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The Metabolic Profile of Azimilide in Man: In Vivo and in Vitro Evaluations
The Metabolic Profile of Azimilide in Man: In Vivo and In Vitro Evaluations P. RILEY,1 P.C. FIGARY,2 J.R. ENTWISLE,1 A.L. ROE,1 G.A. THOMPSON,3 R. OHASHI,4 N. OHASHI,4 T.J. MOOREHEAD1 1
Drug Safety Assessment, Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason Montgomery Road, Mason, Ohio 2
Bioanalytical Chemistry, Procter & Gamble Pharmaceuticals, Route 320, Woods Corners, Norwich, New York
3
Clinical Pharmacology & Pharmacokinetics, Procter & Gamble Pharmaceuticals, 8700 Mason Montgomery Rd, Mason, Ohio
4
Exploratory Toxicology & Drug Metabolism and Pharmacokinetics, Tanabe Seiyaku, 2-2-50, Kawagishi, Toda, Saitama 335-8505, Japan
Received 28 February 2005; accepted 24 May 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20429
ABSTRACT: The metabolic fate of azimilide in man is unusual as it undergoes a cleavage in vivo resulting in the formation of two classes of structurally distinct metabolites. During a metabolite profiling study conducted in human volunteers to assess the contribution of all pathways to the clearance of 14C-azimilide, greater than 82% of radioactivity was recovered in urine (49%–58%) and feces (33%). Urine, feces, and plasma were profiled for metabolites. A cleaved metabolite, 4-chloro-2-phenyl furoic acid was present at high concentration in plasma (metabolite/parent AUC ratio approx. 4), while other plasma metabolites, azimilide N-oxide (metabolite/parent AUC ratio 0.001), and a cleaved hydantoin metabolite (metabolite/parent AUC ratio ¼ 0.3) were present at lower concentrations than azimilide. In urine, the cleaved metabolites were the major metabolites, (>35% of the dose) along with phenols (as conjugates, 7%–8%), azimilide N-oxide (4%–10%), a butanoic acid metabolite (2%–3%), and desmethyl azimilide (2%). A limited investigation of fecal metabolites indicated that azimilide (3%–5%), desmethyl azimilide (1%–3%), and the butanoic acid metabolite (<1%) were present. Contributing pathways for metabolism of azimilide, identified through in vitro and in-vivo studies, were CYPs 1A1 (est. 28%), 3A4/5 (est. 20%), 2D6 (<1%), FMO (est. 14%), and cleavage (35%). Enzyme(s) involved in the cleavage of azimilide were not identified. ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2084–2095, 2005
Keywords: mass balance; metabolism; cytochrome P450; drug metabolizing enzymes; flavin monooxygenases; monoamine oxidase; xanthine oxidase
Azimilide is a novel class III antiarrhythmic drug, currently in clinical development for the
Abbreviations: MAO-B, monoamine oxidase B; XO, xanthine oxidase; FMO, human flavin monooxygenases; MPO, myeloperoxidase; CYP, cytochromes P450; NMR, nuclear magnetic resonance; AUC m/AUCp, Area under the curve in plasma concentration–time curve of metabolite/area under the curve in plasma concentration–time curve of parent. Correspondence to: P. Riley (Telephone: 513-622-0587; Fax: 513-622-1652; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 94, 2084–2095 (2005) ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association
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treatment of supraventricular arrhythmias1 and as an adjunctive therapy for use with implantable cardioverter defibrillators.2 It has a unique chlorophenylfuranyl structure (Fig. 1), and acts by blocking multiple ion channels, in particular the rapidly and slowly activated components of the delayed rectifier cardiac potassium current, IKr and IKs, respectively.3 By blocking IKs as well as IKr, azimilide maintains efficacy under the clinically important conditions of b-adrenergic stimulation and high heart rates.4 The recently completed ALIVE study demonstrated that azimilide has no
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EXPERIMENTAL Materials and Methods Figure 1. Structure of azimilide denoting positions of radiolabel. *denotes the 14C azomethine radiolabel. d denotes the 14C hydantoin radiolabel.
adverse effect on mortality in post-MI patients,5 and is generally well tolerated.5,6 The disposition of azimilide has been extensively studied in multiple animal species including mice, rats, rabbits, dogs, and monkeys (unpublished data). In these species, biliary clearance of azimilide and its metabolites dominated the excretion profile. In contrast, metabolism studies of azimilide in man have shown renal excretion of metabolites to account for a much larger proportion of the administered azimilide dose. A glucuronide conjugate of 4-chlorophenylfuroic acid (4-CPFA) formed following cleavage of the azomethine bond was the dominant metabolite, accounting for approximately 30% of the dose. This paper describes an analysis of the metabolic fate of azimilide in the urine, feces, and plasma of volunteers following a single oral dose of azimilide dihydrochloride. Since cleavage of the azomethine bond is a major feature of azimilide metabolism in humans, two radiolabeled forms of azimilide were synthesized, labeled with 14C at either the azomethine carbon, or on the hydantoin ring (Fig. 1), allowing the fate of both the left and right side of the cleaved molecule to be followed. The enzymes involved in azimilide cleavage were investigated with in vitro incubations of azimilide with possible enzyme candidates Flavin Monooxygenase (FMO), xanthine oxidase (XO), Monoamine Oxidase form B (MAO-B), and Myeloperoxidase (MPO). In addition, the role of CYPs, and other liver enzymes, was determined by incubating azimilide with a human liver microsome preparation. Based on the current literature,7 MAO-B, FMO-1, and FMO-3 were considered potential candidates for cleavage of azimilide. Unpublished rat studies in vivo suggest that CYPs do not play a role in the cleavage of azimilide. These hypotheses were investigated in the current study. In addition, the roles of CYPs and FMO in N-oxidation and N-demethylation of azimilide were investigated in vitro.
Internal standards, d8-azimilide dihydrochloride and 14C-azimilide dihydrochloride labeled either on the azomethine carbon atom or in the hydantoin ring and selected metabolites were synthesized by Chemical Development, Procter & Gamble Pharmaceuticals Inc., Norwich, NY. Various inhibitors of enzyme activity and components of the NADPH regenerating system were purchased from SigmaAldrich (Milwaukee, WI) unless otherwise noted. All chromatography solvents were supplied by JT Baker (Phillipsburg, NJ) and were of chromatographic grade. Scintillation supplies were obtained from Packard Instruments (PE Life Sciences, Shelton, CT). Enzymes were purchased from several commercial suppliers. In Vivo Study Design Twelve healthy, non-smoking male volunteers, aged 21–38 years (weight range 61.3–85.4 kg), were included in this study conducted at BioClin, Inc., Richmond, Virginia. Subjects received a single dose of 280 mg azimilide dihydrochloride labeled with 14C at either the azomethine carbon (n ¼ 6), or in the hydantoin ring (n ¼ 6) (final specific activity of dose 0.36 mCi/mg, each patient receiving 100 mCi total radioactivity) (Fig. 1). As only a small fraction of the dose was radioactive, d8-azimilide dihydrochloride was incorporated into the dose as an aid in the identification of metabolites by mass spectrometry. Blood samples were drawn for analysis of plasma at 0 (pre-dose), 3, 6, 12, 24, 36, and 48 h, and then every 24 h until two successive samples were below quantifiable limits of radiometric detection. At each time point, 10 mL blood was collected, processed to plasma and analyzed for metabolites. Twenty-four hour pooled urine and feces samples were collected until two successive measurements were below quantifiable limits. Assessment of radioactivity by scintillation counting of urinary and fecal samples provided the mass balance for the study. Samples from four subjects from each group were analyzed for metabolites. Mass Balance Assessment Total mass balance was assessed at the clinical site (BioClin, Inc.). Urine was pooled as 24 h samples and each pooled sample was analyzed for JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
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total 14C activity by liquid scintillation counting. Pooled fecal samples (24 h) were homogenized and aliquots taken for oxidation and scintillation counting. Analysis of Plasma The quantification of azimilide and its metabolites in plasma by HPLC/radiometric quantification (HPLC/RAD) was not possible as the concentrations were below the limits of detection for this method. Therefore an HPLC/MS assay method was developed using authentic standards of various metabolites for constructing the calibration curve. Due to incompatible chemical stabilities, and the diverse nature of the products it was necessary to prepare one set of samples with the internal standards IS1 (the hydroxyethyl analogue of azimilide) and IS2 (5-(4-fluorophenyl)-furan-2carboxylic acid) (allowing analysis of azimilide and azimilide N-oxide, and 5-(4-chlorophenyl)furan-2-carboxylic acid (4-CPFA), respectively), and another set of samples with IS3 (1-amino-3-[4(4-acetyl-1-1-piperazinyl)butyl]-2,4-imidazolidinedione) for the analysis of the cleaved hydantoin metabolites. For analysis of azimilide, azimilide N-oxide, and 4-CPFA plasma samples (500 mL) from four volunteers were mixed with internal standards (50 mL). To these mixtures, 500 mL 0.1 N ammonium hydroxide was added, followed by 20% methanol/acetonitrile (5 mL), before centrifugation at 3000 rpm for 10 min. The supernatant was carefully removed, and evaporated to dryness using a Savant Evaporator under medium heat. Dried samples were reconstituted in 500 mL 25 mM ammonium formate pH 5.5/10% methanol prior to HPLC/MS analysis. Plasma samples from two volunteers were analyzed for cleaved hydantoin metabolites (M1). When processing these samples, the drying stage was performed under a stream of nitrogen at 508C, and the resulting dried sample was dissolved in 500 mL 50 mM ammonium bicarbonate, pH 8.5/ 20% methanol. Control samples were analyzed in the same manner, using human plasma collected pre-dose. Chromatography Azimilide and azimilide N-oxide were assayed by HPLC/MS, using an Inertsil ODS-3 HPLC column (50 2.1 mm) on a Hitachi 6200A pump run at JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
