Introduction

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Acute allergic reactions, also known as immediate (type I) hypersensitivity reactions, including anaphylaxis with a potentially fatal outcome, are triggered by three major classes of proinflammatory mediators, namely preformed granule-associated bioactive amines (e.g., histamine, serotonin) and acid hydrolases (e.g., -hexosaminidase), newly synthesized arachidonic acid metabolites (e.g., leukotriene C₄, prostaglandin D₂, and platelet activating factor), and a number of proinflammatory vasoactive cytokines [e.g., tumor necrosis factor (TNF¹)- and interleukin-6] (Galli, 1993). These proinflammatory mediators are released from sensitized mast cells upon activation through the antigen-mediated cross-linking of their high-affinity cell surface IgE receptors/Fc epsilon () RI (Galli, 1993; Malaviya et al., 1993). IgE receptor/FcRI is a multimeric receptor with -, -, and homodimeric -chains. Both - and -subunits of the IgE receptor/FcRI contain immunoreceptor tyrosine-based activation motifs, which allow interaction with protein tyrosine kinases (PTK) and PTK substrates via their SH2 domains (Scharenberg et al., 1995). The engagement of IgE receptors by antigen triggers a cascade of biochemical signal transduction events, including activation of multiple PTK (Scharenberg et al., 1995). The activation of PTK and subsequent tyrosine phosphorylation of their downstream substrates have been implicated in the pathophysiology of type I hypersensitivity reactions (Scharenberg et al., 1995). The elucidation of the PTK-dependent signal transduction events that lead to FcRI-mediated mast cell degranulation and mediator release may provide the basis for the rational design of potent mast cell inhibitors for prevention and treatment of allergic reactions.

In a recent study, we found that the IgE/antigen induced degranulation and mediator release are substantially reduced with Jak3^/ mast cells from JAK3-null mice that were generated by targeted disruption of the Jak3 gene in embryonic stem cells (Malaviya and Uckun, 1999), indicating that JAK3 plays a pivotal role in IgE receptor/FcRI-mediated mast cell responses both in vitro and in vivo. We subsequently reported that treatment of rodent and human mast cells with 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) (WHI-P131; JANEX-1), a rationally designed potent and specific inhibitor of JAK3 that does not affect the enzymatic activity of other protein tyrosine kinases, including the Src family tyrosine kinase LYN (lyn-gene product), the ZAP/SYK family tyrosine kinase SYK (spleenic tyrosine kinase), the TEC family tyrosine kinase BTK (Bruton's tyrosine kinase), the receptor family tyrosine kinase IRK (insuline receptor kinase), or Janus kinases JAK1 and JAK2 (Goodman et al., 1998; Sudbeck et al., 1998, 1999), inhibited degranulation and proinflammatory mediator release after IgE receptor/FcRI cross-linking. In vivo administration of this potent JAK3 inhibitor prevented mast cell degranulation and development of cutaneous and systemic fatal anaphylaxis in mice. More recently, JANEX-1 was also found to be a potent inhibitor of asthmatic reactions in mice (Malaviya et al., 2001). Thus, targeting JAK3 with a specific inhibitor, such as JANEX-1, may provide the basis for new and effective treatment and prevention programs for mast cell-mediated allergic reactions and asthma.

In addition to its anti-allergic activity, JANEX-1 may also be useful for treatment of JAK3-positive hematologic malignancies (e.g., acute lymphoblastic leukemia) (Sudbeck et al., 1999) and inflammatory disorders (e.g., graft versus host disease after allogeneic bone marrow transplantation) mediated by JAK3-positive lymphocytes (Cetkovic-Cvrlje et al., 2001). Furthermore, recent studies have indicated that JANEX-1 may also be useful for prevention of fatal thromboembolism (Tibbles et al., 2001) and treatment of amyotrophic lateral sclerosis (Trieu et al., 2000). The pharmacokinetic features of JANEX-1 in rodents and cynomolgus monkeys have been studied using a high-performance liquid chromatography (HPLC)-based quantitative detection method (Chen et al., 1999a; Uckun et al., 1999b). JANEX-1 was very well tolerated by mice and monkeys, and therapeutic plasma concentrations of JANEX-1 could be achieved at nontoxic dose levels. The biological activity profile and lack of significant systemic toxicity of JANEX-1 suggest that this JAK3 inhibitor may be useful in the treatment of allergic and inflammatory disorders and hematologic malignancies.

