Abstract
[14C]7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine ([14C]-TPA023; 99 μCi/dose) was administered to five young, healthy, fasted male subjects as a single oral dose (3.0 mg) in solution (propylene glycol/water, 10:90 v/v). The parent compound was rapidly absorbed (plasma Tmax ∼2 h), exhibited an apparent terminal half-life of 6.7 h, and accounted for approximately 53% of the total radioactivity in plasma. After 7 days of collection, the mean total recovery of radioactivity in the excreta was 82.6%, with 53.2% and 29.4% in urine and feces, respectively. Radiochromatographic analysis of the excreta revealed that TPA023 was metabolized extensively, and only trace amounts of unchanged parent were recovered. Radiochromatograms of urine and feces showed that TPA023 underwent metabolism via three pathways (t-butyl hydroxylation, N-deethylation, and direct N-glucuronidation). The products of t-butyl hydroxylation and N-deethylation, together with their corresponding secondary metabolites, accounted for the majority of the radioactivity in the excreta. In addition, approximately 10.3% of the dose was recovered in urine as the triazolo-pyridazine N1-glucuronide of TPA023. The t-butyl hydroxy and N-desethyl metabolites of TPA023, the TPA023 N1-glucuronide, and the triazolo-pyridazine N1-glucuronide of N-desethyl TPA023 were present in plasma. In healthy male subjects, therefore, TPA023 is well absorbed and is metabolized extensively (t-butyl hydroxylation and N-deethylation > glucuronidation), and the metabolites are excreted in urine and feces.
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and is essential for the overall balance between neuronal excitation and inhibition (Young and Chu, 1990; Chebib and Johnston, 2000). GABA influences neurons via three classes of receptors, GABAA and GABAC, which are ligand-gated ion channels, and GABAB, which is a G-protein-coupled receptor (Chebib and Johnston, 2000). The most widely studied of these, in terms of both its molecular biology and pharmacology, is the GABAA receptor. GABAA receptors are heteromeric pentamers of subunits expressed by a large multigene family, currently consisting of 6α, 3β, 3γ, δ, ϵ, θ, 3ρ, and π genes (Graham et al., 1996; Johnston, 1996; Barnard et al., 1998; Chebib and Johnston, 2000; McKernan et al., 2000). Modulation of the GABAA receptor has a number of effects on behavior and is the site of action of a number of drugs, including ethanol, neurosteroids, general anesthetics, barbiturates, and benzodiazepines, which mediate their action through binding sites on various subunits.
Generalized anxiety disorder (GAD) is a disease state characterized by excessive anxiety and persistent worry for a period of 6 months or more (Syramek et al., 2002). The ideal agent for the treatment of GAD would have a rapid onset of action, elicit few adverse effects, and show no tolerance (Syramek et al., 2002). Benzodiazepines, which modulate their activity by binding to the α1, 2, 3, and 5 subunits of the GABAA receptor and increase the potency of GABA by increasing the frequency of channel openings in response to GABA binding (Chebib and Johnston, 2000), are the most commonly prescribed drugs used to treat GAD. They are extremely effective anxiolytics with a rapid onset of action, although several adverse effects, including sedation, impaired psychomotor performance, memory impairment, and the potential for abuse and dependence (Syramek et al., 2002), limit their use. However, not all of these effects are mediated through the same α subunits; therefore, novel subunit-specific GABAA receptor modulators may result in compounds with an improved side effect profile (McKernan et al., 2000; Rudolph et al., 2001; Blackaby et al., 2006; Goodacre et al., 2006a).
TPA023 (Fig. 1) is being developed as an anxiolytic for use in the treatment of GAD. The compound behaves as a potent GABAA receptor agonist with functional selectivity toward α2 and α3 receptor subtypes (Atack et al., 2006; Carling et al., 2006; Goodacre et al., 2006b; Jennings et al., 2006; Russell et al., 2006). In animal models, it is an efficacious anxiolytic that lacks the side effects experienced with more nonselective GABAA receptor agonists. The objectives of the present study were: 1) to investigate the routes of elimination of TPA023 in healthy subjects after a single oral dose; 2) to quantitate the levels of total radioactivity and TPA023 in plasma after the single oral dose; 3) to examine the metabolism of TPA023 in humans; and 4) to demonstrate mass balance for [14C]TPA023 in humans.