600 mL/min, linked to Sciex API III mass spectrometer. The HPLC mobile phase consisted of Buffer A (90% 25 mM ammonium formate, pH 4.0/10% acetonitrile) and Buffer B (25% 25 mM ammonium formate, pH 4.0/75% acetonitrile), and material was eluted using a linear gradient of 60% Buffer A, 40% Buffer B to 10% Buffer A, 90% Buffer B over 0.5 min. Mass spectrometry typically involved ionization in the positive mode with APCI, using a probe temperature of 5008C, nebulizer gas at 80 psi, auxiliary gas at 3 L/min, and curtain gas at 1.2 L/min. Azimilide was monitored at 458/253 m/z, IS1 at 474/344 m/z, and azimilide N-oxide at 474/253 m/z. The hydantoin metabolite was assayed using a separate, but similar HPLC-MS procedure. The experimental setup used for measuring 4-CPFA was similar, except that a 100 2.1 mm ODS-3 HPLC column was used with isocratic elution using 30% acetonitrile/70% 12.5 mM ammonium formate pH 5.5. 4-CPFA and IS2 were monitored in the negative mode at 221/117 and 205/161 m/z, respectively. The concentrations of azimilide, azimilide Noxide, hydantoin metabolite, and 4-CPFA in the plasma samples were calculated from standard curves generated from the internal standards and authentic standards of metabolites. Weighted linear regression analyses were carried out for the observed signals (expressed as the ratio of peak area for each analyte to its corresponding internal standard). Analyte concentrations were converted to mg equivalents of azimilide/mL using the following equation. mgEq azimilide=mL ¼ ng=mL analyte ðmw azimilideÞ=ðmw analyteÞ mg=1000 ng Analysis of Urine Aliquots of urine (9.4 mL) were evaporated to dryness in a Savant evaporator (set at a cool temperature) before resuspending in 1 mL HPLC mobile phase (98% ammonium acetate [50 mM, pH 5.2], 2% methanol). Three 10 mL samples of the resuspended solution were mixed with scintillation fluid, before counting. Solutions of authentic standards were injected onto the column every fourth sample as a check of the column performance. HPLC was undertaken using a Waters C18 Symmetry Cartridge Column (4.6 250 mm) on a
METABOLIC PROFILE OF AZIMILIDE
Perkin Elmer 410 Quaternary pump using UV detection at 315 nm (Perkin Elmer 235c Detector Model LC235) and radiometric detection (Packard Radiometric Model A515A Flo-One Detector). The mobile phase consisted of Buffer A (98% 50 mM ammonium acetate, pH 5.2/2% methanol), Buffer B (66% 50 mM ammonium acetate, pH 4.6/26% acetronitrile/8% methanol), and Buffer C (73% 50 mM ammonium acetate, pH 6.0/22% acetonitrile/5% tetrahydrofuran). The column was eluted using a linear gradient from 100% Buffer A to 100% Buffer B over 10 min, followed by a linear gradient from 100% Buffer B to 100% Buffer C over 5 min. Eluted peaks were identified by comparison of retention times with standards, and confirmed by mass spectrometry. Recovery of radioactivity after HPLC was measured by passing a known quantity of radioactivity onto the column and measuring the radioactivity in the eluent over a complete HPLC run. The areas under peaks of interest were determined electronically, and expressed as a fraction of the total area under all peaks. The number of counts in each peak was calculated as the fraction of the total number of counts injected onto the column (determined by scintillation counting). Using an efficiency value of 0.88 (determined experimentally), cpm was converted to dpm and to mCi. The mg Equivalents of azimilide (base) were then calculated from the known specific activity (in this case, 0.36 mCi/mg). Loss of sample at the initial evaporation/resuspension stage was calculated by scintillation counting before and after this step. Analysis of Feces Samples (1 g) of previously prepared fecal homogenates were combined with 25 mL of extraction solvent (72.7% acetonitrile/18.2% methanol/9.1% 32 mM ammonium acetate pH 5.0) and shaken for 10 min. The extract was centrifuged and a 2.5 mL sample of the supernatant was combined with 200 mL of the internal standard (4.8 mg/mL IS1). The combined sample was mixed and evaporated to dryness under a stream of nitrogen. The dried sample was reconstituted with 0.5 mL of mobile phase (pH 5.4 buffer containing 32% acetonitrile/ 5% methanol/63% 32 mM ammonium acetate pH 5.0). Particulate matter was removed by centrifugation and a 50 mL sample of supernatant was injected into the HPLC system. HPLC was conducted on a Metasil C-8 column (particle size was 5 mm, column dimensions 25 cm 4.6 mm). The mobile phase flow rate was 1.5 mL/
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min. Absorbance was monitored at 340 nm. Isocratically eluted metabolite peaks (desmethyl azimilide and the butanoic acid metabolite) and azimilide were identified by comparison of retention times with standards.
In Vitro Determination of Enzymic Pathways Cleaved Metabolites Human liver microsome incubations. Human liver microsomes were purchased from BD/Gentest Inc. (Woburn, MA). Reaction tubes containing 25 mg 14C-azimilide (labeled at the azomethine position) were incubated at 378C with 0.5 or 1 mg microsomal protein for 10–20 min in a final volume of 250 mL. Reactions were buffered in 50 mM potassium phosphate buffer (pH 7.4), 0.8 mM diethylenetriaminepentaacetic acid (DETAPAC), 0.5 mM NADPþ, 0.4 mM glucose-6-phosphate, and 1 IU glucose-6-phosphate dehydrogenase, and were terminated by rapid freezing in a dry ice/ acetone bath. Inactivated, boiled microsomes were used as a negative control. Microsome-catalyzed oxidation of 10-(N,N-dimethylaminopentyl)2-(trifluoromethyl) phenothiazine (5-DTP) provided a positive control. In addition, the activity of the microsome preparations for, CYP1A1(7-ethoxyresorufin), CYP1A2 (caffeine),8 CYP2A6 (coumarin),9 CYP2B6 (benzphetamine),10 CYP2C9 (tolbutamide),11 CYP2C19 (S-mephenytoin),12 CYP2D6 (dextromethorphan),13 CYP2E1 (chlorzoxazone), CYP3A4 (testosterone),14 CYP4A (lauric acid), and Cyt b515 were assessed (probe substrates indicated). Flavin monooxygenase (FMO) incubations. Recombinant human FMO-1 and FMO-3 were expressed from E. coli as maltose-binding proteins.16 Incubations with 14C-azimilide were performed at pH 7.4, but otherwise under the conditions described for microsomal incubations, using 25 or 50 mg of human FMO enzymes for 20 min. Inactivated, boiled FMO-1 and FMO-3 (25 mg/mL protein) were used as negative controls, and oxidation of 5-DTP was followed as a positive control. Monoamine oxidase B (MAO-B) incubations. Incubations were performed under the conditions described for microsomal incubations, using 2.5 or 5 mg of MAO-B for 10–20 min. Inactivated, boiled MAO-B (2.5 mg) incubations were used as negative JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
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controls, and the conversion of benzylamine (3 mM) to benzaldehyde monitored as a positive control.17 Xanthine oxidase (XO) incubations. XO (0.2 or 1.0 units/mL), obtained from Sigma Chemical Co. was incubated with azomethine labeled 14C-azimilide (10 mM, 0.061 mCi) in 50 mM sodium phosphate buffer (pH 7.5) at 378C in triplicate, for 0, 10, 30, and 90 min. The activity of XO was verified by monitoring its ability to metabolize hypoxanthine, and its inhibition by allopurinol.18 Myeloperoxidase (MPO) incubations. Azimilide (500 mM) was incubated with MPO (Cortex, 10 units/ mL) in 100 mM phosphate buffered saline, pH 6.0. The enzymatic reaction was initiated by the addition of hydrogen peroxide (final concentration 10 mM) and left at room temperature for 45 min. The reaction mixture was passed through C18 Sep peaks for concentration and eluted with methanol. Other Metabolic Products Phenolic metabolites. Phenolic metabolites were not observed in microsomal incubations with azimilide and further investigation with Supersomes 1 (BD/Gentest) expressing CYP1A1 and CYP1A2 (50 mg) in a buffer containing 10 mM glucose-6-phosphate, 5 mM MgCl2, 0.5 mM bNADPþ, 0.2 units glucose-6-phosphate dehydrogenase, 0.1 M sodium phosphate buffer (pH 7.4, total volume 500 mL) at concentrations of 20 and 200 mM azimilide were undertaken. Reaction mixtures were pre-incubated at room temperature for 2 min prior to the addition of Supersomes1. The reactions were quenched at 30 min by addition of 100 mL acetonitrile, and precipitated protein was removed by centrifugation. Inhibition of CYP1A1 and CYP1A2 with furafylline provided a positive control. Desmethyl and azimilide N-oxide. Human liver microsomes (1 mg/mL protein), with known cytochrome P-450 activities, were mixed with 14Cazimilide (azomethine label, 200 and 400 mM) in a buffer containing 50 mM potassium phosphate (pH 7.4), 0.1 mM EDTA, 3 mM MgCl2 and preincubated at 378C for 3 min before addition of glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and 0.5 mM NADPþ in a final volume of approximately 250 mL. After 30 min, reactions were quenched by addition of 250 mL acetonitrile. The samples were centrifuged and the resulting JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
supernatant analyzed by HPLC/RAD. Control incubations in the presence of furaphylline (30 mM), sulfaphenazole (10 mM) (Ultrafine Chemicals), quinidine (10 mM), or triacetyloleandomycin21 (TAO, 0.1–500 mM) to specifically inhibit CYP1A2, CYP2C9/10, CYP2D6, and CYP3A4/5, respectively were included.19,20 Negative controls for FMO by inclusion of methimazole (200 mM, competitive inhibitor), and after heat inactivation of the thermally-labile FMO enzyme by heating to 508C for 90 s were also assessed in microsomal incubations. Recombinant cytochrome P-450 enzymes (Gentest Corporation), specifically CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 were conducted using 2 mg protein and 400 mM azimilide per reaction, with reactions terminated at 3 h by quenching with acetonitrile. Analysis of In Vitro Enzyme Pathway Studies Analysis of in vitro reaction mixtures for cleavage products. After quenching FMO-1, FMO-3, MAOB, XO, microsomal and Supersome1 incubations were centrifuged at 10000 rpm for 5 min, before aliquots of supernatant were removed for liquid scintillation and HPLC/RAD analyses. Chromatographic resolution was achieved with a Waters C8 5 mm, 3.9 150 mm column, on a Waters 600 LC pump, with UV detection at 310 nm (Perkin Elmer, s/n 92568) and radiometric detection using a Packard Flo One Beta, model 525TR series radioactivity detector. The mobile phase consisted of 30% acetonitrile/70% 25 mM ammonium formate buffer, pH 3.2, delivered isocratically at 1 mL/min (with supplemental scintillant at 3 mL/min). The quantities of 14C-azimilide, and the cleavage products 14C-F-1054 (an aldehyde, which is rapidly converted to 4-CPFA in vivo) and 14 C-4-CPFA (the corresponding carboxylic acid) formed in the reactions were calculated by comparison of HPLC retention times of reaction mixtures with those of synthesized authentic standards. Analysis of MPO reactions. Samples from the incubation of azimilide with MPO/H2O2 were analyzed by LC/MS performed on a Sciex API III mass spectrometer with an ion spray interface. Analysis of in vitro reaction mixtures for phenolic metabolites. The polar nature of the phenolic metabolites required modification of standard
METABOLIC PROFILE OF AZIMILIDE
analytical methods for this compound. HPLC was undertaken at 408C using a Waters C18, 5 mm, 4.6 150 mm column, on a Waters 626 HPLC pump, with fluorescence detection using a Waters 474 fluorescence detector (excitation l ¼ 340 nm; emission l ¼ 400 nm). Mobile phases consisted of Buffer A (acetonitrile/water/formic acid [95/5/ 0.1,]) and Buffer B (acetonitrile/water/formic acid [5/95/0.1). Material was eluted from the column by running a linear gradient at 1 mL/min from 90% Buffer A/10% Buffer B to 10% Buffer A/90% Buffer B, over 30 min. The column was linked to an LTC mass spectrometer with electrospray ionization and time of flight analysis to identify eluted material. NMR spectroscopy was used to determine the position of the hydroxyl group on the benzene ring. Material was evaporated to dryness under a stream of N2, before resuspending in D2O. 1 H-NMR was undertaken at 600 MHz on a Bruker DRX-600 instrument, with presaturation of the residual water peak during the relaxation delay.