The purpose of the present study was to investigate the phase I metabolism of JANEX-1. Here, we show that JANEX-1 is metabolized by the cytochrome P450 enzymes CYP1A1 and CYP1A2 in a regioselective fashion to form the biologically inactive 7-O-demethylation product 4-(4'-hydroxyphenyl)-amino-6-methoxy-7-hydroxyquinazoline (JANEX-1-M). Our molecular modeling studies indicated that the cytochrome P4501A family enzymes bind and demethylate JANEX-1 at the C-7 position of the quinazoline ring since the alternative binding conformation with demethylation at the C-6 position would result in a severe steric clash with the binding site residues.

	Materials and Methods

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Chemicals. Acetonitrile and acetone were purchased from Fisher Chemicals (Fair Lawn, NJ). Furafylline and (S)-(+)-mephenytoin were purchased from Ultrafine Chemicals (Manchester, UK). Ketoconazole was from ICN Biomedicals, Inc. (Aurora, OH), and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). The reagents for synthesis were purchased from Aldrich (Milwaukee, WI). The human liver microsomes (individuals: H003, H006, H023, H030, H042, H043, H056, H066, H070, H089, H093, and H112; pooled: H161) were purchased from GENTEST (Woburn, MA). The protein concentrations and specific activities of each P450 isoform in the human microsomes were provided in the data sheets by the manufacturer.

Synthesis and Characterization of Putative JANEX-1 Metabolites. Sodium ethanethiolate (1.26 g, 12.0 mmol) was added to a suspension of JANEX-1 · HCl (1.00 g, 3.0 mmol) in N,N-dimethylformamide (14 ml) in a dry round-bottom flask under N₂. The reaction was heated to 100°C and stirred 16 h. The mixture was concentrated in vacuo, and the isomers were separated by flash chromatography on silica (MeOH/CH₂Cl₂, 10:90, v/v). The two products (compound 1 and compound 2) were recrystallized from methanol, yielding compound 1 · 0.5 MeOH (275 mg, 31%) and compound 2 · 0.5 MeOH (80 mg, 9%).

1

1

X-Ray Structure of JANEX-1-M. Colorless rectangular plates of JANEX-1-M were grown from methanol by slow evaporation at room temperature. A crystal was mounted on a glass fiber using epoxy, and X-ray diffraction data for a 0.6 × 0.5 × 0.1-mm crystal were collected at room temperature using a SMART 1K CCD X-ray detector (Bruker AXS, Madison, WI). Structure solution and refinement were performed using the SHELXTL suite of programs (Bruker AXS), and absorption correction was applied using the SADABS program (Sheldrick, 2001).

Incubation of JANEX-1 with Liver Microsomes. All incubation mixtures contained the following ingredients (a modified procedure of Chen et al., 1995, 1999b) at the indicated final concentrations (200 µl): 1× PBS, microsomes (1 mg/ml), glucose 6-phosphate (10 mM), glucose-6-phosphate dehydrogenase (2 units/ml), and MgCl₂ (5 mM). The mixtures were preincubated at 37°C for 5 min. The metabolism reaction was initiated by the addition of JANEX-1 (various concentrations). After incubation for 10 or 15 min, the reaction was stopped by addition of 800 µl of acetone and thoroughly mixed using a vortex device. The mixtures were extracted and separated through the HPLC system, as described below.

Inhibiton Experiments. The effects of various cytochrome P450 substrates/inhibitors on the formation of JANEX-1-M in pooled human liver microsomes (catalog no. H161) were studied by measuring JANEX-1-M formation after preincubation with the following compounds: -naphthoflavone, furafylline, coumarin, diethyldithiocarbamic acid, orphenadrine, sulfaphenazole, (S)-mephenytoin, quinidine, p-nitrophenol, ketoconazole, troleandomycin, and lauric acid. All inhibitors were dissolved in DMSO and added to the incubations at a final DMSO concentration of 0.5% (v/v). Control incubations (without inhibitors) also contained 0.5% DMSO. The final concentration of JANEX-1 was 50 µM, and final concentrations of P450 substrates/inhibitors were those recommended in previous publications (Kajita et al., 2000; Wynalda, 2000).