Materials and Methods
Chemicals. [14C]TPA023 (>99% radiochemical purity) and authentic standards of TPA023, M11 (t-butyl hydroxy), M15 (aldehyde), M10 (carboxylic acid), M12 (N-desethyl metabolite), M9 (alcohol of N-desethyl metabolite), M14 (O-dealkylation), and M5 (N1-glucuronide of TPA023) were synthesized at Merck Research Laboratories (Rahway, NJ). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO), or Aldrich Chemical Co. (Milwaukee, WI). Pooled human liver microsomes were purchased from Xenotech LLC (Kansas City, KS).
Human Studies. The study protocol was approved by the Southern Institutional Review Board (Miami, FL). All subjects understood the procedures and agreed to participate in the study by giving written informed consent (Declaration of Helsinki). No concomitant medication was taken during the study.
[14C]TPA023 (99 μCi, 3 mg; 0.05 mg/ml) was administered to five healthy, fasted male subjects as a single oral dose in solution (propylene glycol/water; 10:90, v/v). The dose solution was followed by two separate rinses of water (125 ml), which were completely ingested within 2 min of dose consumption. Thereafter, heparinized blood was collected at intervals through 168 h (7 days). The plasma was separated and stored at -20°C. Urine and feces were collected at various intervals for 7 days and stored at -20°C.
Measurement of Total Radioactivity. Aliquots of plasma and urine (0.2-1.0 ml) were transferred to polyethylene vials, and 15 ml of scintillation cocktail (Ready-Safe; Beckman Coulter, Fullerton, CA) was added for counting in a Packard 2500 TR liquid scintillation counter (PerkinElmer Life and Analytical Sciences, Boston, MA). Aliquots of the fecal homogenate and red blood cells (two times, ∼0.2 g) were combusted in a Packard robotic tissue oxidizer (model 307). The resultant carbon dioxide was trapped in a Carbosorb/Permafluor mixture (Packard Instrument Co). Radioactivity in all samples was determined with a Packard 2500 TR liquid scintillation counter.
LC-MS/MS Analysis of Plasma for TPA023. Plasma samples were centrifuged (3000 rpm, 5 min) in a Beckman GS-6KR refrigerated centrifuge (Beckman Coulter Inc., Fullerton, CA), and a 1-ml aliquot of the supernatant was transferred to clean test tubes. To each sample tube was added 1:1 acetonitrile/water (50 μl), internal standard working solution (0.05 μg/ml MRK-2 in 1:1 acetonitrile /water, 50 μl), and 0.15 M phosphate solution (pH 2; 1 ml). The tubes were vortexed after each addition. An Ansys SPEC Plus 96-well solid phase extraction plate (MP3 Mixed Phase Cation, 15 mg; Varian, Inc., Palo Alto, CA) was conditioned with methanol (800 μl), water (800 μl), and 0.1 M phosphate buffer (pH 2; 800 μl), each drawn through the plate wells with low negative pressure (<10 inches Hg). Two 1-ml aliquots of each tube were then added to the plate wells, followed by the addition of water (1 ml), 1.0 M acetic acid (800 μl), and methanol (800 μl), each drawn through with low negative pressure (<10 inches Hg). After removing any excess wash solvent, the plate was placed onto a disposable microtiter tray and centrifuged for 5 min at 1000 rpm to remove any residual liquid from the plate. The plate was then placed on top of a 650-μl 96-position deep-well sample collection plate, and 300 μl of 78:20:2 (v/v/v) methylene chloride/isopropanol/ammonium hydroxide was added into each well. The assembly was then centrifuged for 5 min at 1000 rpm to elute the analyte and internal standard into the collection plate. The eluate was evaporated to dryness under nitrogen using an EvapArray evaporator (Jones Chromatography, Lakewood, CO) set at 40°C. The residues were reconstituted with 100 μl of reconstitution solvent (85:15 v/v; 10 mM ammonium acetate/acetonitrile), and the plate was sealed, vortexed, sonicated, vortexed a second time, and placed into a PerkinElmer series 200 autosampler tray for analysis (PerkinElmer Life and Analytical Sciences, Boston, MA).
The samples were run on a PerkinElmer LC series 200 system. TPA023 and the internal standard, MRK-2, were separated on a Restek Ultra IBD column (30 × 4.6 mm, 3 μm; Phenomenex, Torrance, CA) with a mobile phase eluting at a flow rate of 1 ml/min. The mobile phase consisted of 35% acetonitrile in 10 mM ammonium acetate (pH 4) held isocratically over the 5-min run time. Under these conditions, [14C]TPA023 and [12C]TPA023 coeluted at a retention time of 2.1 min.