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by comparison with known retention times for azimilide N-oxide and desmethyl azimilide.
RESULTS Mass Balance Assessment Label stability studies were conducted in rats prior to commencement of the preclinical assessment of azimilide. As loss via 14CO2 in these studies was less than 0.5%, no further assessment of loss of 14CO2 in humans was undertaken. Samples of urine and feces, collected as pooled 24 h samples were assessed for radioactivity by scintillation counting (Tab. 1) for all subjects. Mean total recovery was good with >82% (79– 86)% recovered in the hydantoin group and >91% (87–95)% recovered in the group receiving azomethine label. Radioactivity was recovered primarily in the urine (49% vs. 58% for hydantoin vs. azomethine label) with approximately 33% of the administered dose being recovered in feces. As no 14 CO2 was released from either radiolabel in label stability studies (unpublished data) the difference between the urinary values obtained likely reflects differences in the disposition (ratio of renal to non renal extraction) of the structurally distinct cleaved metabolites.
Analysis of in vitro reaction mixtures for desmethyl azimilide and azimilide N-oxide. HPLC was undertaken using an Exsil C8 5 or 10 mm, 4.6 250 mm column, on a Perkin Elmer 410 quaternary pump, with UV detection at 340 nm (Perkin Elmer LC235) and radiometric detection using a Packard Flo One, model A515 radioactivity detector. The column was eluted isocratically in 60% buffer A (50 mM ammonium acetate, pH 5.5/ 40% buffer B (10% tetrahydrofuran in acetonitrile), or 50% Buffer A/50% Buffer B, and peaks identified
Characterization and Quantitation of Metabolites From preclinical studies it was established that metabolites could be classified by structure into six groups of metabolites and these were assigned
Table 1. Mass Balance and Excretion Pattern of Radioactivity Following a Single Oral Dose (280 mg, 100 mCi) 14C-azimilide Dihydrochloride to Healthy Male Volunteers Azomethine Label Volunteer ID 47550001 47550002 47550003 47550004 47550005 47550006 Mean (% CV) Hydantoin label 47550007 47550008 47550009 47550010 47550011 47550012 Mean (% CV)
% Administered Radioactive Dose Excreted Urine 61.00 61.53 51.46 59.48 57.86 57.58 58.05 (6.3)
Feces 29.62 33.84 39.79 33.18 29.57 36.79 33.60 (11.9)
Total 90.62 95.37 91.25 92.67 87.44 94.37 91.91 (3.1)
51.69 49.15 52.08 50.46 44.66 49.01 49.44 (5.4)
28.45 37.00 33.04 32.40 37.80 30.00 32.94 (11.3)
80.14 86.15 85.11 82.86 82.45 79.01 82.58 (3.3)
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Figure 2. Representative radiochromatogram of urine showing retention order of metabolites (chromatograms were generated with urine from rats dosed with a mixture of hydantoin and azomethine labeled azimilide and is used for illustrative purposes only to show retention time of metabolites).
the notation as M1–M6 based on retention time in the radiochromatogram (Fig. 2). Metabolites, where multiple isomers existed, within each group were quantified separately and then combined to give the total for each group. For the cleaved metabolites, M1 and M3, where quantitation was based on radioactivity, one group was not present as the label remained with the other portion of the molecule. Characterization of Plasma Metabolites The concentration of radioactivity present in plasma at any given time was insufficient to measure plasma concentrations of metabolites by HPLC/RAD and thus an HPLC/MS assay was developed to measure metabolites for which authentic standard was available. Three metabolites were monitored and the relative ratios of AUC of metabolite to parent (AUCm/AUCp) were calculated and are presented in Figure 3. The maximal concentration (Cmax) of azimilide was reached within 3 to 6 h post dose, whereas the Cmax value for the cleaved metabolite, 4-CPFA (M3), was not reached until 36–48 h post dose (Fig. 4). No glucuronide or glycine conjugates of 4CPFA were observed in plasma. Characterization of Urinary Metabolites Urinary excretion of azimilide and its metabolites was quantitated by HPLC/RAD, with subsequent identification by mass spectrometry and NMR. For subjects who received the azomethine labeled material, no hydantoin metabolites (M1) were observed as the radiolabel remained with the furoic acid metabolites. Similarly, for subjects who received the hydantoin radiolabeled azimiJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
lide no furoic acid metabolites were observed. The percent dose excreted as each metabolite is presented in Figure 3. Characterization of Fecal Metabolites The metabolic profile in feces was not determined for all metabolite groups (M1–M6) as limited authentic samples were available for defining analytical parameters. Thus, only the concentrations of azimilide, desmethyl azimilide and the butanoic acid metabolite (M6) were measured. Of the approximated 33% of dose excreted in feces, only ca. 6% of the dose was accounted for by these metabolites (Fig. 3). In preclinical studies, only traces of the cleaved metabolites (M1 and M3) were observed in bile and thus, it is likely that the remaining 27% of dose is azimilide N-oxide (M4) or the conjugated phenols (M2). Ratios of M2 to M4 in bile collected from animals studies ranged from 0.5 (rat) to 3 (monkey). A value of 1 was used to estimate fecal excretion for estimation of pathways (Table 2). In Vitro Analysis of Cleavage Products Human Liver Microsomal Metabolism of Azimilide Human liver microsomes were shown to be active in all positive control tests. Low concentrations of 4-CPFA were present in some samples but were matched in negative controls suggesting that some low background hydrolytic activity was occurring. Reaction of Azimilide With FMO-1, FMO-3, and MAO-B All enzymes were active in positive control reactions, but had no effect on the cleavage of
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Figure 3. Proposed metabolic pathways of azimilide in man.
azimilide with the apparent low level of FMO-1 and FMO-3 activity matched in negative controls. XO Incubations 4-CPFA was formed in the presence of XO in low quantities (up to 1.84% of azimilide was converted to 4-CPFA). However, there was no evidence of time, or enzyme concentration dependence of the reaction, and 4-CPFA was formed in similar quantities in the presence of the XO inhibitor allopurinol. Myeloperoxidase Incubations Figure 4. Plasma concentration versus time curves for measured metabolites and azimilide following a single oral dose (280 mg; 100 mCi) 14C-azimilide dihydrochloride to healthy male volunteers.
Azimilide underwent degradation under the conditions of the assay to 4-chlorophenyl-2-furfural, a potential precursor to 4-CPFA. Further investigation showed that similar degradation could be JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
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achieved under acidic conditions, with the rate being optimum at pH 4, and thus, it is likely that the MPO provided the chemical environment rather than the catalytic environment for this degradation to occur. In Vitro Analysis of Other Metabolic Products Phenolic Metabolites Incubation of azimilide with CYP1A1 (20 and 200 mM), but not CYP1A2 (200 mM), resulted in hydroxylation of the chlorophenyl moiety of azimilide to the extent of 2.59, 1.01, and 0.0% respectively. 1H NMR spectroscopy demonstrated that in these experiments, the hydroxyl group was substituted at the ortho position relative to the chlorine atom. From in vivo studies, multiple monohydroxylated species (excreted as glucuronide and sulfate conjugates) were observed. The Km for the conversion of azimilide to the phenolic metabolites was determined to be 5.2 mM. Desmethyl Azimilide and Azimilide N-Oxide Human liver microsomes were found to catalyze the conversion of azimilide to both azimilide Noxide (Km ¼ 259 mM, Vmax ¼ 0.26 nmol/mg/min, Clint 1.02 mL/min/mg) desmethyl azimilide (Km ¼ 179 mM, Vmax ¼ 0.17 nmol/mg/min, Clint 0.93 mL/min/mg) in a time- and concentrationdependent manner. Specific inhibition of the CYP3A4/5 isoforms using TAO (500 mM) resulted in 64% and 92% inhibition of N-demethylation and N-oxidation, respectively. Inhibition of other isoforms had little noticeable effect. Inhibition of microsomal FMO activity by heat inactivation resulted in approximately 45% inhibition of Noxidation, while only slightly (7%) affecting Ndemethylation. Incubations using recombinant cytochrome P-450 isoforms resulted in notable formation of desmethyl azimilide in the presence of CYP2D6 (yielding approximately 0.8 nmoles/ reaction) and CYP3A4 (yielding approximately 2.8 nmoles/reaction), and in the formation of azimilide N-oxide in the presence of CYP3A4 (yielding approximately 1.7 nmoles/reaction). Taken together, these results suggest that azimilide N-oxide is formed through the action of CYP3A4/5 and FMO, and desmethyl azimilide is formed through the action of FMO, CYP3A4/5, and CYP2D6. One approach to define the potential for drug interactions is based on the relative importance of different metabolic pathways as outlined by Thompson.22 For azimilide, metabolites include JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
M2 (catalyzed by CYP1A1), M4 (catalyzed by CYP3A4/5 and FMO), M5 (catalyzed by CYP 3A4/ 5, FMO and CYP 2D), M6 (catalyzed by an unknown pathway), and the cleaved metabolites, M1 and M3. Although no mechanism for the cleavage of azimilide has been identified, approx. 35% of the dose is metabolized via this pathway (Fig. 3). For M2 (excreted as phenolic conjugates) formation is via a single enzyme. Although metabolic profiling for M2 in feces was not conducted in this study, inference from biliary studies in higher animal species (unpublished data) where 10–20% of the dose was consistently recovered for both M2 and M4, with no M3 being excreted in bile. Since approximately 30% of human fecal radioactivity could not be attributed to measured metabolites or parent drug, recovery of 15% of the dose in feces as M2 and 15% as M4 was assumed in man. Since M2 is formed via a single enzyme, CYP1A1 contribution to total clearance is 28%. For metabolites M4 and M5 multiple enzymes contribute to the formation of each metabolite. Based on in vitro studies, heat inactivation of FMO decreases formation of M4 by 45%. Since 30% of the dose is recovered as M4, FMO accounts for 13.5% of total clearance, with the remaining 16.5% attributed to CYP 3A4. Both enzymes also contribute to the formation of M5, which is only a minor metabolite (4% total recovery). From the in vitro studies, FMO contributes less than 10% to M5 formation, and the relative rates of formation of M5 between CYP3A4 and CYP2D6 suggest that CYP 2D6 accounts for <1% of the administered dose. Thus the total contribution from CYP3A4 is approximately 20%, while FMO contributes approximately 14% and CYP2D6 <1%. These estimates are summarized in Table 2. The contributions of enzymes responsible for the formation of M6 are unlikely to be major contributors to potential interactions as only 2–3% of dose is metabolized through this pathway.