Incubation of JANEX-1 with Microsomes Containing Baculovirus-Expressed Enzymes. The metabolism of JANEX-1 by specific cytochrome P450 isoforms was studied using incubations conducted as described above, except that baculovirus-expressed P450 enzymes were used at a concentration of 50 pmol/ml for the incubation. JANEX-1 was used at a final concentration of 50 µM in these experiments.

Identification of Metabolite(s) by LC-MS. Mass spectrum analysis was carried out using atmospheric pressure ionization-electrospray and a high-energy-dynode electron multiplier (Hewlett Packard, Palo Alto, CA), which is connected to the LC system (Chen et al., 1999b). The conditions for mass spectrum analysis were set at a fragmentor of 75 V, a drying gas flow of 10 l/min, a nebulizer pressure of 25 psig, and a drying gas temperature of 350°C.

Pharmacokinetic Studies in Mice. Female CD-1 mice (6-8 weeks old) from Charles River Laboratories, Inc. (Wilmington, MA) were housed in a USDA-accredited animal care facility under standard environmental conditions. All rodents were housed in microisolator cages (Lab Products, Inc., Maywood, NJ) containing autoclaved bedding. Mice were allowed free access to autoclaved pellet food and tap water throughout the study. All animal studies were approved by the Parker Hughes Institute Animal Care and Use Committee, and all animal care procedures conformed to the principles outlined in the Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC).

In Vitro Mast Cell Biology Experiments. RBL-2H3 cells [a rat origin mucosal mast cell line kindly provided by Dr. R. P. Siraganian (NIH, Bethesda, MD)] in 48-well tissue culture plates were sensitized with a monoclonal anti-dinitrophenyl (DNP)-IgE antibody (0.24 mg/ml) by overnight incubation at 37°C in a humidified 5% CO₂ atmosphere. Unbound IgE was removed by washing cells with PIPES-buffered saline containing 1 mM calcium chloride.

Anaphylaxis Models. To examine the effect of JANEX-1 and JANEX-1-M on passive cutaneous anaphylaxis in mice, dorsal sides of the ears of BALB/c mice were injected intradermally with 20 ng of DNP-IgE (left ears) or PBS (right ears) in a 20-µl volume using a 30-gauge needle, as previously described (Miyajima et al., 1997). After 20 h, mice were treated with JANEX-1 or JANEX-1-M (20 mg/kg i.p.) twice at 1-h intervals before the antigen challenge. Control mice were treated with an equal volume of vehicle. Thirty minutes after the last dose of JANEX-1 or JANEX-1-M or vehicle, mice were challenged with 100 µg of antigen (DNP-BSA) in 200 µl of 2% Evans blue dye intravenously. Mice were sacrificed by cervical dislocation 30 min after the antigen challenge. For quantitation of Evans blue dye extravasation, as a measure of anaphylaxis-associated vascular hyperpermeability, 8-mm skin specimens were removed from the ears of mice, minced in 2 ml of formamide, and incubated at 80°C for 2 h in a water bath to extract the dye. The absorbance was read at 590 nm.

HPLC Determination of JANEX-1 and Its Metabolites. The levels of JANEX-1 and its metabolite in the microsome systems and plasma were determined using a quantitative HPLC method (Chen et al., 1999a). In brief, 800 µl of acetone was added to the microsome system to terminate the reaction. Following centrifugation (300g, 5 min), the supernatant was transferred to a clean tube and dried under a slow, steady stream of nitrogen gas. The residue was reconstituted in 50 µl of methanol/water (9:1, v/v). A 35-µl aliquot of the reconstituted solution was injected for HPLC analysis using a Hewlett Packard series 1100 instrument equipped with a quaternary pump, an autosampler, an auto electronic degasser, an automatic thermostatic column compartment, a diode array detector, and a computer with Chemstation software for data analysis. A 250 × 4-mm Lichrospher 100 RP-18 (5 µm) analytical column and a 4 × 4-mm Lichrospher 100 RP-18 (5 M) guard column were obtained from Hewlett Packard. The mobile phase contained acetonitrile/water (0.1% of TFA and 0.1% TEA) (28:72, v/v), which was degassed automatically by the electronic degasser system. The column was equilibrated and eluted under isocratic conditions using a flow rate of 0.6 ml/min at ambient temperature. The wavelength of detection was set at 340 nm for JANEX-1 and its metabolite.