The HPLC system was interfaced with a SCIEX API 3000 tandem mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a heated nebulizer interface. Mass spectral analyses were carried out in the positive ion mode. The heated nebulizer probe temperature was 500°C. Tandem mass spectrometry (MS/MS) monitoring of the precursor/product ion transitions was carried out. The precursor → product ion combinations of m/z 396 → 287, 398 → 289, and 362 → 334 were used to quantify [12C]TPA023, [14C]TPA023, and the internal standard, respectively.
Using this procedure, the assay for the total concentration of TPA023 in human plasma was validated in the range of 0.1 to 50 ng/ml. The precision (%CV) was better than 6% at all concentrations within the standard curve range, except at the lower limit of quantitation (10.1% at 0.1 ng/ml). Accuracy was within 5% of nominal at all standard concentrations.
Preparation of Plasma, Urine, and Fecal Samples for Metabolite Profile Analysis.Plasma. For each subject, aliquots of plasma were pooled (based on total C-14 in sample) to produce a representative 0.5 to 8 h sample for each subject. The plasma proteins in the samples were precipitated by the addition of four volumes of acetonitrile while vortexing vigorously, followed by centrifugation (3000 rpm, 10 min). The supernatants were transferred to clean tubes, and an aliquot (0.5 or 1-ml) was taken for liquid scintillation counting. Recovery of radioactivity after extraction was >90%. The supernatants were evaporated to dryness in a centrifugal vacuum concentrator (Speed-Vac, Forma Scientific Inc., Marietta, OH). The residues were reconstituted in 300 μl of 30% acetonitrile in water, and the solutions were transferred to autosampler vials for radiochromatography. The injection volume was 60 μl for HPLC-MS/MS radiochromatography.
Urine. Representative 0- to 48-h samples for each subject were obtained by combining aliquots from each time interval (up to and including 48 h) in proportion to their respective volumes. A 0.1- or 0.2-ml aliquot of the pooled mixture was taken for scintillation counting. To concentrate the urine samples before radiochromatography, aliquots of the 0- to 24-h (1- or 2-ml) and 24- to 48-h (5- to 10-ml) urine samples were transferred to glass centrifuge tubes, and 3 volumes of acetonitrile was added while vortexing vigorously. The samples were then treated as the plasma samples. The reconstituted samples were transferred to autosampler vials for radiochromatography (100-μl injection volume). The remaining material was submitted for evaluation by mass spectrometry.
Feces. To concentrate the fecal samples before radiochromatography, aliquots (1, 2, or 10 ml) were transferred to glass centrifuge tubes and prepared for analysis as the plasma and urine samples. One hundred-microliter aliquots of the resulting supernatant were taken for liquid scintillation counting. Recovery of radioactivity was acceptable (>60%).
Radiochromatographic Analysis of Plasma, Urine, and Fecal Extracts. The above samples were subjected to HPLC on a PerkinElmer LC 235C system. The metabolites were separated on a Zorbax RX C8 column (4.6 × 250 mm, 5 μm; Agilent Technologies, Palo Alto, CA) with mobile phase eluting at a flow rate of 1 ml/min. A linear gradient started at 10% acetonitrile in 25 mM ammonium formate (pH 3) and increased to 50% acetonitrile over 30 min, followed by a 10-min isocratic run at 50% acetonitrile for a total run time of 40 min. A postrun time of 5 min under starting mobile phase conditions permitted reequilibration of the column. The effluent was monitored using a photodiode array detector (260 and 300 nm) and by an in-line radiochemical detector (β-RAM model 2B; INUS Systems, Inc., Tampa, FL). A 3 ml/min flow rate was used for the scintillation cocktail (total flow rate of 4 ml/min through the radiochemical detector).
