added and the sample was thoroughly vortexed for 15 s. After centrifugation at 10°C with 19,800 relative centrifugal force, the supernatant was transferred into high-performance liquid chromatography vials and directly used for analysis.
Nine standard solutions (2.5, 5, 10, 50, 500, 1000, and 2500 ng/ml) and three quality controls (3.75, 75, and 1250 ng/ml) were prepared by spiking human plasma of different donors. Blank human plasma and predose rat plasma were used to confirm the specificity of the method.
Online SPE was performed on an Oasis HLB cartridge (Waters, Eschborn, Germany) with an isocratic clean-up flow of H2O/ACN/FA (98:2:0.1) at 2.5 ml/min. For the chromatographic separation, a Grom 120 SIL-ODS 4 HE (50 x 2 mm, 5 µm) with a precolumn of the same material (Grom Analytik, Rottenburg, Germany) was used with a linear binary gradient of solvent A (water/0.1% FA) and B (ACN/0.1% FA) at a flow rate of 500 µl/min during 5 min, beginning with 20% B for 0.5 min and an increase to 100% B at 4.5 min until the end. After each injection, the SPE cartridge was regenerated with an injection of 100 µl of methanol/ACN/H2O/FA (10:10:4:0.1 v/v) within 1.5 min. The gradient for the wash step was 100% B for 0.5 min, changing to 20% B in 0.05 min, holding to the end. The detection was performed on a TSQ Quantum triple quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA) in the electrospray ionization-positive SRM mode. This method was validated with respect to matrix effects, linearity, selectivity, intra- and interday precision, and accuracy. The linear range of the method extends from 2.5 ng/ml (limit of quantitation) to 2500 ng/ml. All metabolite areas of the SRM traces were related to the area of the 100 ng/ml internal standard ML3596 and multiplied by 100. This procedure also served for semiquantification of metabolites for which reference material was not available.
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Detection was performed either on a Surveyor PDA (Thermo Fisher Scientific) at 320 nm or on the TSQ Quantum triple quadrupole mass spectrometer in electrospray ionization-positive mode. Spray voltage was set at 3.2 kV and the heated capillary operated at 320°C. Sheath gas and auxiliary gas flow rate worked at 40 and 10 arbitrary units, respectively. Eluates from the first and the last 8 min were diverted to waste to prevent contamination of the interface of the mass spectrometer by matrix components.
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Results |
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The main metabolite of ML3403 was the corresponding sulfoxide, ML3603. Kinetic analyses demonstrated that ML3603 reached a concentration maximum between 5 and 30 min. Further degradation led to the formation of the corresponding N-oxide (M-437-pyr), M-sulfone, and the dealkylation product M-317 (Fig. 2). All of the identified metabolites could be found in different amounts in liver microsomes of all the examined species. In comparison to the other species, microsomes from cynomolgus monkey converted ML3403 much faster into the pyridine N-oxide metabolites M-421-pyr and M-437-pyr.
In Vitro Metabolism in Baculovirus-Expressed Human P450 Isoenzymes. To identify the enzymes involved in the biotransformation of ML3403, incubations with six cytochrome P450 isoenzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) were performed. For the evaluation of the concentration-time profiles of substrate and metabolites, a mixture of the six most abundant individual baculovirus cDNA-expressed cytochrome P450 isoenzymes (Supermix) was compared with the time profiles produced by HLMs.
To this end, individual Supersomes containing CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were combined to simulate the average relative enzyme activities described for HLMs. In this Supermix assay, the rate of degradation of ML3403 as well as the metabolite profile was similar to that observed with HLMs. Thereafter, the potential involvement of FMO3 in the metabolism of ML3403 was tested. Incubations without NADPH and with cytochrome P450 reductase/b5 without P450 enzymes served to exclude nonenzymatic metabolite formation.
Incubations with FMO3, with control microsomes and without NADPH, did not lead to an increase in any of the metabolites. Incubations with microsomes that were heated to 45°C for 5 min to inactivate FMOs showed no difference from incubations with freshly thawed microsomes. This result demonstrates that the cytochrome P450 enzyme system is responsible for the phase I biotransformation of ML3403.