Enzyme Kinetic Analysis and Pharmacokinetic Analysis. Enzymatic kinetic analysis and pharmacokinetic analysis were performed using WinNonlin program V 3.0 (Pharsight, Mountain View, CA) (Chen et al., 1999b, 2001; Uckun et al., 1999a,b). Kinetic parameters such as V_max (maximum reaction velocity) and K_m (the substrate concentration that corresponds to 50% V_max) were estimated by fitting the data to the Michaelis-Menten model (V = V_max * C^r/(K_m + C^r), where C is the drug concentration and r is the Hill coefficient. Metabolic clearance was estimated as (CL_met = V_m/K_m), and Eadie-Hofstee plots were constructed to determine whether the kinetics was mono- or biphasic. The relationship between the formation of JANEX-1-M and various cytochrome P450 activities was examined by calculating the nonparametric Spearman rank correlation coefficient using Instat computer software, version 3.0 (GraphPad Software, Inc., San Diego, CA).

Homology Model of Human CYP1A1 and Docking of JANEX-1-M. A homology model of human CYP1A1 was constructed based on the crystal structure of CYP2C5 (PDB accession number: 1dt6) using homology and docking modules with INSIGHT II software (Molecular Simulations, Inc., San Diego, CA). The homology modeling of CYP1A1 was carried out in two steps summarized as follows: 1) the most reasonable sequence alignment between the CYP1A1 and a coordinate template was determined; and 2) new coordinates were assigned to the CYP1A1 residues according to the template coordinates (based on the sequence alignment), followed by the determination of loop coordinates and an energy minimization of the entire structure. The coordinates for several loop regions where sequence insertions occur were chosen from a limited number of possibilities automatically generated by the program and manually adjusted to a more ideal geometry using the program CHAIN (Sack, 1988). Finally, the constructed model was subjected to energy minimization using the X-PLOR program (Brunger, 1992) so that any steric strain introduced during the model-building process could be relieved. The model was screened for unfavorable steric contacts, and if necessary, such side chains were adjusted either by using a rotamer library database or by manually rotating the respective side chains. Initially, JANEX-1 was manually docked into the three-dimensional model of CYP1A1. The initial coordinates of JANEX-1 was modified directly from its X-ray structure, rotated, and translated as a rigid body into the active site with the O-7 group fixed at a distance from the heme iron of 3.0 Å. After the manual maneuver, major steric interactions with nearby residues were avoided. Otherwise, an even sampling of various initial positions was used within the general binding region that is known from crystal structures of P450 enzyme complexes. Subsequently, an automatic docking procedure was followed and evaluated using the Ludi score function (Bohm, 1992) for maximum binding affinity. Finally, the docked position of the molecule was inspected and compared with known compounds in superimposed homologous protein complexes using templates with PDB access codes 1BVV, 1F4T, 1E9X, and 1EA1.

	Results

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Metabolism of JANEX-1 in Mice and Human Microsomes. We used analytical HPLC to examine mouse urine samples collected from a group of 10 mice between 0 to 6, 6 to 24, 24 to 30, 30 to 48, and 48 to 72 h after i.v. injection of a 100 mg/kg bolus dose of JANEX-1 lactobionate for the presence of JANEX-1 and potential JANEX-1 metabolites. The parent compound JANEX-1 was detected at a retention time (R_T) of 10.2 min in urine samples collected between 0 to 6 and 6 to 24 h but not at later time points (Fig. 1, A and B). A potential metabolite peak with a retention time of 7.9 min was also detected in the same urine samples (Fig. 1B). The on-line LC-MS yielded m/z values of 298 for the parent compound and 284 for the potential metabolite (Fig. 1C), prompting the hypothesis that the putative metabolite is a demethylated form (M-14) of JANEX-1.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Detection of a metabolite in urine of JANEX-1-treated mice. A, representative chromatograms from blank mice urine; B, urine from mice administered i.v. with JANEX-1; C, on-line MS spectrum. HPLC column, Zorbax SB-phenyl; mobile phase, acetonitrile/water containing 0.1% TFA (28:72, v/v); flow rate, 0.6 ml/min; detected with UV at 340 nm and selected ion monitoring at m/z of 284 and 298 (positive ion).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Metabolism of JANEX-1 in human liver microsomes. A, formation of JANEX-1-M in human liver microsomes. HPLC column, Lichrospher 100 RP-18; mobile phase, acetonitrile/water containing 0.1% TFA and 0.1% TEA (28:72, v/v); flow rate, 0.6 ml/min; detected with UV at 340 nm; B, on-line LC-MS spectrum for JANEX-1-M; C, effects of incubation conditions on JANEX-1-M formation. JANEX-1 was used at a final concentration of 50 µM in all experiments. a, data are presented as mean ± S.D. from at least 3 experiments; b, the complete activating systems are described under Materials and Methods; c, microsomal proteins (catalog no. H30) was inactivated at 90°C for 5 min.