Incubation of Urine with β-Glucuronidase. Aliquots of the pooled urine (0-24 h) were transferred to 13 × 100 mm test tubes and dried under nitrogen in a Pierce (Rockford, IL) N-Evaporator. The tube contents were resuspended in 1 ml of 0.2 M sodium phosphate buffer (pH 7.0), and Escherichia coli β-glucuronidase enzyme (1000 units, Type VII-A; Aldrich Chemical Co.) was added to each tube. A second set of control samples was prepared to which no enzyme was added. All the samples were incubated overnight (∼17 h) at 37°C in a shaking water bath. The reactions were stopped with the addition of 5 volumes of acetonitrile, and the samples were prepared for analysis as described above. The reconstituted samples were sonicated and transferred to autosampler vials for radiochromatography (100-μl injection volume). The remaining material of selected samples was submitted for evaluation by mass spectrometry.
LC-MS/MS Analysis of Plasma, Urine, and Fecal Extracts. Samples were injected onto a Hewlett-Packard HP1100 gradient high-performance liquid chromatography system (Agilent Technologies) interfaced to a Finnigan TSQ 7000 tandem mass spectrometer (Thermo Electron, Waltham, MA) and a β-RAM radiochemical detector in parallel. Ten percent of the HPLC effluent was split into the mass spectrometer. Metabolite separation was carried out on a Phenomenex (Torrance, CA) Luna C18 (2) column (4.6 m × 25 cm, 5 μm) at a constant flow rate of 1 ml/min. A linear gradient starting at 10% of 0.1% formic acid in acetonitrile in 25 mM ammonium formate (pH 3) was maintained for 5 min, increased to 50% of 0.1% formic acid in acetonitrile over 30 min, and followed by a 10-min isocratic run at 50% of 0.1% formic acid in acetonitrile for a total run time of 45 min. A postrun of 3 min at 90% of 0.1% formic acid in acetonitrile was run before the next injection.
Mass spectral analyses were carried out with electrospray ionization in the positive ion mode. The capillary temperature was 220°C and the electrospray ionization ionizing voltage was maintained at 5.0 kV for all analyses. MS/MS was based on collision-induced dissociation (CID) of ions entering the radiofrequency-only octapole region where argon was used as the collision gas at a pressure of 1.7 mtorr. A combination of collision offsets, ranging from -25 eV to -45 eV, were used for all MS/MS analyses.
Incubation of N-Desethyl TPA023 (M12) with UDP-Glucuronic Acid-Fortified Human Liver Microsomes. Incubations were performed at 37°C in a shaking water bath, using 12 × 75 mm borosilicate glass disposable culture tubes. In brief, the final assay volume was 1.0 ml and consisted of 0.1 M potassium phosphate buffer (pH 7.4), magnesium chloride (10 mM), d-saccharic acid 1,4 lactone (1.25 mM), microsomal protein (1.0 mg/ml), and alamethicin (25 μg). The reactions were initiated by addition of UDP-glucuronic acid (4 mM) and M12 (40 μM) dissolved in DMSO (<1% v/v final volume in assay) after a 3-min preincubation period (37°C, open to air) and then stopped by addition of 1 ml of acetonitrile after a 6-h incubation (37°C, open to air). In each case, the samples were centrifuged for 10 min at 3000 rpm and the supernatant was transferred into clean tubes. After evaporation to dryness under nitrogen, the residue was reconstituted in 400 μl of 10% acetonitrile in 25 mM ammonium formate (pH 3).
A series of injections was made onto an HPLC system (using the radiochromatography conditions described above) interfaced with a Foxy fraction collector (ISCO Inc., Lincoln, NE). The effluent was collected as 30-s fractions (1 ml/min). Fractions corresponding to the retention times of the glucuronide tubes were pooled and concentrated for NMR analysis.
NMR Analysis of Microsomal Metabolites. NMR experiments were performed on a 500 MHz spectrometer (Inova; Varian Inc.) fitted with a 3-mm inverse detection probe (MIDG-3 probe; Nalorac, Martinez, CA). Samples of M12 and each of the isolated metabolite fractions were dissolved in DMSO-d6 and NMR data sets were acquired at 25°C. All 1H chemical shifts are reported on the δ scale (parts per million) downfield from tetramethyl silane using the DMSO-d6 lock signal for reference at 2.51 ppm. One-dimensional and two-dimensional total correlation spectroscopy (to trace through bond connectivity; Summers et al., 1986), and two-dimensional transverse rotating Overhauser spectroscopy (ROESY; to show through space proximity; Hwang and Shaka, 1992) data sets were acquired for all of the M12 metabolites.
