Drug Metabolism and Disposition Fast Forward
First published on June 24, 2004; DOI: 10.1124/dmd.104.000349
0090-9556/04/3210-1092-1095$20.00
DMD 32:1092-1095, 2004
EFFECTIVE DOSING REGIMEN OF 1-AMINOBENZOTRIAZOLE FOR INHIBITION OF ANTIPYRINE CLEARANCE IN GUINEA PIGS AND MICE USING SERIAL SAMPLING
Suresh K. Balani,
Ping Li,
Joanne Nguyen,
Kym Cardoza,
Hang Zeng,
Dun-Xue Mu,
Jing-Tao Wu,
Liang-Shang Gan, and
Frank W. Lee
Drug Safety and Disposition, Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts
(Received April 20, 2004;
accepted June 23, 2004)
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Abstract
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Single-dose pharmacokinetics of 1-aminobenzotriazole (ABT), a potent nonspecific inhibitor of cytochromes P450 (P450s), were characterized after oral administration to mice and guinea pigs at doses of 50, 100, and 150 mg/kg using serial sampling in both species. Only 30-µl blood samples were drawn from jugular vein-cannulated mice using Microvette capillary tubes containing lithium heparin. A comparison of the pharmacokinetics of antipyrine (AP) administered i.v. at 20 mg/kg to mice followed by serial and terminal sampling techniques yielded similar results. The ABT concentrations in plasma were sustained at high levels (5-100 µM) for at least 12 h in both species. Pretreatment of animals with ABT 2 h prior to AP administration decreased the plasma AP clearance by about 95% in mice at all ABT doses studied and 84, 95, and 95% in guinea pigs at a dose of 50, 100, and 150 mg/kg ABT, respectively. In vitro, the dissociation constants (KI) for ABT as the P450 mechanism-based inactivator were determined to be 45.6 and 193 µM, and the maximal inactivation rate constants (kinact) were determined to be 0.089 and 0.075 min-1 for the mouse and guinea pig liver microsomes, respectively. The projected P450 inactivations at the plasma Cmax of ABT agreed with the inhibitions of P450-mediated AP clearance observed in vivo. For mechanistic studies in vivo overall, a 2-h prior oral pretreatment with ABT at 50 mg/kg in mice and 100 mg/kg in guinea pigs would provide significant systemic concentrations of the inhibitor over 24 h and inhibition of P450-dependent clearance of test compounds.
In our last communication (Balani et al., 2002
), the dosing regimen of 1-aminobenzotriazole (ABT), a nonspecific inhibitor of cytochromes P450 (P450s) (Huijzer et al., 1989; Constan et al., 1999), was established in rats, dogs, and monkeys to effectively inhibit P450s and hence decrease the plasma clearance and increase the exposure of antipyrine (AP), a nonspecific probe substrate of P450s (Engel et al., 1996; Sharer and Wrighton, 1996; Matzke et al., 2000). All P450s were shown to be affected by ABT treatment (Balani et al., 2002). Due to wide distribution of ABT in rats, P450 inhibition is expected to be general in the body tissues (Town et al., 1993). The literature previously contained varied treatment of animals with ABT (e.g., dosing route, dose level, pretreatment time, and frequency of dosing). The current study extends our previous studies to mice and guinea pigs, which are routinely used for mechanistic PK, toxicity, and pharmacology studies; thus, it is intended to provide guidelines for the pretreatment of animals with ABT to significantly alter the oxidative metabolism of test compounds (e.g., to evaluate metabolite versus parent compound-based toxicities or boost compound concentration available for a target enzyme or receptor). Although the safety assessment of chronic dosing of ABT in mice and guinea pigs has not been reported, its safety in rats has clearly been demonstrated (Mico et al., 1988). This report also highlights the use of serial sampling in mice, thus saving cost and labor and reducing the number of animals in studies. In vitro studies were also conducted in mouse and guinea pig hepatic microsomes to gauge the relative effect of ABT pretreatment on AP intrinsic clearance.
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Materials and Methods
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Materials. Antipyrine, 4-Hydroxyantipyrine, ABT, and NADPH were purchased from Sigma-Aldrich (St. Louis, MO). Mouse and guinea pig liver microsomes were purchased from XenoTech (Lenexa, KS). All other chemicals were of analytical grade.
