Dmd057570 1252..1260

Abstract

KRO-105714 [N-(5-benzoyl-2-(4-(2-methoxyphenyl)piperazin-1-yl) thiazol-4-yl)pivalamide] is a 2,4,5-trisubstituted 1,3-thiazole derivative that exerts anti–atopic dermatitis activity via robust suppression of the sphingosylphosphorylcholine receptor. This study used high-resolution/high-accuracy tandem mass spectroscopy (HRMS) and recombinant cDNA–expressed cytochrome P450 (P450) isoforms to identify the metabolic pathway and metabolites of KRO105714 in human liver microsomes (HLMs) as therapeutic agents for inflammation. The incubation of KRO-105714 with pooled HLMs in the presence of NADPH generated four metabolites (M1–M4). The metabolites were identified using HRMS and confirmed using synthetic standards for M2 and M4. M1 and M2 were identified as monohydroxylated metabolites, and M3 and M4 were identified as O-demethyl KRO-105714 and C-demethyl KRO-105714, respectively. In the inhibition study with selective CYP3A4 inhibitors and incubation in recombinant cDNA–expressed P450 enzymes, all the metabolites of KRO-105714 were formed by CYP3A4 in HLMs. The CYP3A4-mediated formation of M4 from M2 was confirmed via incubation of M2 in HLMs. These results showed that the unusual C-demethylated metabolite M4 was generated from monohydroxyl metabolite M2 via a CYP3A4-mediated enzymatic reaction in HLMs. Introduction Sphingosylphosphorylcholine (SPC) is structurally similar to sphingosine 1-phosphate, which along with lysophosphatidic acid is classified as a lysophospholipid. These substances act as important signaling intermediates in the cell proliferation, migration, and inflammatory responses of immune function (Nixon et al., 2008). SPC is generated by the action of sphingomyelin deacylase from sphingomyelin, a structural component of cell membranes (Higuchi et al., 2000; Nixon et al., 2008). Furthermore, SPC displays a wide range of biologic activities such as angiogenesis, activation of calcium signaling, protection of cells from apoptosis, and stimulation of nitric oxide production (Desai and Spiegel, 1991; Chen et al., 1998; Boguslawski et al., 2000; Higuchi et al., 2000; Jeon et al., 2005). Atopic dermatitis is an example of an SPC-related disease (Imokawa, 2009). Patients with the disease experience pain, itching, and poor quality of life. Direct intradermal injection of SPC has shown that SPC causes itching owing to its structural similarity to lysophosphatidic acid (Hashimoto et al., 2004). KRO-105714 [N-(5-benzoyl-2-(4-(2-methoxyphenyl)piperazin-1-yl) thiazol-4-yl)pivalamide] is a 2,4,5-trisubstituted 1,3-thiazole derivative and therapeutic agent for anti-inflammatory diseases induced by SPC (Gong et al., 2009). The SPC receptor is inhibited by these novel derivatives, and the anti-inflammatory effect of KRO-105714 has been confirmed using dermal cells derived from humans and mice. As a part of the preclinical study of KRO-105714, we investigated its in vitro metabolic pathway in human liver microsomes (HLMs) using highresolution/high-accuracy tandem mass spectroscopy (HRMS) coupled with high-performance liquid chromatography (HPLC) and cDNAexpressed cytrochrome P450 (P450) enzymes. A number of analytical techniques have been developed for detection and determination in drug metabolism studies. For example, HRMS techniques such as quadrupole time-of-flight, Fourier transform ion cyclotron resonance mass spectrometry (MS), and hybrid high-resolution tandem mass spectrometry (MS/MS; Orbitrap) have been applied to identify metabolite structure. Orbitrap, the newest addition to the HRMS This work was supported by a National Research Foundation grant funded by the Korean government [NRF-2012R1A4A1028835] and a Korea Health Technology R&D Project grant funded by Ministry of Health & Welfare, Republic