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Chem. Res. Toxicol. 2006, 19, 1650-1659
Inhibition of Cytochrome P450 3A4 by a Pyrimidineimidazole: Evidence for Complex Heme Interactions J. Matthew Hutzler,* Roger J. Melton, Jeanne M. Rumsey, Mark E. Schnute,† Charles W. Locuson,‡ and Larry C. Wienkers§ Pharmacokinetics, Dynamics and Metabolism (PDM), Department of Medicinal Chemistry, Pfizer Global Research and DeVelopment, 700 Chesterfield Parkway West T3A, Chesterfield, Missouri 63017, and Department of Experimental and Clinical Pharmacology, UniVersity of Minnesota, Minneapolis, Minnesota 55455 ReceiVed August 18, 2006
PH-302 inhibits the inducible form of nitric oxide synthase (iNOS) by coordinating with the heme of the monomeric form and preventing formation of the active dimer. Inherent with the mechanism of pharmacology for this compound was the inhibition of cytochrome P450 3A4 (P450 3A4), observed from early ADME screening. Further investigation showed that PH-302 inhibited P450 3A4 competitively with a Ki of ∼2.0 µM against both midazolam and testosterone hydroxylation in human liver microsomes. As expected, spectral binding analysis demonstrated that inhibition was a result of type II coordination to the P450 heme with the imidazole moiety of PH-302, although only 72% of the maximal absorbance difference was achievable with PH-302 compared to that of the smaller ligand imidazole. Time-dependent inhibition of P450 3A4 by PH-302 was also observed because of metabolite-inhibitory (MI) complex formation via metabolism of the methylenedioxyphenyl group. The profile for time-dependent inhibition in recombinant P450 3A4 was biphasic, and was kinetically characterized by a kinact of 0.08 min-1 and a Ki of 1.2 µM for the first phase (0-1.5 min) and a kinact of 0.06 min-1 and a Ki of 23.8 µM for the second phase (1.5-10 min). Spectral characterization of the PH-302 MI complex demonstrated that formation began to plateau within 3 min, consistent with the kinetic profile of inactivation by PH-302. Meanwhile, spectral evidence for the imidazole-derived type II difference spectrum of PH-302 was captured simultaneously with the formation of the MI complex. The presence of simultaneously operable type II coordination and rapidly saturable MI complex formation with heme by PH-302 indicates the presence of complex heme interactions with this unique molecule. Information from these mechanistic studies adds to our understanding of the nature of P450 3A4 inhibition by PH-302 and provides a potentially useful tool compound for future studies investigating binding interactions in this important drugmetabolizing enzyme. Introduction PH-302 is a pyrimidineimidazole molecule (Figure 1) that was investigated in preclinical studies for potency and selectivity for inhibiting the inducible form of nitric oxide synthase (iNOS1). This class of compounds have been shown to possess a novel mechanism, inhibiting the iNOS enzyme by direct interaction of the imidazole with the heme, and thus preventing the dimerization of iNOS monomers to the active dimer (1). Similar to azole antifungals, such as ketoconazole and itraconazole, which target the sterol 14R-demethylase P450 (CYP51) by heme coordination, it was determined upon early ADME screening that PH-302 was also a potent inhibitor of cytochrome P450 3A4 (P450 3A4). P450 3A4 represents the most abundant and important P450 in the human liver and intestine and is quite * Corresponding author. Tel: 314-274-0261. Fax: 314-274-4426. Email:
[email protected]. † Pfizer Global Research and Development. ‡ University of Minnesota. § Current address: Amgen, Inc., Pharmacokinetics & Drug Metabolism, AW2-D / 2381, 1201 Amgen Court West, Seattle, WA 98119-3105. 1 Abbreviations: iNOS, inducible nitric oxide synthase; P450 3A4, cytochrome P450 3A4; ADME, absorption, distribution, metabolism, excretion; SSRI, selective serotonin reuptake inhibitor; MIC, metaboliteinhibitory complex; MDP, methylenedioxyphenyl; 7-BFC, 7-benzyloxy-4(trifluoromethyl)-coumarin; 7-MFC, 7-methoxy-4-(trifluoromethyl)-coumarin; AMMC, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy4-methylcoumarin.
Figure 1. Structure of PH-302, containing both an imidazole and a methylenedioxyphenyl (MDP) functional group in its chemical structure.