Identification and Quantitation of Metabolites in Plasma, Urine, and Feces. Authentic standards of TPA023 and its metabolites (M5, M9, M10, M11, M12, and M14) were available to support the characterization of TPA023 metabolites in urine, feces, and plasma by similarity of chromatographic retention time and their respective UV and MS spectra. The N-glucuronide conjugates (M2, M6, and M7) of M12, generated in human liver microsomes (structures confirmed by NMR) also served as standards. For each sample of urine and fecal homogenate, all metabolites were quantitated based on the percentage of total radioactivity for each peak observed.
Pharmacokinetic Methods. Plasma concentrations of TPA023 and radioactivity were used to estimate pharmacokinetic parameters for each subject. The Cmax and Tmax values were obtained by inspection of the concentration-time data. The apparent terminal t1/2 was estimated from the best-fit parameters of a single exponential to the log-linear portion of the plasma concentration-time curve. The best-fit parameters were obtained using nonlinear regression by the Simplex algorithm with weight = 1 (Yeh and Remphrey, 1990). The area under the plasma concentration-time curve (AUC∞) was calculated using the linear trapezoidal method up to the last measured concentration. The additional area was estimated from the last measured concentration and the value of apparent terminal rate constant.
Results
Absorption and Excretion of [14C]TPA023. The mean concentration-time profiles of TPA023 and radioactivity in plasma after oral administration are shown in Fig. 2, and the geometric mean AUC ratio of TPA023 to total radioactivity (95% confidence interval) was 0.53 (0.47, 0.62), indicating that metabolites accounted for an appreciable amount of the radioactivity in plasma (data not shown). The single oral dose was rapidly absorbed and the mean plasma Tmax and Cmax for TPA023 were 2.0 h and 28 ng/ml, respectively (Table 1). The harmonic mean apparent terminal t1/2 was 6.7 h. The mean plasma Tmax and Cmax values for the total radioactivity in plasma were 2.2 h and 42.9 ng/ml, respectively (Table 2). The harmonic mean apparent terminal t1/2 was 7.7 h.
The mean total recovery of total radioactivity in the excreta after 7 days was 82.6% (Table 3, Fig. 3). Excretion was primarily via the renal route, with 53.2% of the dose eliminated in urine and 29.4% recovered in the feces. The majority of the urinary and fecal radioactivity was recovered within 48 and 96 h, respectively.
Metabolism of [14C]TPA023. Metabolites of TPA023 were identified based on protonated molecular ions and their CID fragmentation. A number of TPA023 ([M + H]+ion at m/z 396) metabolites were observed in plasma, urine, and fecal extracts, and they were identified by LC-MS/MS. The major fragmentation pathway of TPA023 occurs between the carbon and the oxygen at the ethyl triazole methoxy moiety, which gives rise to two major fragments, m/z 287 (from the substituted triazole pyridazine region of the molecule) and m/z 110 (ethyl triazole ring).
The retention times (tR), [M + H]+ion at m/z, and major CID fragments are listed in Table 4. The tR and CID fragmentations of M2, M6, and M7 were similar to those obtained with M2, M6, and M7 isolated from human liver microsomes (see below).
1H NMR data of M2 determined it to be a conjugate at nitrogen N1 in the triazolo-pyridazine ring. The anomeric proton of the glucuronide appears as a doublet with a scalar coupling constant of 8.8 Hz at 5.95 ppm. Correlations are seen from proton 8 in the triazolopyridazine moiety to glucuronide anomeric protons and also to the t-butyl protons in the ROESY spectrum. Metabolite M6 was determined to be a conjugate at nitrogen N4 of the triazole moiety on the bottom portion of M12 by NMR. In the ROESY spectrum, cross peaks are seen from the anomeric proton of the glucuronide (at 5.21 ppm, doublet with J = 9.5 Hz) to the methylene connected to the triazole, the triazole at proton 3, and the t-butyl group protons. M7 was determined to be a conjugate at nitrogen N1 of the triazole by NMR. The anomeric proton of the glucuronide (at 5.39 ppm, doublet with J = 8.9 Hz) shows cross-peaks to bridge methylene proton to the triazole and the t-butyl group protons in the ROESY spectrum.