In the sulfoxidation reaction to ML3603 and further oxidation to M-sulfone, four cytochrome isoenzymes are primarily involved: CYP1A2, CYP2C19, CYP2D6, and CYP3A4. Figure 3 shows the percentage of ML3603 and M-sulfone formation after an incubation of 10 min in relation to the amount of ML3403 in the control incubation.
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Involvement of Different rP450s of the Sulfoxidation Reaction by Percentage TNR. Michaelis-Menten kinetics of ML3403 degradation was determined for the P450 isoforms 1A2, 2C19, 2D6, and 3A4, which are predominantly responsible for S-oxidation (Fig. 5). The kinetic constants km and Vmax of each P450 isoform were derived by nonlinear regression of volume/concentration data pairs and used for calculation of their contribution in vivo using the TNR approach. In the sulfoxidation reaction to ML3603, CYP3A4 was the major isoenzyme calculated to contribute 50% to the reaction, followed by CYP1A2 with 32% and CYP2D6 with about 16% (Table 1).
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In Vitro Metabolism in Female and Male Rat and Human Liver Microsomes. To examine gender-specific differences, incubations of male and female pooled RLMs were compared with incubations with male and female pooled HLMs. The time profile of the RLMs showed clearcut differences in metabolite formation rates. Male RLMs produced ML3603 and M-sulfone faster, compared with female RLMs (Fig. 3). In HLMs, gender differences were much less distinct, especially when considering the higher P450 enzyme activity of the female microsomes. Michaelis-Menten kinetic measurements in pooled male and female HLMs resulted in km values for ML3403 sulfoxidation that did not differ significantly (Fig. 5).
Intrinsic Clearance Estimation in Different Species by Substrate Depletion Approach. For the prediction of in vivo intrinsic clearance by the substrate depletion approach, the disappearance rate of ML3403 was measured in microsomes from different species at a low substrate concentration of 2 µM. The determined in vitro half-time (t) was very fast in mouse (t = 6 min) and rat (t = 11 min), whereas a strong difference between female (t = 166 min) and male (t = 9 min) rats was observed. The calculated plasma clearances (Cp) using the well stirred model were slightly lower than the Cp values using the parallel-tube model. A compilation of the parameters is given in Table 2.
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The individual and derived mean concentration-time profiles reflect a rapid absorption of the parent drug and rapid appearance of the metabolite. The concentrations of the metabolite ML3603 were substantially higher than those of ML3403. Monophasic elimination kinetics were observed for both compounds. In female animals, concentration-time profiles could be followed for 24 h. In contrast, plasma concentrations in male rats were below the lower limit of quantitation of 2.5 ng/ml at 24 h postdose.
The kinetic parameters of animals 1 to 4 were derived from data measured at 0 to 11 h and for animals 5 to 8 at 0 to 24 h. Therefore, AUC0-t values determined for animals 1 to 4 cannot be directly compared with those of animals 5 to 8. To allow the calculation of the total exposure toward ML3403 plus ML3603, the parameters were converted into µM for Cmax and µM · h for AUC0-t. Calculated pharmacokinetic parameters for ML3403 and ML3603 based on mean concentration-time profiles are given in Table 3 and the calculated female/male ratios of the parameters Cmax and AUC are given in Table 4.
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). All these metabolites could be identified in plasma samples of Wistar rats after oral gavage of ML3403. Identification of Phase II Metabolites in Wistar Rats. Besides the major metabolites, two dominant peaks could be detected in rat plasma samples with protonated molecule peaks at 331 m/z (M-331) and 345 m/z (M-345), respectively. The MS/MS spectra of M-317 versus M-331 and M-345 revealed characteristic shifts of 14 Da and 28 Da (Fig. 7). The proposed fragment ions confirm the suggested structures of the two phase II metabolites as N-methylated derivatives of the sulfoxide from which the phenylethyl group is removed (M-317).
Additionally, two minor unknown peaks with a [M + H]+ at 359 Da (M-359) and 361 Da (M-361) could be identified in the chromatogram. According to MS/MS and in-source pseudo-MS3 spectra, the two metabolites could be characterized as two more phase II metabolites. The chemical structure of M-359 could be assigned as the acetyl-conjugated derivative (+42 Da) of M-317 with a characteristic loss of CH3CO (Levsen et al., 2005) in MS, and M-361 was identified as the dimethyl-conjugated metabolite of M-333 (+28 Da). The structures and characteristics of all identified in vivo metabolites are given in Fig. 2 and Table 5.