Animal Pharmacokinetic Studies. Male, jugular vein-cannulated and noncannulated C57Bl/6 mice and jugular vein-cannulated Hartley SJVC guinea pigs were obtained from Hilltop Laboratory Animals, Inc. (Scottdale, PA). For terminal sampling, mice were dosed intravenously with AP at 20 mg/kg and dissolved in normal saline (1 ml/kg). Three animals per time point of 0, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h were sacrificed, and maximum blood was collected by cardiac puncture into tubes containing lithium heparin as an anticoagulant. Plasma was harvested after centrifugation of the blood. For serial sampling, 5 jugular vein-cannulated mice were given 20 mg/kg AP, and 30 µl of blood was collected from each mouse at the above time points; the collected blood was deposited directly into Microvette capillary blood tubes (Sarstedt, Inc., Newton, NC) containing lithium heparin. Samples were placed on wet ice and quickly transferred into an Eppendorf Micro Centrifuge (model 5415 D; Eppendorf - 5 Prime, Inc., Boulder, CO) and centrifuged for 2 min at 2000g at the end of each collection interval. Exactly 10 µl of plasma was transferred into polypropylene conical tubes (VWR International, Bridgeport, NJ). Plasma samples were frozen at -20°C immediately following each collection interval. Serial samples of 0.3 ml of blood per time point were drawn from guinea pig (n = 3) for the intravenous AP study, and plasma was separated. This was the same as those for mice, plus 46 and 48 h.
Single oral dose studies of ABT were conducted in jugular vein-cannulated mice (n = 4) and guinea pigs (n = 3) at 50, 100, and 150 mg/kg using 0.5% methyl cellulose as a vehicle (5 ml/kg). Serial plasma samples were collected as described above from mice at 0, 0.5, 1, 3, 6, 9, 24, 32, and 48 h and guinea pigs at 0, 0.5, 1, 3, 6, 9, 24, 32, 56, and 72 h.
For the interaction studies, animals were dosed intravenously with AP at 20 mg/kg 2 h after an oral administration of ABT at 50, 100, or 150 mg/kg (n = 3 or 4). Serial plasma sampling ensued as described above for 48 h from both species. Plasma samples from all studies were frozen until analysis.
Inactivation of Antipyrine Metabolism by ABT. Inactivation rates were obtained using the conventional two-step incubation methods. All incubations were conducted in 96-well plates. Mouse and guinea pig liver microsomes (2.5 mg/ml) were preincubated with ABT at 0, 10, 50, 100, and 200 µM with or without 2 mM NADPH in a total volume of 100 µl with 0.1 M potassium phosphate buffer, pH 7.4, and 3 mM magnesium chloride. At selected time intervals (0, 5, and 15 min) (at and above 30 min, the kinetics were not in the linear range), 15-µl aliquots of the incubation mixture were transferred into plates containing 135 µl of 0.1 M potassium phosphate buffer, pH 7.4, containing antipyrine (final concentration, 200 µM) and NADPH (2 mM). The samples were incubated for 20 min at 37°C, and the reaction was terminated by the addition of 100 µl of acetonitrile containing 1 µM carbutamide (internal standard). The sample plates were centrifuged at 3000g for 10 min, and the supernatant was analyzed by LC/MS/MS for 4-hydroxyantipyrine concentration.
Bioanalysis. In vivo plasma samples were protein precipitated and analyzed by LC/MS/MS using a method similar to the one reported previously (Balani et al., 2002). In general, the LC/MS/MS system consisted of a binary high-performance liquid chromatography pump (1100; Agilent Technologies, Palo Alto, CA), an HTS PAL autosampler (LEAP Technologies, Carrboro, NC), and a triple-quadrupole mass spectrometer (API-4000; Applied Biosystems, Foster City, CA). Separation was performed on a YMC ODS AQ column (2 x 50 mm; Waters, Milford, MA) using formic acid (0.1%) in water and acetonitrile as mobile phases A and B, respectively. The mass spectrometer was operated in multiple reaction monitoring mode using positive ion electrospray ionization. A major difference in this method compared with the previously reported one was that the sample volume was reduced to 10 µl of plasma for the analysis of AP and ABT concentrations in serially sampled mouse studies. Accordingly, a lower limit of quantitation of 10 nM was achieved. The quantitation limit for guinea pigs for the two analytes was 1 nM. In vitro hepatic microsomal incubation samples were analyzed for 4-hydroxyantipyrine by LC/MS/MS using a SCIEX API-4000 instrument (Applied Biosystems). The LC conditions involved a Supelco Discovery C18 column (10 x 3 mm; Supelco, Bellefonte, PA), an Agilent pump, and a LEAP autosampler. The mobile phase consisted of a gradient mixture of 0.1% formic acid and acetonitrile.