DISCUSSION Clearance of azimilide is mediated via both metabolic and renal pathways. Due to the diversity of pathways involved, a priori dosage regimen adjustments may not be required for patients with renal or hepatic impairment. This is supported by recent pharmacokinetic/pharmacodynamic studies of azimilide in man, where no adjustments in dose were required in patients in renal failure23 or with mild to moderate hepatic failure.24
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Table 2. Estimates of the Different Pathways Involved in the Metabolism of Azimilide
Pathway CYP1A1a CYP3A4/5a CYP2D6 FMOa Cleavage
Approximate % Metabolized Through Pathway 28 20 <1 14 35
a Based on an estimate of 15% recovery of each M4 (azimilide N-oxide) and M2 (phenolic metabolites excreted as conjugates) in feces which is in the range of that observed in higher animal species (unpublished data).
The metabolic changes observed in this study are consistent with those observed in animal studies (unpublished data) and oxidative pathways are predicted by typical cytochrome P-450 catalyzed reactions. Formation of the cleaved products is not mediated by CYP enzymes as no cleaved products were observed in incubations of azimilide with microsomes or hepatocytes (humans, rats, dogs, unpublished data) or liver slices (dog, unpublished data). The extent of formation of the cleaved metabolites (M1 and M3) in humans is much higher than that observed in animals, with significantly higher plasma concentrations of 4-CPFA than azimilide at all time points measured (Fig. 4), as shown by the relative AUC of metabolite to parent (4.10, Fig. 3). However, this metabolite shows no antiarrhythmic activity, as measured by increase in action potential duration 90%, and therefore does not contribute to the overall antiarrhythmic profile of azimilide. The low concentration of azimilide N-oxide (M4) and desmethyl azimilide (M5) in plasma also suggests that these metabolites contribute little to the overall pharmacological activity although they do possess some antiarrhythmic activity (20 and 11% relative to parent). Because of concerns about the potential toxicity of acyl glucuronides25 an assay was developed to determine the plasma concentration of the acyl glucuronide of 4-CPFA. No acyl glucuronide was detected in plasma, although the primary excretion product of 4-CPFA in urine was the acyl glucuronide, with traces of the glycine conjugate and free acid. These cleaved metabolites account for approximately 30%–35% of the administered dose. Chemically the azomethine bond is unstable under acid conditions and thus it might be anticipated that acid catalyzed hydrolysis might occur in the stomach after oral administra-
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tion. However, bioavailability of azimilide in man following oral administration approaches 100% and the appearance of 4-CPFA in plasma parallels azimilide concentration, i.e., there is no initial spike as might be expected from absorption of the acid from the stomach. Azimilide possesses detergent properties and an oral dose exceeds the critical micelle concentration in the stomach. It is possible that the azomethine bonds may be internalized within the micelle and protected from hydrolysis. To further understand the metabolism of azimilide via the cleavage pathway several enzyme systems were considered as potential metabolizing systems for formation of the cleaved metabolites. Selection of these enzymes was based on substrate specificities, mechanistic considerations, or as major enzyme systems that could potentially result in drug-drug interactions if involved in the cleavage of azimilide. Despite incubations with cytochromes P-450, MAO-A and B, FMO 1 and 3, XO and MPO, no conclusive evidence was obtained to suggest any of the enzymes tested was involved in the formation of 4-CPFA. Another compound containing an azomethine bond, 4-[4-fluorophenoxy]-benzaldehyde semicarbazone (Co 102862) has been reported as being extensively metabolized with the major metabolites being a carboxylic acid and its glucuronide conjugate. The carboxylic acid metabolite of Co 102862 was presumed to be formed through an aldehyde intermediate by hydrolysis of the azomethine bond.26 The corresponding aldehyde formed from azimilide, 5-(4-chlorophenyl)-2-furaldehyde, is rapidly oxidized to the carboxylic acid in fresh human blood suggesting that if formation occurs through hydrolysis, the observable end product would be the carboxylic acid. Although the mechanism of formation is unknown for the cleaved metabolites other metabolites of azimilide were clearly established as being mediated by various CYPs (predominantly CYP3A4 and 1A1) and for some metabolites, FMO was also implicated. The level of involvement of these isoforms was considered unlikely to lead to significant clinical interactions, and recent pharmacokinetic studies of azimilide in man27,28 supports these findings, with pharmacokinetic parameters unaffected by co-administration of azimilide with digoxin, warfarin, and CYP3A4 inhibitors and inducers. Furthermore, the involvement of CYP1A1 (for formation of the phenolic metabolites, Km ¼ 5.2 mM) was consistent with a change in clearance among smokers.27 The multiJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
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plicity of mechanisms utilized in the metabolism of azimilide indicates that the clearance of azimilide in humans is not excessively reliant on any one particular pathway. In conclusion, azimilide and its metabolites are excreted both in urine and feces, thereby reducing the dependence on a single route of excretion. Metabolism is extensive with less than 15% of parent being excreted unchanged. Multiple pathways involving CY3A4, CYP1A1, CYP2D6, and FMO were identified as contributing to azimilide metabolism and, although the mechanism of formation of the cleaved metabolites has not been clearly identified, the lack of involvement of major metabolizing enzymes such as the CYPs, MAO, FMO, etc. suggests that the likelihood of a clinically relevant drug interaction through known pathways is minimal.
ACKNOWLEDGMENTS We thank our colleagues, Dr. Tom Huggins and Dr. Roy Dobson for their invaluable assistance and discussions concerning metabolite identification. We are also indebted to our collaborators, Dr. John Cashman (Human Biomolecular Research Institute, San Diego) and Jack Uetrecht (University of Toronto) for their help with in vitro preparations. Finally, our thanks go to The Medical Knowledge Group (London) for their assistance in preparation of this manuscript.