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Discussion |
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CYP3A4 was identified also as the main contributor for mediating the N-dealkylation of the phenylethyl moiety. The resulting primary amine undergoes conjugation reactions in rats: N-methylation, N-dimethylation, and N-acetylation.
In an extension of the in vitro study, the in vivo pharmacokinetics of ML3403 in Wistar rats were examined. After oral gavage, ML3403 showed a fast and high turnover into its active sulfoxide metabolite ML3603 comparable to the in vitro findings. In the plasma samples, the tmax values of ML3403 and ML3603 were identical (1 h postdose). The fast metabolic conversion in vitro and the high involvement of the P450 isoenzyme 3A4, known to be present also in the small intestine (Kolars et al., 1992; Hall et al., 1999), are suggestive of a first-pass metabolism.
The calculated pharmacokinetic data clearly demonstrate a substantially higher systemic exposure of female animals to both the parent drug ML3403 and the sulfoxide metabolite ML3603. However, the systemic elimination of both compounds shows highly similar kinetics in male and female animals. Therefore, gender-specific differences of processes responsible for systemic elimination are unlikely (Ripp et al., 1999). The influence of gender-specific metabolism in rat was shown in incubations with male and female RLMs. Male rats showed a faster conversion into all metabolites; in particular, the oxidative removal of the phenylethyl moiety proceeded faster than in female rats. Besides gender-specific mechanisms during drug absorption and/or presystemic elimination, this could be a reason for the higher systemic exposure to ML3603 in female rats. For HLMs gender-specific differences could not be shown in vitro.
In conclusion, all in vitro metabolites could be detected in rat plasma samples; four new conjugated in vivo metabolites (M-331, M-345, M-359, and M-361) were described. The main oxidative pathway is predominantly catalyzed by CYP3A4, CYP1A2, and CYP2D6; for the desalkylation and pyridine N-oxidation, CYP3A4 is nearly solely responsible.
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Acknowledgments |
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Footnotes |
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B.K. and H.S. contributed equally to this work.
ABBREVIATIONS: MAP, mitogen-activated protein; p38, p38 MAP kinase; ACN, acetonitrile; P450, cytochrome P-450 enzyme; FA, formic acid; p.a., proanalysi; LC, liquid chromatography; MS/MS, tandem mass spectrometry; rP450, recombinant P450 isoenzyme; SPE, solid-phase extraction; SRM, selective reaction monitoring; TNF, tumor necrosis factor; SB203580, 4-[5-(4-fluorophenyl)-2-(4-methanesulfinyl-phenyl)-3H-imidazol-4-yl]-pyridine; SB242235, 5-[5-(4-fluorophenyl)-3-piperidin-4-yl-3H-imidazol-4-yl]-2-methoxy-pyrimidine; HLM, human liver microsome; ML3603, {4-[5-(4-fluorophenyl)-2-methylsulfinyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine; ML3403, {4-[5-(4-fluorophenyl)-2-methylsulfa-nyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine; M-sulfone, {4-[5-(4-fluorophenyl)-2-methylsulfonyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine; ML3435, {4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-phenyl-amine; ML3596, {4-[5-(4-fluorophenyl)-2-methylsulfinyl-3H-imidazol-4-yl]-pyridin-2-yl}-phenyl-amine; RLM, rat liver microsome; M-437-pyr, {4-[5-(4-fluorophenyl)-2-methanesulfinyl-3H-imidazol-4-yl]-1-oxy-pyridin-2-yl}-(1-phenyl-ethyl)-amine; M-421-pyr, {4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-1-oxy-pyridin-2-yl}-(1-phenylethyl)-amine; M-317, 4-[5-(4-fluorophenyl)-2-methanesulfinyl-3H-imidazol-4-yl]-pyridin-2-ylamine; CL, clearance; TNR, total normalized rate.
Address correspondence to: Dr. Bernd Kammerer, University Hospital Tübingen, Otfried-Müller-Str. 45, D-72076 Tübingen, Germany. E-mail: bernd.kammerer{at}uni-tuebingen.de
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