A representative radiochromatogram of 0- to 24-h pooled urine is shown in Fig. 4A, and mass spectral analysis revealed the presence of 12 metabolites. Only trace amounts of parent compound were detected. The triazolo-pyridazine N1-glucuronide (M5) and t-butyl hydroxy (M11) derivatives of TPA023 were identified as the most abundant metabolites, accounting for 10.3 and 14% of the dose in 0- to 48-h urine, respectively (Table 5). Two additional metabolites (M1 and M2) were not resolved radiochromatographically but together accounted for an additional 10.8% of the dose present in the urine. However, M1 and M2 were resolved mass spectrally and were identified as the glucuronide of M14 (O-dealkyl TPA023) and M12 (N-desethyl TPA023 glucuronidated at the N1 of the triazolopyridazine moiety), respectively. In addition, glucuronides M3, M4, M5, M6, M7, and M8 were identified in the urine. Individually, none of these metabolites accounted for more than 4% of the dose. Collectively, however, they represented about 8% of the dose. In addition to the t-butyl hydroxy metabolite of TPA023 (M11), three other aglycones (M9, M10, and M12) were detected in urine. M9 (1.4% of the dose) was identified as t-butyl hydroxy/N-desethyl TPA023. M10 (3.0% of the dose) was identified as the carboxylic acid product resulting from the oxidation of the t-butyl moiety. M12 (0.3% of the dose) was identified as N-desethyl TPA023. In toto, the data indicated that up to 30% of the dose was recovered in 0- to 48-h urine as the glucuronide conjugates of TPA023 and its oxidative metabolites (Table 5). In agreement, only five metabolites (M9, M10, M11, M12, and M14) were detected in urine after overnight hydrolysis with β-glucuronidase (Fig. 4B). These data showed that TPA023 was largely cleared by oxidative metabolism involving t-butyl hydroxylation and N-deethylation (Fig. 5).
Fecal extracts were also subjected to radio-LC/MS analysis, and only five metabolites were detected (Fig. 6A). Qualitatively, the pattern of metabolism was similar to that obtained with urine after hydrolysis. This is to be expected, because the metabolites would be secreted into bile and hydrolyzed by gut bacteria en route to the lower colon. As in hydrolyzed urine, t-butyl hydroxy (M11) and N-desethyl (M12) metabolites of TPA023 were present in the feces and accounted for 7.8 and 4.8% of the dose, respectively (Table 6). The carboxylate of TPA023 (M10) and t-butyl hydroxy-N-desethyl metabolite (M9) respectively accounted for another 8.5% and 3.6% of the dose. The product of fluoro phenyl ring hydroxylation (M13) seemed unique to feces, but was a relatively minor metabolite (0.9% of the dose).
Plasma samples, pooled over the interval from the first time point through 8 h, were subjected to radio-LC/MS analysis also (Fig. 6B). In agreement with the data presented in Fig. 2, TPA023 was the major component and comprised the majority of the total radioactivity present therein. In addition, the triazolo-pyridazine N1-glucuronide of M12 (M2), the t-butyl hydroxy (M11), N-desethyl (M12), and triazolo-pyridazine N1-glucuronide (M5) metabolites of TPA023 were also observed in the plasma.
Discussion
The purpose of this study was to investigate the absorption, disposition, and mass balance of [14C]TPA023 in healthy male volunteers after a single 3-mg oral dose. Mass balance was reasonably achieved, with total recovery of radioactivity averaging 82% of the dose. Recovery of radioactivity averaged 53% of the dose in urine and 29% in feces. The results of this study also showed that radiolabeled TPA023 was well absorbed after administration of an oral solution in propylene glycol as indicated by the large fraction of the dose (53%) recovered in the urine.
The plasma concentration-time profiles after administration indicated that TPA023 accounted for the majority of the radioactivity in plasma (∼53%, based upon AUCs) and, qualitatively, it seemed as if the remainder of the radioactivity was roughly equally divided among four metabolites (Fig. 6B). One of the metabolites (N-desethyl, M12) is pharmacologically active in vitro, exhibiting affinity and efficacy similar to those of the parent compound at GABA-A α2/3 subunits. However, this metabolite, along with t-butyl hydroxy (M11), has been shown to be poorly brain-penetrant in rats. In fact, the brain-to-plasma ratios are substantially lower than those of parent (∼0.4 versus ∼0.02) (B. Sohal and A. Pike, unpublished data). The remaining two metabolites (M2 and M5), triazolo-pyridazine N1 glucuronides, also would not be expected to be brain-penetrant. Therefore, it is anticipated that the metabolites of TPA023 would not contribute to the overall pharmacological activity of the compound.