Data Analysis. The pharmacokinetic parameters were calculated with Win-Nonlin software (Pharsight, Mountain View, CA) using noncompartmental analysis of the data. The observed inactivation rates (kobs) for 4-hydroxyantipyrine formation were calculated from the initial slopes by linear regression analysis of natural log percentage of remaining activity versus preincubation time plot. The inactivation kinetics parameters were obtained by fitting to the hyperbolic equation kobs = kinact x [I]/([I] + KI) using Prism Software (GraphPad Software, Inc., San Diego, CA). The projected t1/2 for P450 inactivation was then calculated using Cmax for ABT for the [I] value in the above equation to calculate kobs. Then t1/2 was calculated as (Ln 2)/kobs.
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Results
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Mouse PKs. The plasma concentration-time profiles of antipyrine (20 mg/kg i.v.) by the serial and terminal plasma sampling methods are shown in Fig. 1. For the common time points of the curve, the two plots were practically superimposable. The PK parameters by the two methods also were comparable, as shown in Table 1. The ABT concentration-time profiles following single oral doses of 50, 100, and 150 mg/kg and serial plasma sampling are shown in Fig. 2. The AUC increased in a greater than proportional to dose manner, as shown in Table 2. Pretreatment of animals with 50, 100, and 150 mg/kg ABT p.o. 2 h prior to the i.v. administration of 20 mg/kg AP led to a significant drop in the plasma clearance and consequently to an increase in the AUC of AP, as depicted in Fig. 3 and Table 1.
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FIG. 1. Plasma concentration-time profiles of antipyrine in mice dosed i.v. at 20 mg/kg involving terminal and serial sampling.
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TABLE 1 Pharmacokinetic parameters of antipyrine following a 20-mg/kg i.v. dose in mice and guinea pigs and the effect of pretreatment (2 h prior) with oral ABT on the antipyrine pharmacokinetics
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FIG. 3. Plasma concentration-time profiles of antipyrine in mice dosed i.v. at 20 mg/kg along with an oral dose of ABT given 2 h prior. Data from serial sampling.
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Guinea Pig PKs. The ABT concentration-time profiles following single oral doses of 50, 100, and 150 mg/kg are shown in Fig. 4. The AUC increase was proportional to dose (Table 2). The PK profile after a 20 mg/kg intravenous dose of AP is shown in Fig. 5, and the parameters are given in Table 1. Pretreatment of guinea pigs with 50, 100, and 150 mg/kg of ABT given orally 2 h prior to the i.v. administration of 20 mg/kg AP led to a significant drop in the plasma clearance of AP, as depicted in Fig. 5 and Table 1.
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FIG. 4. Plasma concentration-time profiles of ABT dosed orally at 50, 100, and 150 mg/kg in guinea pigs.
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FIG. 5. Plasma concentration-time profiles of antipyrine dosed i.v. at 20 mg/kg in guinea pigs with and without pretreatment (2 h prior) with ABT dosed orally at 50, 100, and 150 mg/kg.
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In Vitro Inhibition. Inhibition of AP metabolism to 4-hydroxy AP by ABT (up to 200 µM) was found to be time- and concentration-dependent in both species. The dissociation constants (KI) for ABT as P450 mechanism-based inactivators were determined to be 45.6 and 193 µM. The maximal inactivation rate constants (kinact) were determined to be 0.089 and 0.075 min-1 for the mouse and guinea pig liver microsomes, respectively (Table 3).