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evaluation (ALIVE): Azimilide does not affect mortality in post-myocardial infarction patients. Circulation 104:1B. Connolly SJ, Schnell DJ, Page RL, Wilkinson WE, Marcello SR, Pritchett ELC. 2001. Dose–response relations of azimilide in the management of symptomatic, recurrent, atrial fibrillation. Am J Cardiol 88:974–979. Gibson GG, Skett P. 1994. Introduction to drug metabolism. 2nd edn. London: Blackie Academic & Professional. Tassaneeyakul W, Birkett DJ, Veronese ME, McManus ME, Tukey RH, Quattrochi LC, Gelboin HV, Miners JO. 1993. Specificity of substrate and inhibitor probes for human cytochromes P4501A1 and 1A2. J Pharmacol Exp Ther 265:401–407. Greenlee WF, Poland A. 1978. An improved assay of 7-ethoxycoumarin O-deethylase activity: Induction of hepatic enzyme activity in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Pharm Exp Ther 205:596–605. Heyn H, White RB, Stevens JC. 1996. Catalytic role of cytochrome P4502B6 in the N-demethylation of S-mephenytoin. Drug Metab Dispos 24:948–954. Yasar U, Eliasson E, Forslund-Bergengren C, Tybring G, Gadd M, Sjoqvist F, Dahl ML. 2001. The role of CYP2C9 genotype in the metabolism of diclofenac in vivo and in vitro. Eur J Clin Pharmacol 57:729–735. Wrighton SA, Stevens JC, Becker GW, Vanden Branden M. 1993. Isolation and characterization of human liver cytochrome P4502C19: Correlation between 2C19 and S-mephenytoin 40 -hydroxylation. Arch Biochem Biophys 306:240–245. Kronbach T, Mathys D, Gut J, Catin T, Meyer UA. 1987. High-performance liquid chromatographic assays for bufuralol 10 -hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver. Anal Biochem 162:24–32. Buters JTM, Korzekwa KR, Kunze KL, Omata Y, Hardwick JP, Gonzales FJ. 1994. cDNA-directed expression of human cytochrome P450 CYP3A4 using baculovirus. Drug Metab Disp 22:688–692: 1001–1007. Omura T, Sato R. 1964. The carbon monoxidebinding pigment of liver microsomes I. Evidence for its hemoprotein nature. J Biol Chem 239:2370– 2378. Brunelle A, Bi YA, Lin J, Russell B, Luy L, Berkman C, Cashman J. 1997. Characterization of two human flavin-containing monooxygenase (form 3) enzymes expressed in Escherichia coli as maltose binding protein fusions. Drug Metab Dispos 25:1001–1007. Cashman J. 1997. Monoamine oxidase and flavin-containing monooxygenases. In: Sipes IG,
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McQueen CA, Gandolfi AJ, editors. Comprehensive Toxicology. Vol. 3. New York: Elsevier Science. pp 69–96. Fuchs P, Haefeli WE, Ledermann HR, Wenk M. 1999. Xanthine oxidase inhibition by allopurinol affects the reliability of urinary caffeine metabolic ratios as markers for N-acetyltransferase 2 and CYP1A2 activities. Eur J Clin Pharmacol 54:869– 876. Eagling VA, Tjia JF, Back DJ. 1998. Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes. Br Clin Pharmacol 45:107–114. Bourrie M, Meunier V, Berger Y, Fabre G. 1996. Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J Pharmacol Exp Ther 277:321–332. Yamazaki H, Shimada T. 1998. Comparative studies of in vitro inhibition of cytochrome P4503A4-dependent testosterone 6 beta-hydroxylation by roxithromycin and its metabolites, troleandomycin and erythromycin. Drug Metab Dispos 26:1053–1057. Thompson GA, Burgio DE. 2004. The role of metabolite pharmacokinetics and pharmacodynamics in drug development. In: Bonate PL, Howard DR, editors. Pharmacokinetics in Drug
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Development Volume 1: Clinical Study Design and Analysis. Arlington, VA: AAPS Press. pp 321–346. Corey AE, Agnew JR, Valentine SN, Parekh NJ, Powell JH, Thompson GA. 2002. Effect of severe renal impairment on the pharmacokinetics of azimilide following single dose oral administration. Br J Clin Pharmacol 54:449–452. Corey AE, Agnew JR, King EC, Parekh NJ, Powell JH, Thompson GA. 2004. Effect of mild and moderate hepatic impairment on azimilide pharmacokinetics following single dose oral administration. J Pharm Sci 93:1279–1286. Bailey MJ, Dickinson RG. 2003. Acyl glucuronide reactivity in perspective: Biological consequences. Chemico-Biol Interact 145:117. Ramu K, Lam GN, Hughes H. 2000. In vivo metabolism and mass balance of 4-[4-fluorophenoxy]benzaldehyde semicarbazone in rats. Drug Metab Dispos 28:1153–1161. Phillips L, Grasela TH, Agnew JR, Ludwig EA, Thompson GA. 2001. A population pharmacokineticpharmacodynamic analysis and model validation of azimilide. Clin Pharmacol Ther 70:370– 383. El Mouelhi M, Worley DJ, Kuzmak B, Destefano AJ, Thompson GA. 2004. Influence of azimilide on CYP2C19-mediated metabolism. J Clin Pharmacol 44:373–378.
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Drug Safety Assessment, Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason Montgomery Road, Mason, Ohio 2
Bioanalytical Chemistry, Procter & Gamble Pharmaceuticals, Route 320, Woods Corners, Norwich, New York
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Clinical Pharmacology & Pharmacokinetics, Procter & Gamble Pharmaceuticals, 8700 Mason Montgomery Rd, Mason, Ohio
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Exploratory Toxicology & Drug Metabolism and Pharmacokinetics, Tanabe Seiyaku, 2-2-50, Kawagishi, Toda, Saitama 335-8505, Japan
Received 28 February 2005; accepted 24 May 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20429
ABSTRACT: The metabolic fate of azimilide in man is unusual as it undergoes a cleavage in vivo resulting in the formation of two classes of structurally distinct metabolites. During a metabolite profiling study conducted in human volunteers to assess the contribution of all pathways to the clearance of 14C-azimilide, greater than 82% of radioactivity was recovered in urine (49%–58%) and feces (33%). Urine, feces, and plasma were profiled for metabolites. A cleaved metabolite, 4-chloro-2-phenyl furoic acid was present at high concentration in plasma (metabolite/parent AUC ratio approx. 4), while other plasma metabolites, azimilide N-oxide (metabolite/parent AUC ratio 0.001), and a cleaved hydantoin metabolite (metabolite/parent AUC ratio ¼ 0.3) were present at lower concentrations than azimilide. In urine, the cleaved metabolites were the major metabolites, (>35% of the dose) along with phenols (as conjugates, 7%–8%), azimilide N-oxide (4%–10%), a butanoic acid metabolite (2%–3%), and desmethyl azimilide (2%). A limited investigation of fecal metabolites indicated that azimilide (3%–5%), desmethyl azimilide (1%–3%), and the butanoic acid metabolite (<1%) were present. Contributing pathways for metabolism of azimilide, identified through in vitro and in-vivo studies, were CYPs 1A1 (est. 28%), 3A4/5 (est. 20%), 2D6 (<1%), FMO (est. 14%), and cleavage (35%). Enzyme(s) involved in the cleavage of azimilide were not identified. ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2084–2095, 2005
Keywords: mass balance; metabolism; cytochrome P450; drug metabolizing enzymes; flavin monooxygenases; monoamine oxidase; xanthine oxidase
Azimilide is a novel class III antiarrhythmic drug, currently in clinical development for the
Abbreviations: MAO-B, monoamine oxidase B; XO, xanthine oxidase; FMO, human flavin monooxygenases; MPO, myeloperoxidase; CYP, cytochromes P450; NMR, nuclear magnetic resonance; AUC m/AUCp, Area under the curve in plasma concentration–time curve of metabolite/area under the curve in plasma concentration–time curve of parent. Correspondence to: P. Riley (Telephone: 513-622-0587; Fax: 513-622-1652; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 94, 2084–2095 (2005) ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association
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treatment of supraventricular arrhythmias1 and as an adjunctive therapy for use with implantable cardioverter defibrillators.2 It has a unique chlorophenylfuranyl structure (Fig. 1), and acts by blocking multiple ion channels, in particular the rapidly and slowly activated components of the delayed rectifier cardiac potassium current, IKr and IKs, respectively.3 By blocking IKs as well as IKr, azimilide maintains efficacy under the clinically important conditions of b-adrenergic stimulation and high heart rates.4 The recently completed ALIVE study demonstrated that azimilide has no
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EXPERIMENTAL Materials and Methods Figure 1. Structure of azimilide denoting positions of radiolabel. *denotes the 14C azomethine radiolabel. d denotes the 14C hydantoin radiolabel.
adverse effect on mortality in post-MI patients,5 and is generally well tolerated.5,6 The disposition of azimilide has been extensively studied in multiple animal species including mice, rats, rabbits, dogs, and monkeys (unpublished data). In these species, biliary clearance of azimilide and its metabolites dominated the excretion profile. In contrast, metabolism studies of azimilide in man have shown renal excretion of metabolites to account for a much larger proportion of the administered azimilide dose. A glucuronide conjugate of 4-chlorophenylfuroic acid (4-CPFA) formed following cleavage of the azomethine bond was the dominant metabolite, accounting for approximately 30% of the dose. This paper describes an analysis of the metabolic fate of azimilide in the urine, feces, and plasma of volunteers following a single oral dose of azimilide dihydrochloride. Since cleavage of the azomethine bond is a major feature of azimilide metabolism in humans, two radiolabeled forms of azimilide were synthesized, labeled with 14C at either the azomethine carbon, or on the hydantoin ring (Fig. 1), allowing the fate of both the left and right side of the cleaved molecule to be followed. The enzymes involved in azimilide cleavage were investigated with in vitro incubations of azimilide with possible enzyme candidates Flavin Monooxygenase (FMO), xanthine oxidase (XO), Monoamine Oxidase form B (MAO-B), and Myeloperoxidase (MPO). In addition, the role of CYPs, and other liver enzymes, was determined by incubating azimilide with a human liver microsome preparation. Based on the current literature,7 MAO-B, FMO-1, and FMO-3 were considered potential candidates for cleavage of azimilide. Unpublished rat studies in vivo suggest that CYPs do not play a role in the cleavage of azimilide. These hypotheses were investigated in the current study. In addition, the roles of CYPs and FMO in N-oxidation and N-demethylation of azimilide were investigated in vitro.