Identification of 4-(4'-Hydroxyphenyl)-amino-6-methoxy-7-hydroxyquinazoline as the Major Metabolite of JANEX-1. We next sought to determine the structural identity of JANEX-1-M, the major metabolite of JANEX-1. Since demethylation could occur on either one of the two methoxy groups attached to the quinazoline ring of JANEX-1, it was important to determine whether the 6-O-demethylated or the 7-O-demethylated form of JANEX-1 was the actual metabolite (Fig. 3A). To this end, we first used sodium ethanethiolate to demethylate the methoxy groups of JANEX-1. The demethylation reaction yielded two structural isomers, which were separated by flash chromatography on silica and then further purified by recrystallization from methanol. The electrospray ionization-MS yielded a molecular mass of 284 for both synthetic isomers.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   4-(4'-Hydroxyphenyl)-amino-6-methoxy-7-hydroxyquinazoline as the major metabolite of JANEX-1. A, proposed O-demethylation metabolism pathway for JANEX-1. B, representative chromatograms from synthetic compound 1 and compound 2. HPLC column, Lichrospher 100 RP-18; mobile phase, acetonitrile/water containing 0.1% TEA and 0.1% TFA (28:72, v/v); detected at 340 nm. C, on-line LC-MS spectrum for synthetic compound 1; D, X-ray crystal structure of JANEX-1-M (30% ellipsoids; T = room temperature). Disordered solvent molecules not shown (see text).

View this table: [in this window] [in a new window]   TABLE 1 X-ray crystal structure of JANEX-1-M: crystal data and refinement statistics Rint = S|Fo2  Fo2|/S|Fo2|, R1 = SFo|  |Fc/S|Fo|, wR2 = {S[w(Fo2  Fc2)2]/S[w(Fo2)2]}1/2, GooF = S = {S[w(Fo2  Fc2)2]/(n  p)}1/2, where n = reflections, p = parameters.

Activity and Pharmacokinetic Features of JANEX-1-M, 4-(4'-Hydroxyphenyl)-amino-6-methoxy-7-hydroxyquinazoline. Targeting JAK3 in mast cells with JANEX-1 inhibits IgE receptor-mediated mast cell responses (Malaviya et al., 1999). To examine the effect of JANEX-1-M on IgE receptor/FcRI-mediated mast cell degranulation and cytokine release, IgE-sensitized RBL-2H3 mast cells were preincubated with increasing concentrations of JANEX-1-M, JANEX-1, or vehicle for 1 h before challenge with antigen (DNP-BSA). Notably, JANEX-1 prevented mast cell degranulation and release of preformed granule-associated -hexosaminidase (Fig. 4A) and release of the proinflammatory cytokine TNF- (Fig. 4B) in a concentration-dependent fashion, with near to complete inhibition at 30 µM. Unlike JANEX-1, its 7-O-demethylated metabolite JANEX-1-M did not inhibit mast cell degranulation or TNF- release after IgE receptor/FcRI cross-linking (Fig. 4, A and B). These results demonstrate that JANEX-1-M is >10-fold less potent than JANEX-1 in inhibiting IgE receptor-mediated mast cell responses.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Biologic activity and pharmacokinetics of JANEX-1-M. A and B, RBL-2H3 cells were sensitized with monoclonal anti-DNP IgE, treated with JANEX-1, JANEX-1-M, or vehicle, and then challenged with DNP-BSA, as described in detail under Materials and Methods section. A, mast cell degranulation (-hexosaminidase release, percentage of total) was assessed by measuring the -hexosaminidase levels in cell-free supernatants and Triton X-100-solubilized pellets using the formula: -hexosaminidase release, % of total = 100 × (-hexosaminidase level in supernatant/-hexosaminidase level in supernatant + solubilized pellet). B, TNF- levels in cell-free supernatants were measured. The results are expressed as a percentage of maximum control release from vehicle-treated control mast cells. The data points represent the mean ± S.E.M. values obtained from three to six independent experiments. C, prevention of passive cutaneous anaphylaxis in mice using JANEX-1. The effects of JANEX-1 and JANEX-1-M on anaphylaxis-associated vascular hyperpermeability were examined by evaluating the cutaneous extravasation of albumin-bound Evans blue dye in mice, and the plasma exudation indices were determined for vehicle- and JANEX-1- or JANEX-1-M-treated mice. , P < 0.05. D, plasma pharmacokinetics of JANEX-1 and JANEX-1-M. Plasma JANEX-1-M (JANEX-1) concentrations in mice following intravenous injection of JANEX-1-M (JANEX-1) at a dose level of 40 mg/kg (four mice per time point).