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Discussion
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ABT has generally been used as a nonspecific P450 inhibitor. Most laboratories use varied ABT dosing schedules for such studies. Pharmacokinetics of ABT and its interaction with AP were reported for rats, dogs, and monkeys (Balani et al., 2002); based on those data, an effective dosing regimen of ABT was established. The current report extends those studies to other species (mice and guinea pigs), because ABT continues to be used successfully for mechanistic studies in mice (Adler et al., 2000; Hoffler et al., 2003; van Ravenzwaay et al., 2003) and guinea pigs (Knickle and Bend, 1992; Xu et al., 1995; Woodcroft et al., 1997). Thus, the pharmacokinetic parameters were generated for AP in mice using the usual terminal sampling approach and in guinea pigs using serial sampling. The AP pharmacokinetics were also assessed in mice following serial blood sampling from either jugular vein-cannulated or noncannulated animals using tail vein sampling. Only 30 µl of blood was collected at each time point, and after separation, 10 µl of plasma was used for the AP and ABT concentration measurements. The tail vein bleeding frequently led to a degree of hemolysis after the initial couple of time points; as anticipated, this led to the overestimation of plasma concentrations (data not shown) because of the possible partitioning of AP into red blood cells. Sampling from cannulated mice did not cause hemolysis. As shown in Fig. 1 and Table 1, the AP plasma concentration-time profile after the terminal sampling was similar to the one from serial sampling, with low clearance, a volume of distribution similar to that of body water (data not shown), and a short half-life. The results from this comparative study increased the confidence in using the serial micro sampling and analysis for mice studies. All further mice studies were conducted using the jugular vein-cannulated animals. These studies demonstrated that serial sampling in mice to obtain complete pharmacokinetic profiles is practical, saves time and cost, reduces the number of animals required, and increases throughput compared with studies where three animals are sacrificed at each time point in building the PK profile. This serial blood sampling has become possible with the availability of highly sensitive LC/MS/MS assays for microsamples and the commercial availability of jugular vein-cannulated mice. Furthermore, automation in sampling is also available using precise, intelligent automated blood samplers.
As shown in Fig. 2 and Table 2, single 50, 100, and 150 mg/kg oral dose ABT pharmacokinetics in mice were nonlinear, with a greater than dose proportional increase in the exposure. The effect of ABT dosed orally at 50, 100, and 150 mg/kg (2 h prior to AP administration) in mice on 20 mg/kg AP i.v. pharmacokinetics is depicted in Fig. 3, and the parameters are given in Table 1. The plasma AP concentrations stayed at high levels for at least 8 h (and likely longer) compared with that after AP administration alone. The clearance of AP declined by about 95% at all of the ABT doses tested. The magnitude of clearance at the above ABT doses and corresponding AP plasma concentration-time profiles suggested that a single 50-mg/kg oral ABT dose 2 h prior to test the administration of the compound would provide a significant decline in the P450-based clearance in mice. Depending on a need for continuous inhibition of P450, b.i.d. dosing of ABT at 50 mg/kg is suggested.
Pharmacokinetics of ABT given orally at 50, 100, and 150 mg/kg in guinea pigs is described by the parameters shown in Table 2 and the plasma concentration-time profile shown in Fig. 4. The usual serial sampling procedure was followed in this larger animal. The AUC of ABT seemed to increase in a dose-proportional manner in guinea pigs. The plasma clearance of AP in guinea pigs was lower than in mice. Following the 2-h prior treatment of animals with oral doses of 50, 100, and 150 mg/kg ABT, the AP clearance dropped by 84, 95, and 95%, respectively (Table 1; Fig. 5). Thus, in guinea pigs, a higher dose of 100 mg/kg would provide better inhibition of P450 based on the clearance of test compounds. For continuous inhibition of metabolism, once-a-day dosing is suggested.
In vitro inhibition studies with ABT at concentrations similar to the Cmax observed in in vivo studies showed that the AP metabolic inhibition was dependent on the preincubation time and concentration of ABT by both mouse and guinea pig liver microsomes. The respective projected P450 inactivation half-lives in mice (at plasma Cmax) were 9.1, 8.3, and 8.1 min for 50, 100, and 150 mg/kg ABT and 13.3, 12.4, and 11.2 min for guinea pigs. Qualitatively, these t1/2 values indicated that P450s were inactivated very rapidly during the ABT pretreatment period in mice and that this inhibition was faster than in guinea pigs. Thus, because of the long, 2-h pretreatment period for ABT relative to the short inactivation t1/2, there were not big differences in the in vivo plasma clearance values for AP in the two species.
In conclusion, in a single-dose setting, 2-h prior treatment with ABT given orally at 50 mg/kg in mice and 100 mg/kg in guinea pigs is suggested to provide a significant drop in P450-dependent clearance. For chronic, optimal P450 inhibitory effects, 50 mg/kg b.i.d. dosing of ABT in mice and 100 mg/kg q.i.d. in guinea pigs is suggested, considering accumulation of the inhibitor on repeat dosing. The serial plasma sampling in mice was also successfully demonstrated; therefore, it can be used more routinely to reduce the number of mice per study, reduce cost and labor, and increase the throughput.
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Acknowledgments
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We thank Drs. Gerald T. Miwa, J. Scott Daniels, and Chuang Lu for helpful discussions and Susan Colson for excellent proofreading.
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Footnotes
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doi:10.1124/dmd.104.000349.