Internal standards, d8-azimilide dihydrochloride and 14C-azimilide dihydrochloride labeled either on the azomethine carbon atom or in the hydantoin ring and selected metabolites were synthesized by Chemical Development, Procter & Gamble Pharmaceuticals Inc., Norwich, NY. Various inhibitors of enzyme activity and components of the NADPH regenerating system were purchased from SigmaAldrich (Milwaukee, WI) unless otherwise noted. All chromatography solvents were supplied by JT Baker (Phillipsburg, NJ) and were of chromatographic grade. Scintillation supplies were obtained from Packard Instruments (PE Life Sciences, Shelton, CT). Enzymes were purchased from several commercial suppliers. In Vivo Study Design Twelve healthy, non-smoking male volunteers, aged 21–38 years (weight range 61.3–85.4 kg), were included in this study conducted at BioClin, Inc., Richmond, Virginia. Subjects received a single dose of 280 mg azimilide dihydrochloride labeled with 14C at either the azomethine carbon (n ¼ 6), or in the hydantoin ring (n ¼ 6) (final specific activity of dose 0.36 mCi/mg, each patient receiving 100 mCi total radioactivity) (Fig. 1). As only a small fraction of the dose was radioactive, d8-azimilide dihydrochloride was incorporated into the dose as an aid in the identification of metabolites by mass spectrometry. Blood samples were drawn for analysis of plasma at 0 (pre-dose), 3, 6, 12, 24, 36, and 48 h, and then every 24 h until two successive samples were below quantifiable limits of radiometric detection. At each time point, 10 mL blood was collected, processed to plasma and analyzed for metabolites. Twenty-four hour pooled urine and feces samples were collected until two successive measurements were below quantifiable limits. Assessment of radioactivity by scintillation counting of urinary and fecal samples provided the mass balance for the study. Samples from four subjects from each group were analyzed for metabolites. Mass Balance Assessment Total mass balance was assessed at the clinical site (BioClin, Inc.). Urine was pooled as 24 h samples and each pooled sample was analyzed for JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
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total 14C activity by liquid scintillation counting. Pooled fecal samples (24 h) were homogenized and aliquots taken for oxidation and scintillation counting. Analysis of Plasma The quantification of azimilide and its metabolites in plasma by HPLC/radiometric quantification (HPLC/RAD) was not possible as the concentrations were below the limits of detection for this method. Therefore an HPLC/MS assay method was developed using authentic standards of various metabolites for constructing the calibration curve. Due to incompatible chemical stabilities, and the diverse nature of the products it was necessary to prepare one set of samples with the internal standards IS1 (the hydroxyethyl analogue of azimilide) and IS2 (5-(4-fluorophenyl)-furan-2carboxylic acid) (allowing analysis of azimilide and azimilide N-oxide, and 5-(4-chlorophenyl)furan-2-carboxylic acid (4-CPFA), respectively), and another set of samples with IS3 (1-amino-3-[4(4-acetyl-1-1-piperazinyl)butyl]-2,4-imidazolidinedione) for the analysis of the cleaved hydantoin metabolites. For analysis of azimilide, azimilide N-oxide, and 4-CPFA plasma samples (500 mL) from four volunteers were mixed with internal standards (50 mL). To these mixtures, 500 mL 0.1 N ammonium hydroxide was added, followed by 20% methanol/acetonitrile (5 mL), before centrifugation at 3000 rpm for 10 min. The supernatant was carefully removed, and evaporated to dryness using a Savant Evaporator under medium heat. Dried samples were reconstituted in 500 mL 25 mM ammonium formate pH 5.5/10% methanol prior to HPLC/MS analysis. Plasma samples from two volunteers were analyzed for cleaved hydantoin metabolites (M1). When processing these samples, the drying stage was performed under a stream of nitrogen at 508C, and the resulting dried sample was dissolved in 500 mL 50 mM ammonium bicarbonate, pH 8.5/ 20% methanol. Control samples were analyzed in the same manner, using human plasma collected pre-dose. Chromatography Azimilide and azimilide N-oxide were assayed by HPLC/MS, using an Inertsil ODS-3 HPLC column (50 2.1 mm) on a Hitachi 6200A pump run at JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
600 mL/min, linked to Sciex API III mass spectrometer. The HPLC mobile phase consisted of Buffer A (90% 25 mM ammonium formate, pH 4.0/10% acetonitrile) and Buffer B (25% 25 mM ammonium formate, pH 4.0/75% acetonitrile), and material was eluted using a linear gradient of 60% Buffer A, 40% Buffer B to 10% Buffer A, 90% Buffer B over 0.5 min. Mass spectrometry typically involved ionization in the positive mode with APCI, using a probe temperature of 5008C, nebulizer gas at 80 psi, auxiliary gas at 3 L/min, and curtain gas at 1.2 L/min. Azimilide was monitored at 458/253 m/z, IS1 at 474/344 m/z, and azimilide N-oxide at 474/253 m/z. The hydantoin metabolite was assayed using a separate, but similar HPLC-MS procedure. The experimental setup used for measuring 4-CPFA was similar, except that a 100 2.1 mm ODS-3 HPLC column was used with isocratic elution using 30% acetonitrile/70% 12.5 mM ammonium formate pH 5.5. 4-CPFA and IS2 were monitored in the negative mode at 221/117 and 205/161 m/z, respectively. The concentrations of azimilide, azimilide Noxide, hydantoin metabolite, and 4-CPFA in the plasma samples were calculated from standard curves generated from the internal standards and authentic standards of metabolites. Weighted linear regression analyses were carried out for the observed signals (expressed as the ratio of peak area for each analyte to its corresponding internal standard). Analyte concentrations were converted to mg equivalents of azimilide/mL using the following equation. mgEq azimilide=mL ¼ ng=mL analyte ðmw azimilideÞ=ðmw analyteÞ mg=1000 ng Analysis of Urine Aliquots of urine (9.4 mL) were evaporated to dryness in a Savant evaporator (set at a cool temperature) before resuspending in 1 mL HPLC mobile phase (98% ammonium acetate [50 mM, pH 5.2], 2% methanol). Three 10 mL samples of the resuspended solution were mixed with scintillation fluid, before counting. Solutions of authentic standards were injected onto the column every fourth sample as a check of the column performance. HPLC was undertaken using a Waters C18 Symmetry Cartridge Column (4.6 250 mm) on a
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Perkin Elmer 410 Quaternary pump using UV detection at 315 nm (Perkin Elmer 235c Detector Model LC235) and radiometric detection (Packard Radiometric Model A515A Flo-One Detector). The mobile phase consisted of Buffer A (98% 50 mM ammonium acetate, pH 5.2/2% methanol), Buffer B (66% 50 mM ammonium acetate, pH 4.6/26% acetronitrile/8% methanol), and Buffer C (73% 50 mM ammonium acetate, pH 6.0/22% acetonitrile/5% tetrahydrofuran). The column was eluted using a linear gradient from 100% Buffer A to 100% Buffer B over 10 min, followed by a linear gradient from 100% Buffer B to 100% Buffer C over 5 min. Eluted peaks were identified by comparison of retention times with standards, and confirmed by mass spectrometry. Recovery of radioactivity after HPLC was measured by passing a known quantity of radioactivity onto the column and measuring the radioactivity in the eluent over a complete HPLC run. The areas under peaks of interest were determined electronically, and expressed as a fraction of the total area under all peaks. The number of counts in each peak was calculated as the fraction of the total number of counts injected onto the column (determined by scintillation counting). Using an efficiency value of 0.88 (determined experimentally), cpm was converted to dpm and to mCi. The mg Equivalents of azimilide (base) were then calculated from the known specific activity (in this case, 0.36 mCi/mg). Loss of sample at the initial evaporation/resuspension stage was calculated by scintillation counting before and after this step. Analysis of Feces Samples (1 g) of previously prepared fecal homogenates were combined with 25 mL of extraction solvent (72.7% acetonitrile/18.2% methanol/9.1% 32 mM ammonium acetate pH 5.0) and shaken for 10 min. The extract was centrifuged and a 2.5 mL sample of the supernatant was combined with 200 mL of the internal standard (4.8 mg/mL IS1). The combined sample was mixed and evaporated to dryness under a stream of nitrogen. The dried sample was reconstituted with 0.5 mL of mobile phase (pH 5.4 buffer containing 32% acetonitrile/ 5% methanol/63% 32 mM ammonium acetate pH 5.0). Particulate matter was removed by centrifugation and a 50 mL sample of supernatant was injected into the HPLC system. HPLC was conducted on a Metasil C-8 column (particle size was 5 mm, column dimensions 25 cm 4.6 mm). The mobile phase flow rate was 1.5 mL/
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min. Absorbance was monitored at 340 nm. Isocratically eluted metabolite peaks (desmethyl azimilide and the butanoic acid metabolite) and azimilide were identified by comparison of retention times with standards.
In Vitro Determination of Enzymic Pathways Cleaved Metabolites Human liver microsome incubations. Human liver microsomes were purchased from BD/Gentest Inc. (Woburn, MA). Reaction tubes containing 25 mg 14C-azimilide (labeled at the azomethine position) were incubated at 378C with 0.5 or 1 mg microsomal protein for 10–20 min in a final volume of 250 mL. Reactions were buffered in 50 mM potassium phosphate buffer (pH 7.4), 0.8 mM diethylenetriaminepentaacetic acid (DETAPAC), 0.5 mM NADPþ, 0.4 mM glucose-6-phosphate, and 1 IU glucose-6-phosphate dehydrogenase, and were terminated by rapid freezing in a dry ice/ acetone bath. Inactivated, boiled microsomes were used as a negative control. Microsome-catalyzed oxidation of 10-(N,N-dimethylaminopentyl)2-(trifluoromethyl) phenothiazine (5-DTP) provided a positive control. In addition, the activity of the microsome preparations for, CYP1A1(7-ethoxyresorufin), CYP1A2 (caffeine),8 CYP2A6 (coumarin),9 CYP2B6 (benzphetamine),10 CYP2C9 (tolbutamide),11 CYP2C19 (S-mephenytoin),12 CYP2D6 (dextromethorphan),13 CYP2E1 (chlorzoxazone), CYP3A4 (testosterone),14 CYP4A (lauric acid), and Cyt b515 were assessed (probe substrates indicated). Flavin monooxygenase (FMO) incubations. Recombinant human FMO-1 and FMO-3 were expressed from E. coli as maltose-binding proteins.16 Incubations with 14C-azimilide were performed at pH 7.4, but otherwise under the conditions described for microsomal incubations, using 25 or 50 mg of human FMO enzymes for 20 min. Inactivated, boiled FMO-1 and FMO-3 (25 mg/mL protein) were used as negative controls, and oxidation of 5-DTP was followed as a positive control. Monoamine oxidase B (MAO-B) incubations. Incubations were performed under the conditions described for microsomal incubations, using 2.5 or 5 mg of MAO-B for 10–20 min. Inactivated, boiled MAO-B (2.5 mg) incubations were used as negative JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