Kinetics of JANEX-1 Metabolism in Liver Microsomes and Its Relationship to CYP1A2 Activity. The metabolism of JANEX-1 to JANEX-1-M in pooled human liver microsomes followed Michaelis-Menten kinetics with V_max and K_m values (mean ± S.D.) of 34.6 ± 9.8 pmol/min/mg and 107.3 ± 66.3 µM, respectively (Fig. 5A). The corresponding rate of metabolic clearance for JANEX-1 was 0.3 µl/min/mg.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   A, kinetics of formation of JANEX-1-M in human liver microsomes; B, Eadie-Hofstee plot for the O-demethylation by human liver microsomes; and C, correlation of the JANEX-1-M formation with CYP1A2 activities in individual human liver microsomes. JANEX-1 was used at a final concentration of 50 µM in these experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   A, CYP1A1 (ethoxyresorufin O-deethylase) activities; B, the formation of JANEX-1-M in the induced-rat liver microsomes; C, formation of JANEX-1-M in different animal liver microsomes and its correlation with CYP1A activities. , P < 0.001.

Metabolism of JANEX-1 by Recombinant Human CYP1A1 and CYP1A2. We next sought to determine the identity of the cytochrome P450 isoform, which is responsible for the metabolism of JANEX-1 by using microsome preparations containing baculovirus-expressed recombinant cytochrome P450 isoforms (Crespi, 1995). The results presented in Fig. 7A indicate that metabolism of JANEX-1 to form JANEX-1-M is mainly mediated by cytochrome P450 1A1 and 1A2.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Kinetics of formation of JANEX-1-M in the presence of recombinant human P450. A, formation of JANEX-1-M in the presence of various baculovirus-expressed P450 isoforms. ND, not detectable. B, kinetics of the formation of JANEX-1-M in the presence of CYP1A1 versus CYP1A2.

Structural Basis for the Regioselective O-Demethylation of JANEX-1 by CYP1A1/CYP1A2. We next set out to elucidate the structural basis for the regioselective demethylation of JANEX-1 by enzymes of the CYP1A family. To this end, we constructed a series of three-dimensional models of human cytochrome P450 enzymes based on their amino acid sequence similarity with several cytochrome P450 proteins with known crystal structures, including rabbit CYP2C5 (see Materials and Methods). A homology model of CYP1A1 was established using the HOMOLOGY module within the INSIGHT II program (Molecular Simulations, Inc.) (see Materials and Methods). The binding mode of JANEX-1 was determined by a docking procedure using the AFFINITY module within INSIGHT II, with a fixed distance between the O-7 atom and the heme iron atom. Next, the homology model of CYP1A1 was superimposed with crystal structures of a number of P450 enzymes and their complexes with small molecules (PDB access codes: 1DT6, 2C17A, 1FAG, and 2BMH) (Hasemann et al., 1994; Hishiki et al., 2000).