ABBREVIATIONS: ABT, 1-aminobenzotriazole; AP, antipyrine; PK, pharmacokinetic; LC, liquid chromatography; MS/MS, tandem mass spectometry; AUC, area under the curve.
Address correspondence to: Dr. Suresh K. Balani, Millennium Pharmaceuticals, Inc., 45 Sidney Street, Cambridge, MA 02139. E-mail: suresh.balani{at}mpi.com
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References
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Adler ID, Baumgartner A, Gonga H, Friedman MA, and Skerhut M (2000) 1-Aminobenzotriazole inhibits acrylamide-induced dominant lethal effects in spermatids of male mice. Mutagenesis 15: 133-136.[Abstract/Free Full Text]
Balani SK, Zhu T, Yang TJ, Liu Z, He B, and Lee FW (2002) Effective dosing regimen of 1-aminobenzotriazole for inhibition of antipyrine clearance in rats, dogs, and monkeys. Drug Metab Dispos 30: 1059-1062.[Abstract/Free Full Text]
Constan AA, Sprankle CS, Peters JM, Kedderis GL, Everitt JI, Gonzalez FL, and Butterworth BE (1999) Metabolism of chloroform by CYP 2E1 is required for induction of toxicity in the liver, kidney and nose of male mice. Toxicol Appl Pharmacol 160: 120-126.[CrossRef][Medline]
Engel G, Hofmann U, Heidemann H, Cosme J, and Eichelbaum (1996) Antipyrine as a probe for human oxidative drug metabolism: identification of the cytochrome P450 enzymes catalyzing 4-hydroxyantipyrine, 3-hydroxymethylantipyrine and norantipyrine formation. Clin Pharmacol Ther 59: 613-623.[CrossRef][Medline]
Hoffler U, El-Masri HA, and Ghanayem BI (2003) Cytochrome P450 2E1 is the principal enzyme responsible for urethane metabolism: comparative studies using CYP2E1-null and wild-type mice. J Pharmacol Exp Ther 305: 557-564.
Huijzer JC, Adams JD, Jaw JY, and Yost GS (1989) Inhibition of 3-methylindole bioactivation by cytochrome P450 suicide substrate 1-aminobenzotriazole and alpha-methylbenzylaminobenzotriazole. Drug Metab Dispos 17: 37-42.[Abstract]
Knickle LC and Bend JR (1992) Dose-dependent, mechanism-based inactivation of cytochrome P450 monooxygenases in vivo by 1-aminobenzotriazole in liver, lung, and kidney of untreated, phenobarbital-treated and beta-naphthoflavone-treated guinea pigs. Can J Physiol Pharmacol 70: 1610-1617.[Medline]
Matzke GR, Frye RF, Early JJ, Straka RJ, and Carson SW (2000) Evaluation of the influence of diabetes mellitus on antipyrine metabolism and CYP1A2 and CYP2D6 activity. Pharmacotherapy 20: 182-190.[CrossRef][Medline]
Mico BA, Federowicz DA, Ripple MG, and Kerus W (1988) In vivo inhibition of oxidative drug metabolism by and acute toxicity of 1-aminobenzotriazole. A tool for biochemical toxicology. Biochem Pharmacol 37: 2515-2519.[CrossRef][Medline]
Sharer JE and Wrighton SA (1996) Identification of the human hepatic cytochromes P450 involved in the in vitro oxidation of antipyrine. Drug Metab Dispos 24: 487-494.[Abstract]
Town C, Henderson L, Chang D, Mortillo M, and Garland W (1993) Distribution of 1-aminobenzotriazole in male rats after administration of an oral dose. Xenobiotica 23: 383-390.[Medline]
van Ravenzwaay B, Gamer AO, Leibold E, and Kaufmann W (2003) Effect of cytochrome P-450 inhibition of tetrahydrofuran-induced hepatocellular proliferation in female mice. Arch Toxicol 77: 459-464.[CrossRef][Medline]
Woodcroft KJ, Webb CD, Yao M, Weedon AC, and Bend JR (1997) Metabolism of the cytochrome P450 mechanism-based inhibitor N-benzyl-1-aminobenzotriazole to products that covalently bind with protein in guinea pig liver and lung microsomes: comparative study with 1-aminobenzotriazole. Chem Res Toxicol 10: 589-599.[CrossRef][Medline]
Xu D, Voigt JM, Mico BA, and Colby HD (1995) Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs. J Steroid Biochem Mol Biol 54: 281-285.[CrossRef][Medline]
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Abstract
Materials and Methods
