In Vitro Inhibitory Effect of 1-Aminobenzotriazole on Drug Oxidations in Human Liver Microsomes: a Comparison with SKF-525A

Drug Metab. Pharmacokinet. 20 (5): 351–357 (2005).

Regular Article In Vitro Inhibitory EŠect of 1-Aminobenzotriazole on Drug Oxidations in Human Liver Microsomes: a Comparison with SKF-525A Chie EMOTO, Shigeo MURASE, Yasufusa SAWADA, and Kazuhide IWASAKI* Department of Pharmacokinetics Dynamics Metabolism, Nagoya Laboratories, Pˆzer Japan Inc., Aichi, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: 1-Aminobenzotriazole (ABT) is extensively used as a non-speciˆc cytochrome P450 (CYP) inhibitor. In this study, the inhibitory eŠect of ABT on CYP-dependent drug oxidations was investigated in human liver microsomes (HLM) and compared with that of SKF-525A, another non-speciˆc inhibitor. The following probe activities for human CYP isoforms were determined using pooled HLM: phenacetin O-deethylation (CYP1A2); diclofenac 4?-hydroxylation (CYP2C9); S-mephenytoin 4?-hydroxylation, (CYP2C19); bufuralol 1?-hydroxylation (CYP2D6); chlorzoxazone 6-hydroxylation (CYP2E1); midazolam 1?-hydroxylation, nifedipine oxidation, and testosterone 6b-hydroxylation (CYP3A). ABT had the strongest inhibitory eŠect on the CYP3A-dependent drug oxidations and the weakest eŠect on the diclofenac 4?-hydroxylation. SKF-525A potently inhibited the bufuralol 1?-hydroxylation, but weakly inhibited chlorzoxazone 6-hydroxylation. The inhibitory eŠects of ABT and SKF-525A were increased by preincubation in some probe reactions, and this preincubation eŠect was greater in ABT than in SKF-525A. The remarkable IC50 shift (À 10 times) by preincubation with ABT was observed on the phenacetin O-deethylation, chlorzoxazone 6-hydroxylation, and midazolam 1?-hydroxylation. In conclusion, ABT and SKF525A had a wide range of IC50 values in inhibiting the drug oxidations by HLM with and without preincubation.

Key words: Cytochrome P450 (CYP); human liver microsomes; 1-aminobenzotriazole (ABT); SKF-525A CYP1, CYP2, and CYP3.3) CYP enzymes play an important role in the drug metabolism, one of the major clearance routes in the body. Therefore, estimation of the contribution of CYP enzymes to metabolic reactions in human liver microsomes (HLM) is very important in the drug development process. 1-Aminobenzotriazole (ABT) shows the non-speciˆc CYP inhibition, while it does not inhibit other enzymes such as cytochrome b5, NADPH cytochrome c reductase, glutathione S-transferase, and UDP-glucuronosyl transferase.4) The selectivity of ABT in inhibiting the drug oxidations catalyzed by human CYP enzymes had not been fully investigated. Therefore, we investigated previously the inhibitory eŠect of ABT on probe drug oxidations catalyzed by several human recombinant CYP isoforms and found that ABT is a potent inhibitor for CYP3A4-dependent drug oxidations, but weakly inhibits CYP2C9-dependent drug oxidations.5) ABT is also known as a mechanism-based inactivator of CYP enzymes by N-alkylation of heme moiety.6,7) Recently,

Introduction The cytochrome P450 (CYP) enzymes constitute a superfamily of heme-containing monooxygenase, and multiple forms of CYP enzymes exist in mammals. These CYP enzymes are responsible for the oxidation of many drugs, environmental chemicals and endogenous substrates.1) CYP enzymes mainly exist in the liver as well as in the extrahepatic tissues, such as the intestines, lungs and kidneys.2) In humans, xenobiotics are metabolized primarily by three CYP families; namely,

Abbreviations used are: CYP, cytochrome P450; ABT, 1-aminobenzotriazole; HLM, human liver microsomes; LC W MS W MS, high-performance liquid chromatographic separation with tandem mass spectrometric detection; POD, phenacetin O-deethylation; DFOH, diclofenac 4?-hydroxylation; S-MPOH, S-mephenytoin 4?-hydroxylation; BFOH, bufuralol 1?-hydroxylation; CZXOH, chlorzoxazone 6hydroxylation; MDZ1?OH, midazolam 1?-hydroxylation; NIFOX, nifedipine oxidation; and TESOH, testosterone 6b-hydroxylation.