View larger version (51K): [in this window] [in a new window]   Fig. 8.   Model of JANEX-1-M bound to the catalytic site of P450 CYP1A1. A, the JANEX-1 (green, the 7-methyl group is not shown for clarity) molecule is demethylated at the 7-position, mainly by CYP1A1 and CYP1A2, generating JANEX-1-M, shown here docked into the active site of P450 CYP1A1. The quinazoline group of JANEX-1 is situated on top of the heme group (space-filling model, iron in red, nitrogen atoms in blue and carbon atoms in white), with the O-7 atom close to the iron in the center. In this model JANEX-1 can form VDW contacts with multiple residue side chains (pink) and is near the I helix, containing residues A317 and D320. The hydroxyphenyl ring of JANEX-1-M is sandwiched between the I helix, containing G225 and F123 from the BC loop. The 4-hydroxyl group hydrogen bonds to T111 near V228. B, the alternative binding conformation of JANEX-1 (blue dashed lines) involves demethylation of the methoxy group on the C-6 carbon of the quinazoline ring. This product, with the resulting 6-methoxy group interacting with the heme, is unlikely to be favored because of steric hindrance between the hydroxyphenyl ring and the heme and residues nearby. In the JANEX-1 model, the 4-hydroxyl group on the phenyl ring could form a hydrogen bond with T111, and a small residue, 225 (a glycine in CYP1A1 and a valine in CYP1A2), has close VDW contact with the 4-hydroxyphenyl ring, both of which are a unique combination only found in CYP1A1 and CYP1A2, but not for other P450 enzymes that we examined. This observation is consistent with the experimental results indicating that JANEX-1 is selectively demethylated by CYP1A1 and CYP1A2. Prepared using INSIGHT II.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our experimental data presented here provides unprecedented evidence that the JAK3 inhibitor JANEX-1 is metabolized by cytochrome P450 enzymes CYP1A1 and CYP1A2 in a regioselective fashion to form the biologically inactive 7-O-demethylation product 4-(4'-hydroxyphenyl)-amino-6-methoxy-7-hydroxylquinazoline. Our molecular modeling studies indicated that the cytochrome 1A family enzymes must bind and demethylate the molecule at the C-7 position of the quinazoline ring since the alternative binding conformation with demethylation at the C-6 position would result in a severe steric clash with the residues that are associated with the heme iron of the enzyme at the center of a bottom plateau of a valley-shaped binding region. Our model further indicated that the combination of the hydrogen bond with T111 and the presence of a small residue at 225 makes the cytochrome 1A family most suitable to bind JANEX-1 and demethylate the molecule at the C-7 position of the quinazoline ring.

O-Demethylation has been reported for other 6,7-dimethoxy-quinazoline compounds, including prazosin (Taylor et al., 1977), doxazosin (Kaye et al., 1986), and DDQ (Yamato et al., 1982). The metabolic pathway of JANEX-1 is similar to that of DDQ, which was biotransformed in vivo to form only the 7-demethylation metabolite, but it is different from the metabolism of prazosin and doxazosin, which were metabolized in vivo to form both 6- and 7-demethylation metabolites. However, the detailed metabolic pathways of prazosin, doxazosin, and DDQ have not been reported.

CYP3A is the most abundant cytochrome P450 enzyme subfamily in human liver, which is abundantly expressed in the intestinal mucosa (Kronbach et al., 1989; Crespi, 1995). CYP3A did not appear to be involved in the demethylation of JANEX-1. This may account for good oral bioavailability of JANEX-1 in animals (Uckun et al., 1999b). In human tissues from nonsmokers, the CYP1A1 gene is expressed at very low levels (Crespi, 1995). Due to the low abundance of CYP1A1 in human tissues, CYP1A1 would be unlikely to control the metabolism of JANEX-1 in patients. CYP1A2 activity varies considerably from individual to individual and appears to be modulated by environmental factors (Crespi, 1995). Therefore, the O-demethylation of JANEX-1 is likely to show a significant patient-to-patient variation in clinical settings. It is also likely that the metabolism of JANEX-1 in patients will be affected by other drugs that are metabolized by or induce CYP1A subfamily members. Finally, further characterization of the other routes of excretion and metabolism pathways is required to better understand the metabolite pharmacokinetics of JANEX-1-M.