of Korea [A111345]. M.S. and D.L. contributed equally to this work. dx.doi.org/10.1124/dmd.114.057570. s This article has supplemental material available at dmd.aspetjournals.org. ABBREVIATIONS: CLint, apparent intrinsic clearance; HLM, human liver microsome; HPLC, high-performance liquid chromatography; HRMS, high-resolution/high-accuracy tandem mass spectroscopy; KRO-105714, N-(5-benzoyl-2-(4-(2-methoxyphenyl)piperazin-1-yl)thiazol-4-yl)pivalamide; LC, liquid chromatography; LC-MS/MS, liquid chromatography–tandem mass spectroscopy; MS, mass spectrometry; NGS, NADPHgenerating system; P450, cytochrome P450; SPC, sphingosylphosphorylcholine. 1252 http://dmd.aspetjournals.org/content/suppl/2014/05/14/dmd.114.057570.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from family (Zubarev and Makarov, 2013), is based on high resolution in excess of 1,000,000 full width at half maximum. In investigations of drug metabolites, accurate measurement using HRMS is beneficial in that the change in elemental compositions is performed with accurate masses and charge states below 5 ppm (Lee et al., 2013). In the present study, KRO-105714 was metabolized to four metabolites including C-demethyl, O-demethyl, and two monohydroxyl KRO-105714’s. Among these reactions, C-demethyl KRO-105714 is an uncommon product of metabolic enzymatic reactions; therefore, further investigation of the metabolism of KRO-105714 was undertaken. The purpose of the present study was to characterize the in vitro metabolism of KRO-105714 and identify the metabolic pathway of enzymatic C-demethyl KRO-105714 in HLMs. Materials and Methods Chemicals. KRO-105714 (purity, .99%) was prepared via chemical synthesis. HLMs (150-donor-pooled mixed-gender) and human recombinant cDNA–expressed P450 isoforms were purchased from BD Gentest (Woburn, MA). Glucose 6-phosphate, b-glucose-6-phosphate dehydrogenase, potassium phosphate dibasic, and potassium phosphate monobasic were obtained from Sigma-Aldrich (St. Louis, MO). b-NADPH was obtained from Oriental Yeast Co. (Tokyo, Japan). Solvents were HPLC-grade (Merck KGaA, Darmstadt, Germany), and the other chemicals were of the highest quality available. Biotransformation of KRO-105714. KRO-105714 (50 mM final concentration) was incubated with 1 mg/ml pooled HLMs at 37°C for 90 minutes in the presence of an NADPH-generating system (NGS) containing 0.1 M glucose 6-phosphate, 10 mg/ml b-NADPH, and 1.0 unit/ml b-glucose-6-phosphate dehydrogenase at a final volume of 100 ml. The reaction was stopped via addition of 1 ml of ethyl acetate, and after mixing for 1 minute, the samples were centrifuged at 13,000 rpm for 5 minutes at 20°C. Then, 900 ml of supernatant was separated and removed via evaporation. The samples were reconstituted with 100 ml of acetonitrile in 0.1% formic acid and centrifuged at 13,000 rpm for 10 minutes at 20°C. The supernatants were diluted with 400 ml liquid chromatography (LC)-grade water and injected into an HPLC column for linear trap quadrupole Orbitrap analysis. Chemical Inhibition Studies and Metabolism with cDNA-Expressed P450 Isoforms. The inhibitory effects of known P450 isoform–selective inhibitors and the metabolism of KRO-105714 were evaluated to determine the P450 isoforms involved in the metabolic pathway. Well characterized P450selective inhibitors—i.e., a-naphthoflavone for CYP1A2 (1 and 5 mM), tranylcypromine for CYP2A6 (1 and 5 mM), quercetin for CYP2C8 (5 and 20 mM), fluconazole for CYP2C9 (5 and 10 mM), ticlopidine for CYP2C19 (2 and 10 mM), quinidine for CYP2D6 (10 and 50 mM), diethyldithiocarbamate for CYP2E1 (20 and 100 mM), and ketoconazole for CYP3A4 (2 and 10 mM)— were incubated with KRO-105714 (Newton et al., 1995; Ko et al., 2000; Lee et al., 2008; Khojasteh et al., 2011). Incubations