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controls, and the conversion of benzylamine (3 mM) to benzaldehyde monitored as a positive control.17 Xanthine oxidase (XO) incubations. XO (0.2 or 1.0 units/mL), obtained from Sigma Chemical Co. was incubated with azomethine labeled 14C-azimilide (10 mM, 0.061 mCi) in 50 mM sodium phosphate buffer (pH 7.5) at 378C in triplicate, for 0, 10, 30, and 90 min. The activity of XO was verified by monitoring its ability to metabolize hypoxanthine, and its inhibition by allopurinol.18 Myeloperoxidase (MPO) incubations. Azimilide (500 mM) was incubated with MPO (Cortex, 10 units/ mL) in 100 mM phosphate buffered saline, pH 6.0. The enzymatic reaction was initiated by the addition of hydrogen peroxide (final concentration 10 mM) and left at room temperature for 45 min. The reaction mixture was passed through C18 Sep peaks for concentration and eluted with methanol. Other Metabolic Products Phenolic metabolites. Phenolic metabolites were not observed in microsomal incubations with azimilide and further investigation with Supersomes 1 (BD/Gentest) expressing CYP1A1 and CYP1A2 (50 mg) in a buffer containing 10 mM glucose-6-phosphate, 5 mM MgCl2, 0.5 mM bNADPþ, 0.2 units glucose-6-phosphate dehydrogenase, 0.1 M sodium phosphate buffer (pH 7.4, total volume 500 mL) at concentrations of 20 and 200 mM azimilide were undertaken. Reaction mixtures were pre-incubated at room temperature for 2 min prior to the addition of Supersomes1. The reactions were quenched at 30 min by addition of 100 mL acetonitrile, and precipitated protein was removed by centrifugation. Inhibition of CYP1A1 and CYP1A2 with furafylline provided a positive control. Desmethyl and azimilide N-oxide. Human liver microsomes (1 mg/mL protein), with known cytochrome P-450 activities, were mixed with 14Cazimilide (azomethine label, 200 and 400 mM) in a buffer containing 50 mM potassium phosphate (pH 7.4), 0.1 mM EDTA, 3 mM MgCl2 and preincubated at 378C for 3 min before addition of glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and 0.5 mM NADPþ in a final volume of approximately 250 mL. After 30 min, reactions were quenched by addition of 250 mL acetonitrile. The samples were centrifuged and the resulting JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
supernatant analyzed by HPLC/RAD. Control incubations in the presence of furaphylline (30 mM), sulfaphenazole (10 mM) (Ultrafine Chemicals), quinidine (10 mM), or triacetyloleandomycin21 (TAO, 0.1–500 mM) to specifically inhibit CYP1A2, CYP2C9/10, CYP2D6, and CYP3A4/5, respectively were included.19,20 Negative controls for FMO by inclusion of methimazole (200 mM, competitive inhibitor), and after heat inactivation of the thermally-labile FMO enzyme by heating to 508C for 90 s were also assessed in microsomal incubations. Recombinant cytochrome P-450 enzymes (Gentest Corporation), specifically CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 were conducted using 2 mg protein and 400 mM azimilide per reaction, with reactions terminated at 3 h by quenching with acetonitrile. Analysis of In Vitro Enzyme Pathway Studies Analysis of in vitro reaction mixtures for cleavage products. After quenching FMO-1, FMO-3, MAOB, XO, microsomal and Supersome1 incubations were centrifuged at 10000 rpm for 5 min, before aliquots of supernatant were removed for liquid scintillation and HPLC/RAD analyses. Chromatographic resolution was achieved with a Waters C8 5 mm, 3.9 150 mm column, on a Waters 600 LC pump, with UV detection at 310 nm (Perkin Elmer, s/n 92568) and radiometric detection using a Packard Flo One Beta, model 525TR series radioactivity detector. The mobile phase consisted of 30% acetonitrile/70% 25 mM ammonium formate buffer, pH 3.2, delivered isocratically at 1 mL/min (with supplemental scintillant at 3 mL/min). The quantities of 14C-azimilide, and the cleavage products 14C-F-1054 (an aldehyde, which is rapidly converted to 4-CPFA in vivo) and 14 C-4-CPFA (the corresponding carboxylic acid) formed in the reactions were calculated by comparison of HPLC retention times of reaction mixtures with those of synthesized authentic standards. Analysis of MPO reactions. Samples from the incubation of azimilide with MPO/H2O2 were analyzed by LC/MS performed on a Sciex API III mass spectrometer with an ion spray interface. Analysis of in vitro reaction mixtures for phenolic metabolites. The polar nature of the phenolic metabolites required modification of standard
METABOLIC PROFILE OF AZIMILIDE
analytical methods for this compound. HPLC was undertaken at 408C using a Waters C18, 5 mm, 4.6 150 mm column, on a Waters 626 HPLC pump, with fluorescence detection using a Waters 474 fluorescence detector (excitation l ¼ 340 nm; emission l ¼ 400 nm). Mobile phases consisted of Buffer A (acetonitrile/water/formic acid [95/5/ 0.1,]) and Buffer B (acetonitrile/water/formic acid [5/95/0.1). Material was eluted from the column by running a linear gradient at 1 mL/min from 90% Buffer A/10% Buffer B to 10% Buffer A/90% Buffer B, over 30 min. The column was linked to an LTC mass spectrometer with electrospray ionization and time of flight analysis to identify eluted material. NMR spectroscopy was used to determine the position of the hydroxyl group on the benzene ring. Material was evaporated to dryness under a stream of N2, before resuspending in D2O. 1 H-NMR was undertaken at 600 MHz on a Bruker DRX-600 instrument, with presaturation of the residual water peak during the relaxation delay.
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by comparison with known retention times for azimilide N-oxide and desmethyl azimilide.
RESULTS Mass Balance Assessment Label stability studies were conducted in rats prior to commencement of the preclinical assessment of azimilide. As loss via 14CO2 in these studies was less than 0.5%, no further assessment of loss of 14CO2 in humans was undertaken. Samples of urine and feces, collected as pooled 24 h samples were assessed for radioactivity by scintillation counting (Tab. 1) for all subjects. Mean total recovery was good with >82% (79– 86)% recovered in the hydantoin group and >91% (87–95)% recovered in the group receiving azomethine label. Radioactivity was recovered primarily in the urine (49% vs. 58% for hydantoin vs. azomethine label) with approximately 33% of the administered dose being recovered in feces. As no 14 CO2 was released from either radiolabel in label stability studies (unpublished data) the difference between the urinary values obtained likely reflects differences in the disposition (ratio of renal to non renal extraction) of the structurally distinct cleaved metabolites.
Analysis of in vitro reaction mixtures for desmethyl azimilide and azimilide N-oxide. HPLC was undertaken using an Exsil C8 5 or 10 mm, 4.6 250 mm column, on a Perkin Elmer 410 quaternary pump, with UV detection at 340 nm (Perkin Elmer LC235) and radiometric detection using a Packard Flo One, model A515 radioactivity detector. The column was eluted isocratically in 60% buffer A (50 mM ammonium acetate, pH 5.5/ 40% buffer B (10% tetrahydrofuran in acetonitrile), or 50% Buffer A/50% Buffer B, and peaks identified
Characterization and Quantitation of Metabolites From preclinical studies it was established that metabolites could be classified by structure into six groups of metabolites and these were assigned
Table 1. Mass Balance and Excretion Pattern of Radioactivity Following a Single Oral Dose (280 mg, 100 mCi) 14C-azimilide Dihydrochloride to Healthy Male Volunteers Azomethine Label Volunteer ID 47550001 47550002 47550003 47550004 47550005 47550006 Mean (% CV) Hydantoin label 47550007 47550008 47550009 47550010 47550011 47550012 Mean (% CV)
% Administered Radioactive Dose Excreted Urine 61.00 61.53 51.46 59.48 57.86 57.58 58.05 (6.3)
Feces 29.62 33.84 39.79 33.18 29.57 36.79 33.60 (11.9)
Total 90.62 95.37 91.25 92.67 87.44 94.37 91.91 (3.1)
51.69 49.15 52.08 50.46 44.66 49.01 49.44 (5.4)
28.45 37.00 33.04 32.40 37.80 30.00 32.94 (11.3)
80.14 86.15 85.11 82.86 82.45 79.01 82.58 (3.3)
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Figure 2. Representative radiochromatogram of urine showing retention order of metabolites (chromatograms were generated with urine from rats dosed with a mixture of hydantoin and azomethine labeled azimilide and is used for illustrative purposes only to show retention time of metabolites).
the notation as M1–M6 based on retention time in the radiochromatogram (Fig. 2). Metabolites, where multiple isomers existed, within each group were quantified separately and then combined to give the total for each group. For the cleaved metabolites, M1 and M3, where quantitation was based on radioactivity, one group was not present as the label remained with the other portion of the molecule. Characterization of Plasma Metabolites The concentration of radioactivity present in plasma at any given time was insufficient to measure plasma concentrations of metabolites by HPLC/RAD and thus an HPLC/MS assay was developed to measure metabolites for which authentic standard was available. Three metabolites were monitored and the relative ratios of AUC of metabolite to parent (AUCm/AUCp) were calculated and are presented in Figure 3. The maximal concentration (Cmax) of azimilide was reached within 3 to 6 h post dose, whereas the Cmax value for the cleaved metabolite, 4-CPFA (M3), was not reached until 36–48 h post dose (Fig. 4). No glucuronide or glycine conjugates of 4CPFA were observed in plasma. Characterization of Urinary Metabolites Urinary excretion of azimilide and its metabolites was quantitated by HPLC/RAD, with subsequent identification by mass spectrometry and NMR. For subjects who received the azomethine labeled material, no hydantoin metabolites (M1) were observed as the radiolabel remained with the furoic acid metabolites. Similarly, for subjects who received the hydantoin radiolabeled azimiJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
lide no furoic acid metabolites were observed. The percent dose excreted as each metabolite is presented in Figure 3. Characterization of Fecal Metabolites The metabolic profile in feces was not determined for all metabolite groups (M1–M6) as limited authentic samples were available for defining analytical parameters. Thus, only the concentrations of azimilide, desmethyl azimilide and the butanoic acid metabolite (M6) were measured. Of the approximated 33% of dose excreted in feces, only ca. 6% of the dose was accounted for by these metabolites (Fig. 3). In preclinical studies, only traces of the cleaved metabolites (M1 and M3) were observed in bile and thus, it is likely that the remaining 27% of dose is azimilide N-oxide (M4) or the conjugated phenols (M2). Ratios of M2 to M4 in bile collected from animals studies ranged from 0.5 (rat) to 3 (monkey). A value of 1 was used to estimate fecal excretion for estimation of pathways (Table 2). In Vitro Analysis of Cleavage Products Human Liver Microsomal Metabolism of Azimilide Human liver microsomes were shown to be active in all positive control tests. Low concentrations of 4-CPFA were present in some samples but were matched in negative controls suggesting that some low background hydrolytic activity was occurring. Reaction of Azimilide With FMO-1, FMO-3, and MAO-B All enzymes were active in positive control reactions, but had no effect on the cleavage of
METABOLIC PROFILE OF AZIMILIDE
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Figure 3. Proposed metabolic pathways of azimilide in man.
azimilide with the apparent low level of FMO-1 and FMO-3 activity matched in negative controls. XO Incubations 4-CPFA was formed in the presence of XO in low quantities (up to 1.84% of azimilide was converted to 4-CPFA). However, there was no evidence of time, or enzyme concentration dependence of the reaction, and 4-CPFA was formed in similar quantities in the presence of the XO inhibitor allopurinol. Myeloperoxidase Incubations Figure 4. Plasma concentration versus time curves for measured metabolites and azimilide following a single oral dose (280 mg; 100 mCi) 14C-azimilide dihydrochloride to healthy male volunteers.