Received; April 26, 2005, Accepted; August 23, 2005 *To whom correspondence should be addressed : Kazuhide IWASAKI, Pharmacokinetics Dynamics Metabolism, Nagoya Laboratories, Pˆzer Japan Inc., 5-2 Taketoyo, Aichi 470-2393, Japan. Tel. ＋81-569-74-4330, Fax. ＋81-569-74-4491, E-mail: Kazuhide.Iwasaki＠pˆzer.com

351

352

CHIE EMOTO et al.

Balani et al. have reported that the preincubation of recombinant CYP enzymes with ABT increases the inhibitory eŠect of this inhibitor on the CYP1A2-, CYP2B6-, CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A4-dependent oxidations of ‰uorescent substrates in a dose-dependent manner.8) Taking advantage of the non-speciˆcity in inhibiting the activities of CYP enzymes, ABT has been used in the inhibition study using liver and lung microsomes from mice, rats, rabbits, guinea pigs, and humans for the purpose of assessing the contribution of CYP enzymes to overall metabolism of a compound in question.4,9–12) However, the methods employed in the inhibition studies using ABT varied depending on the laboratories. Some laboratories used ABT without a preincubation, but others used it with the preincubation.13–15) Therefore, we investigated the inhibitory eŠect of ABT with and without preincubation in this study. With respect to CYP3A, it is recommended to use multiple CYP3A probes for the in vitro evaluation of CYP3Adependent drug interactions, and we used midazolam, nifedipine, and testosterone.16) In addition, these inhibitory eŠects of ABT were compared with those of SKF-525A, another non-speciˆc inhibitor. Methods Materials: Phenacetin was obtained from SigmaAldrich (Steinheim, Switzerland). Testosterone, tolbutamide, 4-hydroxymephenytion, 1?-hydroxybufuralol, 6-hydroxychlorzoxazone, 6b-hydroxytestosterone, oxidized nifedipine, and 1?-hydroxymidazolam were from Ultraˆne (Manchester, UK). 4?-Hydroxydiclofenac was from BD Gentest Corp. (Woburn, MA, USA). S-mephenytoin was from Biomol (Plymouth Meeting, PA, USA). ABT, SKF-525A, midazolam, alprazolam, 4-acetamidophenol, buspirone, caŠeine, and diclofenac were from Sigma Chemical Co. (St. Louis, MO, USA). Chlorzoxazone and nifedipine were from Wako Pure Chemical (Osaka, Japan). Bufuralol and [D7] 4-acetamidophenol were synthesized in-house. NADPH was from Oriental Yeast (Tokyo, Japan). Pooled HLM from 59 individuals were obtained from a Pˆzer Global R&D in-house supply. Other reagents were of HPLC grade or better. Inhibition study without preincubation: As probe reactions, the following oxidations were selected: phenacetin O-deethylation (POD) for CYP1A2, diclofenac 4?-hydroxylation (DFOH) for CYP2C9, S-mephenytoin 4?-hydroxylation (S-MPOH) for CYP2C19, bufuralol 1?-hydroxylation (BFOH) for CYP2D6, chlorzoxazone 6-hydroxylation (CZXOH) for CYP2E1, and midazolam 1?-hydroxylation (MDZ1?OH), nifedipine oxidation (NIFOX), and testosterone 6b-hydroxylation (TESOH) for CYP3A. For each of the above oxidations, the standard incuba-