were performed with P450selective inhibitor, pooled HLMs (1 mg/ml) and KR-105714 (50 mM). The activity of all the inhibitors was compared with that of inhibitor-free controls. The incubation mixtures, including 5 ml of recombinant cDNA–expressed P450 [diluted to 10 pmol/ml with phosphate buffer (pH 7.4)] and KRO-105714 (50 mM) reconstituted in 0.1 M phosphate buffer (pH 7.4), were preincubated for 5 minutes at 37°C. The reaction was started by adding the NGS, and the reaction mixtures (final volumes of 100 ml) were incubated for 60 minutes at 37°C in a thermo shaker. The reaction was stopped by adding 500 ml ethyl acetate after mixing and centrifuged at 13,000 rpm for 10 minutes at 20°C. In all experiments, KRO-105714 was dissolved in ethyl acetate; the solvent was subsequently removed via evaporation to dryness under reduced pressure. The residues were reconstituted with 100 ml of 50% acetonitrile (in formic acid 0.1%), and then the internal standard solution (reserpine, 3.0 ng/ml; 5 ml) was added. The supernatants were transferred to autosampler vials, and 10 ml aliquots were used for liquid chromatography–tandem mass spectroscopy (LCMS/MS) analysis. Metabolite Structure Confirmation by Coelution. To confirm structures of M2 (C-hydroxylation) and M4 (C-demethylation), the retention time on HPLC elution and MS and MS spectra were compared between generated metabolites in HLMs and synthetic standards (Supplemental Methods). Metabolites M2 and M4 were prepared from incubation in HLMs, reconstituted to 200 ml in 100% acetonitrile (in formic acid 0.1%), and separated into halves. Synthetic standards for M2 and M4 were mixed to 0.1 mM in 200 ml and separated into halves. Incubated samples were mixed with the synthetic mixtures. The prepared samples were subjected to LC-MS/MS analysis. In Vitro Enzyme Kinetics. Kinetic parameters such as apparent Km (apparent affinity), Vmax (apparent maximum reaction velocity), and CLint (apparent intrinsic clearance) for KRO-105714 C-demethylation were determined in HLMs. The reaction mixture (100 ml) was composed of 0.5 mg protein/ml HLMs containing KRO-105714 (0 to 50 mM) or synthetic standard M2 (0 to 20 mM). The reaction was started with incubation for 60 minutes at 37°C. The kinetic parameters Km, Vmax, and CLint were determined using nonlinear regression analysis (SigmaPlot; Systat Software Inc., San Jose, CA). Instruments. HRMS experiments were conducted on a Finnigan linear trap quadrupole Orbitrap XL hybrid Fourier transform MS system (Thermo Fisher Scientific Inc., Waltham, MA). For LC analysis, an Inertsil ODS-3, 3 mm (2.1 150 mm) (GL Sciences Inc., Tokyo, Japan) column was used. The mobile phases consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in LC-grade water (B). Gradient elution was conducted as follows: B was linearly increased from 30% to 95% for 7 minutes, retained at 95% for an additional 5 minutes, and then directly decreased to 30% and held at 30% for equilibration for 5 minutes at a flow rate of 200 ml/min. The instrument was operated in full-scan mode. Electrospray ionization was performed in positive ion mode. The operating conditions were as follows: spray voltage, 4.5 kV; capillary voltage and temperature, 34 V and 320°C, respectively; sheath and auxiliary gas flow rates, 40 and 15 arbitrary units; and tube lens, 100 V. Data acquisition and analysis were performed using Xcalibur (version 2.2, Thermo Fisher Scientific Inc., Waltham, MA). Fig. 1. Extracted ion chromatograms for KRO-105714 and its metabolites after incubation with HLMs in the presence of an NGS. Characterization of CYP3A4-Mediated C-Demethylation 1253 at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from Results Metabolism of KRO-105714 in HLMs. Representative HPLC chromatograms of metabolism