Azimilide underwent degradation under the conditions of the assay to 4-chlorophenyl-2-furfural, a potential precursor to 4-CPFA. Further investigation showed that similar degradation could be JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
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achieved under acidic conditions, with the rate being optimum at pH 4, and thus, it is likely that the MPO provided the chemical environment rather than the catalytic environment for this degradation to occur. In Vitro Analysis of Other Metabolic Products Phenolic Metabolites Incubation of azimilide with CYP1A1 (20 and 200 mM), but not CYP1A2 (200 mM), resulted in hydroxylation of the chlorophenyl moiety of azimilide to the extent of 2.59, 1.01, and 0.0% respectively. 1H NMR spectroscopy demonstrated that in these experiments, the hydroxyl group was substituted at the ortho position relative to the chlorine atom. From in vivo studies, multiple monohydroxylated species (excreted as glucuronide and sulfate conjugates) were observed. The Km for the conversion of azimilide to the phenolic metabolites was determined to be 5.2 mM. Desmethyl Azimilide and Azimilide N-Oxide Human liver microsomes were found to catalyze the conversion of azimilide to both azimilide Noxide (Km ¼ 259 mM, Vmax ¼ 0.26 nmol/mg/min, Clint 1.02 mL/min/mg) desmethyl azimilide (Km ¼ 179 mM, Vmax ¼ 0.17 nmol/mg/min, Clint 0.93 mL/min/mg) in a time- and concentrationdependent manner. Specific inhibition of the CYP3A4/5 isoforms using TAO (500 mM) resulted in 64% and 92% inhibition of N-demethylation and N-oxidation, respectively. Inhibition of other isoforms had little noticeable effect. Inhibition of microsomal FMO activity by heat inactivation resulted in approximately 45% inhibition of Noxidation, while only slightly (7%) affecting Ndemethylation. Incubations using recombinant cytochrome P-450 isoforms resulted in notable formation of desmethyl azimilide in the presence of CYP2D6 (yielding approximately 0.8 nmoles/ reaction) and CYP3A4 (yielding approximately 2.8 nmoles/reaction), and in the formation of azimilide N-oxide in the presence of CYP3A4 (yielding approximately 1.7 nmoles/reaction). Taken together, these results suggest that azimilide N-oxide is formed through the action of CYP3A4/5 and FMO, and desmethyl azimilide is formed through the action of FMO, CYP3A4/5, and CYP2D6. One approach to define the potential for drug interactions is based on the relative importance of different metabolic pathways as outlined by Thompson.22 For azimilide, metabolites include JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
M2 (catalyzed by CYP1A1), M4 (catalyzed by CYP3A4/5 and FMO), M5 (catalyzed by CYP 3A4/ 5, FMO and CYP 2D), M6 (catalyzed by an unknown pathway), and the cleaved metabolites, M1 and M3. Although no mechanism for the cleavage of azimilide has been identified, approx. 35% of the dose is metabolized via this pathway (Fig. 3). For M2 (excreted as phenolic conjugates) formation is via a single enzyme. Although metabolic profiling for M2 in feces was not conducted in this study, inference from biliary studies in higher animal species (unpublished data) where 10–20% of the dose was consistently recovered for both M2 and M4, with no M3 being excreted in bile. Since approximately 30% of human fecal radioactivity could not be attributed to measured metabolites or parent drug, recovery of 15% of the dose in feces as M2 and 15% as M4 was assumed in man. Since M2 is formed via a single enzyme, CYP1A1 contribution to total clearance is 28%. For metabolites M4 and M5 multiple enzymes contribute to the formation of each metabolite. Based on in vitro studies, heat inactivation of FMO decreases formation of M4 by 45%. Since 30% of the dose is recovered as M4, FMO accounts for 13.5% of total clearance, with the remaining 16.5% attributed to CYP 3A4. Both enzymes also contribute to the formation of M5, which is only a minor metabolite (4% total recovery). From the in vitro studies, FMO contributes less than 10% to M5 formation, and the relative rates of formation of M5 between CYP3A4 and CYP2D6 suggest that CYP 2D6 accounts for <1% of the administered dose. Thus the total contribution from CYP3A4 is approximately 20%, while FMO contributes approximately 14% and CYP2D6 <1%. These estimates are summarized in Table 2. The contributions of enzymes responsible for the formation of M6 are unlikely to be major contributors to potential interactions as only 2–3% of dose is metabolized through this pathway.
DISCUSSION Clearance of azimilide is mediated via both metabolic and renal pathways. Due to the diversity of pathways involved, a priori dosage regimen adjustments may not be required for patients with renal or hepatic impairment. This is supported by recent pharmacokinetic/pharmacodynamic studies of azimilide in man, where no adjustments in dose were required in patients in renal failure23 or with mild to moderate hepatic failure.24
METABOLIC PROFILE OF AZIMILIDE
Table 2. Estimates of the Different Pathways Involved in the Metabolism of Azimilide
Pathway CYP1A1a CYP3A4/5a CYP2D6 FMOa Cleavage
Approximate % Metabolized Through Pathway 28 20 <1 14 35
a Based on an estimate of 15% recovery of each M4 (azimilide N-oxide) and M2 (phenolic metabolites excreted as conjugates) in feces which is in the range of that observed in higher animal species (unpublished data).
The metabolic changes observed in this study are consistent with those observed in animal studies (unpublished data) and oxidative pathways are predicted by typical cytochrome P-450 catalyzed reactions. Formation of the cleaved products is not mediated by CYP enzymes as no cleaved products were observed in incubations of azimilide with microsomes or hepatocytes (humans, rats, dogs, unpublished data) or liver slices (dog, unpublished data). The extent of formation of the cleaved metabolites (M1 and M3) in humans is much higher than that observed in animals, with significantly higher plasma concentrations of 4-CPFA than azimilide at all time points measured (Fig. 4), as shown by the relative AUC of metabolite to parent (4.10, Fig. 3). However, this metabolite shows no antiarrhythmic activity, as measured by increase in action potential duration 90%, and therefore does not contribute to the overall antiarrhythmic profile of azimilide. The low concentration of azimilide N-oxide (M4) and desmethyl azimilide (M5) in plasma also suggests that these metabolites contribute little to the overall pharmacological activity although they do possess some antiarrhythmic activity (20 and 11% relative to parent). Because of concerns about the potential toxicity of acyl glucuronides25 an assay was developed to determine the plasma concentration of the acyl glucuronide of 4-CPFA. No acyl glucuronide was detected in plasma, although the primary excretion product of 4-CPFA in urine was the acyl glucuronide, with traces of the glycine conjugate and free acid. These cleaved metabolites account for approximately 30%–35% of the administered dose. Chemically the azomethine bond is unstable under acid conditions and thus it might be anticipated that acid catalyzed hydrolysis might occur in the stomach after oral administra-
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tion. However, bioavailability of azimilide in man following oral administration approaches 100% and the appearance of 4-CPFA in plasma parallels azimilide concentration, i.e., there is no initial spike as might be expected from absorption of the acid from the stomach. Azimilide possesses detergent properties and an oral dose exceeds the critical micelle concentration in the stomach. It is possible that the azomethine bonds may be internalized within the micelle and protected from hydrolysis. To further understand the metabolism of azimilide via the cleavage pathway several enzyme systems were considered as potential metabolizing systems for formation of the cleaved metabolites. Selection of these enzymes was based on substrate specificities, mechanistic considerations, or as major enzyme systems that could potentially result in drug-drug interactions if involved in the cleavage of azimilide. Despite incubations with cytochromes P-450, MAO-A and B, FMO 1 and 3, XO and MPO, no conclusive evidence was obtained to suggest any of the enzymes tested was involved in the formation of 4-CPFA. Another compound containing an azomethine bond, 4-[4-fluorophenoxy]-benzaldehyde semicarbazone (Co 102862) has been reported as being extensively metabolized with the major metabolites being a carboxylic acid and its glucuronide conjugate. The carboxylic acid metabolite of Co 102862 was presumed to be formed through an aldehyde intermediate by hydrolysis of the azomethine bond.26 The corresponding aldehyde formed from azimilide, 5-(4-chlorophenyl)-2-furaldehyde, is rapidly oxidized to the carboxylic acid in fresh human blood suggesting that if formation occurs through hydrolysis, the observable end product would be the carboxylic acid. Although the mechanism of formation is unknown for the cleaved metabolites other metabolites of azimilide were clearly established as being mediated by various CYPs (predominantly CYP3A4 and 1A1) and for some metabolites, FMO was also implicated. The level of involvement of these isoforms was considered unlikely to lead to significant clinical interactions, and recent pharmacokinetic studies of azimilide in man27,28 supports these findings, with pharmacokinetic parameters unaffected by co-administration of azimilide with digoxin, warfarin, and CYP3A4 inhibitors and inducers. Furthermore, the involvement of CYP1A1 (for formation of the phenolic metabolites, Km ¼ 5.2 mM) was consistent with a change in clearance among smokers.27 The multiJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 9, SEPTEMBER 2005
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plicity of mechanisms utilized in the metabolism of azimilide indicates that the clearance of azimilide in humans is not excessively reliant on any one particular pathway. In conclusion, azimilide and its metabolites are excreted both in urine and feces, thereby reducing the dependence on a single route of excretion. Metabolism is extensive with less than 15% of parent being excreted unchanged. Multiple pathways involving CY3A4, CYP1A1, CYP2D6, and FMO were identified as contributing to azimilide metabolism and, although the mechanism of formation of the cleaved metabolites has not been clearly identified, the lack of involvement of major metabolizing enzymes such as the CYPs, MAO, FMO, etc. suggests that the likelihood of a clinically relevant drug interaction through known pathways is minimal.
ACKNOWLEDGMENTS We thank our colleagues, Dr. Tom Huggins and Dr. Roy Dobson for their invaluable assistance and discussions concerning metabolite identification. We are also indebted to our collaborators, Dr. John Cashman (Human Biomolecular Research Institute, San Diego) and Jack Uetrecht (University of Toronto) for their help with in vitro preparations. Finally, our thanks go to The Medical Knowledge Group (London) for their assistance in preparation of this manuscript.
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