unique from other major P450 enzymes in that it metabolizes molecules with a broad range of shapes and molecular weights (2, 3). As a result of this promiscuous substrate selectivity, P450 3A4 is responsible for metabolizing over 50% of marketed drugs (4). Thus, inhibition of this drug-metabolizing enzyme has led to a high risk of drug-drug interactions with potentially serious clinical implications (5-7). In addition to containing the pharmacologically required imidazole ring, PH-302 also contains a methylenedioxyphenyl (MDP) moiety (Figure 1). Both of these functionalities are known to cause the potent inhibition of P450 enzymes, although via distinct mechanisms. Imidazole-containing compounds are known to coordinate with the heme of P450, inducing a type II spectral shift (8, 9). Well-known azole antifungal compounds
10.1021/tx060198m CCC: $33.50 © 2006 American Chemical Society Published on Web 11/24/2006
Complex P450 3A4 Heme Interactions
that potently inhibit P450 3A4 via this mechanism include ketoconazole (IC50 ) 0.006 µM), itraconazole (IC50 0.15 µM), and clotrimazole (IC50 ) 0.003 µM) (10). In addition, methylenedioxyphenyl (MDP) functional groups have been reported to be metabolized by P450 at the methylenic carbon, leading to ring-opening and the formation of a catechol metabolite (11, 12). The most often cited intermediate in this metabolic pathway is a carbene, containing a free pair of electrons that can form a pseudo-irreversible complex with the heme of P450, a species known as a metabolite-inhibitory complex (MIC) (13, 14). The formation of this complex is characterized spectrophotometrically by an increase in absorbance at 455 nm and typically results in time-dependent inhibition of P450 enzymatic activity. This has been observed with many MDP-containing compounds, including isosafrole (15) and the selective serotonin re-uptake inhibitor (SSRI) paroxetine (16), as well as extracts from the herbal medication goldenseal (17). Cytochrome P450 3A4 is characterized by an especially voluminous active site pocket, reported to be >1300 Å3 (18, 19), with recent crystal structure data showing that upon the binding of larger ligands, the active site volume may approach 2000 Å3 (20). This often complicates the clear interpretation and prediction of substrate/inhibitor binding characteristics with this enzyme. The majority of evidence has suggested that the observed complex binding properties of P450 3A4 ligands derive from multiple binding regions within the spacious active site (21, 22). Current observations of P450 3A4 inhibition by a unique molecule containing two structural moieties known to coordinate with the heme of P450 presented the opportunity to probe the binding interactions directly using spectrophotometric techniques. To this end, studies were carried out in an effort to investigate these potential heme interactions and determine whether both mechanisms were operable. Thus, an understanding of the binding characteristics and mechanisms by which PH-302 inhibits P450 3A4 will be gained.
Experimental Procedures Materials. Potassium phosphate buffer, midazolam hydrochloride, 1′-hydroxy midazolam, testosterone, 6β-hydroxy testosterone, NADPH, NADP+, isocitric acid, isocitric dehydrogenase, magnesium chloride, sodium dithionite, tolbutamide, fluoxetine, piperonyl butoxide, and ketoconazole were purchased from Sigma-Aldrich (St. Louis, MO). Purified cytochrome P450 3A4 (P450 3A4), P450 3A4, 2C9, 2D6, 1A2, and 2C19 Baculosomes and Vivid Blue P450 fluorescent probes were purchased from Invitrogen (Carlsbad, CA), whereas P450 3A4 Supersomes (with coexpressed cytochrome b5) and pooled human liver microsomes (0.33 nmol/mg of protein), along with the fluorescent P450 probes 7-benzyloxy-4-(trifluoromethyl)-coumarin (7-BFC), 7-methoxy-4-(trifluoromethyl)-coumarin (MFC), and 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC), were purchased from BD Biosciences (San Jose, CA). All other chemicals were obtained from commercial sources and were of the highest purity available. Synthesis. PH-302 was synthesized according to methods reported in McMillan et al. (23) (Patent # US6,432,947) and was provided to us by David Davey at Berlex Biosciences (Richmond, CA). The catechol of PH-302 (N-(3-((3,4-dihydroxybenzyl)(methyl)amino) propyl)-N-(2-(1H-imidazol-1-yl)-6-methylpyrimidin-4-yl)glycinamide hydrochloride) was synthesized internally by Pfizer (St. Louis, MO). Briefly, PH-302 (437 mg, 1.0 mmol) was dissolved in CH2Cl2 (40 mL) and cooled to -70 °C. A solution of BCl3 (1.0 M in xylenes, 4.0 mL, 4.0 mmol) was slowly added. The mixture was allowed to slowly warm to room temperature over 8 h and was then stirred an additional 14 h. The reaction was quenched with water (20 mL) and methanol (20 mL). The reaction mixture was concentrated, and the residue was suspended in 1 M aqueous
Chem. Res. Toxicol., Vol. 19, No. 12, 2006 1651 hydrochloric acid (40 mL). The mixture was heated to 50 °C, allowed to cool to room temperature, filtered, and the resulting filtrate was concentrated to afford 485 mg (91%) of the product as a white solid (1H NMR characterization in Supporting Information). [3H]-PH-302 was synthesized with a uniform tritium-radiolabel by an outside vendor and was received as a total of 300 mCi in 0.5 mL of MeOH and then purified internally at Pfizer (St. Louis, MO) to 99.7% radiochemical purity (specific activity: 34.3 µCi/nmol). [3H]-PH-302 was separated using a YMC ODS-AQ S-3 120 A 5 µm 4.6 × 150 mm column and a long gradient of 90% mobile phase A (0.1% TFA)/10% B (acetonitrile) initially flowing at 1.5 mL/min, followed by slow gradient to 100% B over 15 min and holding until 19 min, then returning to initial conditions, and detected by UV (254 nm-retention time 3.06 min) and radiomatic (retention time 3.40 min) methods. P450 Inhibition Screening. PH-302 (3 µM) was screened against P450s 3A4, 2C9, 2D6, 1A2, and 2C19 according to methods previously reported (24) using the fluorescent probe substrates 7-BFC, 7-MFC, AMMC, and Vivid Blue (CYP1A2 and 2C19), respectively, with some minor modifications. Briefly, fluorogenic assays were conducted in 96-well clear-bottom Costar white polystyrene plates, and fluorescence readings were recorded on a TECAN (Durham, NC) Spectra Fluor plate reader. In a typical incubation, the test compound (PH-302) was diluted two times from a 10 mM DMSO stock solution into 40% methanol and then into 15% methanol for a final organic concentration of