Analysis of the excreta demonstrated that TPA023 is metabolized extensively, with less than 1% of the administered oral dose recovered unchanged. Therefore, renal excretion plays a minor role in the clearance of TPA023. In contrast, the primary metabolites of TPA023 (t-butyl hydroxy and N-desethyl), and their respective metabolites, are largely excreted in the urine. The absence of parent in the feces not only indicates good absorption but also indicates that the triazolopyridazine N1-glucuronide (M5) of TPA023 is cleared via the urine, not the bile. Only a relatively small amount of the dose (10%) is recovered in the urine as the N-glucuronide of parent compound, and in the absence of biliary secretion, no enterohepatic recirculation would be expected. In agreement, there was no evidence for secondary peaks in the plasma profiles (Fig. 2).
The observation that t-butyl hydroxylation and N-deethylation are major pathways is in accord with the results of in vitro metabolism studies. The in vitro data show that these two pathways are operative when TPA023 is incubated in the presence of NADPH-fortified human liver microsomes (S. L. Polsky-Fisher, unpublished data). In vitro data also show that the oxidation of TPA023 is catalyzed exclusively by members of the CYP3A subfamily, and other cytochromes P450 (e.g., CYP2C9, 2C8, 2C19, 2D6, and 1A2) play a minor role. The N-glucuronidation of TPA023 in vitro is catalyzed by recombinant UGT1A4 only and was highly correlated with imipramine N-glucuronidation in a bank of human liver microsomes (S. L. Polsky-Fisher, unpublished data).
Because the oxidative metabolism of TPA023 is catalyzed by members of the CYP3A subfamily only, the clearance of TPA023 can be attenuated by potent CYP3A inhibitors such as ketoconazole and itraconazole. In agreement with this hypothesis, itraconazole elicits a clinically significant effect on the oral pharmacokinetics of TPA023 (∼5-fold increase in plasma AUC) (N. G. B. Agrawal, unpublished data). In this regard, TPA023 is similar to agents such as zolpidem, triazolam, and midazolam (Olkkola et al., 1994; Varhe et al., 1994; Greenblatt et al., 1998; Yasui et al., 1998). Moreover, it is highly likely that the clearance of TPA023 will be increased in the presence of CYP3A inducing agents such as rifampicin and St. John's wort (Backman et al., 1996; Villikka et al., 1997; Schmider et al., 1999; Wang et al., 2001).
Based on the results of this study, it is concluded that TPA023, after a single oral dose, is absorbed rapidly and substantially and is metabolized extensively in human subjects (Fig. 5). t-Butyl hydroxylation and N-deethylation represent the major routes of clearance. These metabolites are either excreted directly or are metabolized further to secondary metabolites that are also excreted (urine > feces). In comparison to oxidative metabolism, only a relatively minor fraction of the dose (∼10%) is cleared via direct glucuronidation at the N1 position of the triazolo-pyridazine moiety.
Acknowledgments
We thank Dr. Kevin Moore (Neurosciences Group, Merck Research Laboratories, Terlings Park, UK) for the synthesis of TPA023 metabolite standards, Dr. Matthew Braun (Radiolabeled Compound Synthesis Group, Merck Research Laboratories, Rahway, NJ) for synthesis of M5, Dr. Dennis Dean (Radiolabeled Compound Synthesis Group, Merck Research Laboratories, Rahway, NJ) for the synthesis of [14C]TPA023, and Drs. Andrew Pike (Neurosciences Group, Merck Research Laboratories, Terlings Park, UK) and Kelem Kassahun (Drug Metabolism, Merck Research Laboratories, West Point, PA) for their contributions to the paper.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.008193.
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ABBREVIATIONS: GAD, general anxiety disorder; TPA023, 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; CID, collision-induced dissociation; ROESY, rotating Overhauser spectroscopy; Cmax, maximum concentration in plasma; Tmax, time of occurrence of maximum concentration in plasma; AUC, area under the plasma concentration versus time curve; tR, retention time.
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↵1 Current affiliations: Biotransformation Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey;
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↵2 Department of Drug Metabolism and Pharmacokinetics, Amgen, Thousand Oaks, California;
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↵3 Department of Global Clinical Development General Medicine, Amgen, Thousand Oaks, California;
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↵4 Department of Drug Metabolism and Pharmacokinetics, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey.
- Received January 3, 2006.
- Accepted February 24, 2006.
- The American Society for Pharmacology and Experimental Therapeutics