after incubation of KRO-105714 with HLMs and an NGS were studied, characterized, and experimentally identified using LC-MS/MS. A typical chromatogram of KRO-105714 and its four metabolites (M1–M4) is shown in Fig. 1. Observation of [M+H] ions was performed at m/z 495 for M1, via monohydroxylation (+16 Da) for M2, at m/z 465 for M3, and via demethylation (–14 Da) for M4, with retention times of 9.0, 9.5, 10.7, and 11.2 minutes, respectively (Fig. 1). The [M+H] ions of the metabolites were identified with full-scan MS detection. No metabolites were obtained without the NGS in HLM and human recombinant cDNA–expressed P450 enzymes, thereby showing the involvement of P450 enzymes and indicating that the metabolism of KRO-105714 is NGS-dependent. Characterization of Metabolites in HLMs. Metabolite structures were characterized and assigned based on their HRMS, MS, and MS product ion masses (Figs. 2 and 3). HRMS analysis confirmed the elemental composition of the product ions at ,5 ppm (Table 1). The protonated molecular ion of KRO-105714 was observed at m/z 479.21138 (C26H31O3N4S1) at 11.7 minutes (see Fig. 2). The protonated ion at m/z 395.15397 (C21H23O2N4S1) was generated as a dominant fragment ion of MS, which indicates the loss of an alkoxy group moiety (C5H8O1). MS 3 fragmentation generated a protonated ion at m/z 377.14358, indicating the loss of H2O, and another at m/z 289.11227, which showed the loss of an acetophenone moiety (C7H6O1). The protonated ion at m/z 246.06998 (C12H12O1N3S1) was generated after the loss of cyclohexane (C9H9N1). The precursor ions of M1 and M2 were determined at m/z 495.20715 and 495.20709, respectively, which are 15.9945 Da higher than that of the parent KRO-105714, indicating that the monohydroxylated elemental compositions of protonated M1 and M2 were C26H31O4N4S1 (Table 1). The protonated M1 at m/z 411.14911 was generated as a dominant fragment ion of MS, signaling the loss of an alkoxy group moiety, and MS fragmentation generated ions at m/z 393.13855 (C21H21O2N4S1), thus indicating the loss of H2O; generated ions of m/z 305.10730 (C14H17O2N4S1) indicated the loss of an acetophenone moiety (Fig. 3A). The protonated ion at m/z 246.07007 (C12H12O1N3S1) was generated after cleavage of a cyclohexane (see Fig. 3A). Metabolite M2, the major metabolite peak, gave an [M+H] ion at m/z 495.20715, suggesting that one oxygen atom was inserted from the parent compound. The MS spectrum of M2 characteristic major product ions at m/z 395.15424 (C21H23O2N4S1) suggests the loss of an alkoxy group moiety. The MS spectrum of ions at m/z 289.11237 (C14H17O1N4S1) and m/z 246.07004 (C12H12O1N3S1) was postulated to have been generated by cleavage of an acetophenone moiety and a cyclohexane moiety from the protonated molecular ion (Fig. 3B). Metabolites M3 and M4 had retention times of 10.7 and 11.2 minutes on HPLC and showed protonated ions at m/z 465.19577 and m/z 465.19604, respectively, which are 14.0156 Da lower than that of the parent KRO-105714 (see Table 1). The elemental compositions of protonated M3 and M4 were C25H30O3N4S1, indicating demethylation. Metabolite M3 produced a protonated molecule at m/z 465, indicating an O-demethylated metabolite of KRO-105714. M3 generated characteristic product ions at m/z 381.13864 (C20H21O2N4S1), suggesting the loss of an Fig. 2. MS and MS spectra and proposed mechanism for the formation of the product ions of protonated KRO-105714. 1254 Song et al. at A PE T Jornals on Jne 0, 2017 dm d.aspurnals.org D ow nladed from

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@inproceedings{Song2014Dmd0575701, title={Dmd057570 1252..1260}, author={Min Sun Song and DooHyun Lee and Sun Joo Kim and J S Bae and Jaeick Lee and Young-Dae Gong and Taeho Lee and Sangkyu Lee}, year={2014} }