tion mixture consisted of 1.4 mM NADPH, 3.3 mM MgCl2, 100 mM phosphate buŠer (pH＝7.4), HLM, and a probe substrate in the presence or absence of inhibitor (ABT or SKF-525A) in a ˆnal volume of 200 mL. After a preincubation for 5 min on the air incubator at 379C, the reaction was initiated by adding NADPH. The reaction was terminated by the addition of acetonitrile, which contained an internal standard. The ˆnal HLM concentrations and the incubation time mL and 10 min, respectively, except POD were 0.03 mg W (0.10 mg W mL and 20 min) and S-MPOH (0.15 mg W mL and 30 min). For the kinetic analysis, the range of substrate concentration was 9.4–300 mM for POD, 1.6–50 mM for DFOH, 9.4–300 mM for S-MPOH, 0.16–50 mM for BFOH, 7.8–250 mM for CZXOH, 0.05–100 mM for MDZ1?OH, 2.2–70 mM for NIFOX, and 7.8–250 mM for TESOH. After the removal of proteins by centrifugation at 2,000 rpm for 15 min at 49 C, the supernatants were subjected to high-performance liquid chromatographic separation with tandem mass spectrometric MS W MS). In the NIFOX, incubation was detection (LC W performed in a dark room to avoid the photodegradation of nifedipine.17) Inhibition study with preincubation: The inhibitor (ABT or SKF-525A) was preincubated with 3.3 mM MgCl2, 100 mM phosphate buŠer, and HLM in the presence of 1.3 mM NADPH. After a preincubation for 30 min in the presence of NADPH, an aliquot of the incubation mixture (165 mL) was added to the mixture of NADPH (ˆnal 1.4 mM) and the probe substrate (ˆnal volume, 200 mL). The ˆnal HLM concentrations and incubation time were the same as mentioned above. After the incubation, the reaction was terminated as described above. Time-dependent inhibition assay: ABT was preincubated with 3.3 mM MgCl2, 100 mM phosphate buŠer, and HLM in the presence of 1.0 mM NADPH (ˆnal volume, 300 mL). At selected time interval, aliquots of the preincubation mixture (30 mL) were collected and added to an incubation mixture containing the substrate, 1.0 mM NADPH, 3.3 mM MgCl2, and 100 mM phosphate buŠer (ˆnal volume, 300 mL). The ˆnal HLM concentration and incubation time were the same as mentioned above. After the incubation, the reaction was terminated as described above. MS W MS analysis: All measurements were LC W performed by LC W MS W MS in the multiple reaction monitoring (MRM) mode according to the methods of Emoto et al. except the LC condition for the determination of 4?-hydroxydiclofenac, 6-hydroxychlorzoxazone, 1?-hydroxy bufuralol, oxidized nifedipine, and 1?hydroxymidazolam.5) In the detection of 4?-hydroxydiclofenac, 6-hydroxychlorzoxazone, 1?-hydroxy bufuralol, and oxidized nifedipine, the supernatants of the reaction mixtures

In vitro Inhibitory EŠect of 1-Aminobenzotriazole on Drug Oxidations

Table 1. Probe reactions POD DFOH S-MPOH BFOH CZXOH MDZ1?OH NIFOX TESOH

353

The apparent Km and Vmax values of each drug oxidation by HLM

Km (mM)

Vmax (nmol W min W mg)

CLinta) (mL W min W mg)

Reported Km or S50b) (mM)

36±4 4.9±0.4 21±2 11±1 110±10 0.89±0.05 5.5±0.4 18±1

0.89±0.03 3.5±0.1 0.068±0.001 0.16±0.01 1.4±0.1 0.53±0.01 2.5±0.1 6.6±0.2

0.025 0.71 0.0032 0.015 0.013 0.60 0.45 0.37

7.2－48.13 1.8－22 16－80 2.81－44 22－64.2 1.01－14 23－47 27.8－229

Data are the mean±S.E. (n＝3). a): CLint (intrinsic clearance) was calculated by the following equation; CLint＝Vmax W K m. b): Km or S50 values obtained from the following literature: Wang et al., 200025); Yamazaki et al., 199826); Masimirembwa et al., 199927); Kudo et al., 200028); Patki et al., 200329); Hickman et al., 199830); Nakajima et al., 200231); Shaw et al., 199732); and Ito et al., 200333).

were injected into a YMC ODS L-80 column (2×35 mm; 4 mm, YMC) and eluted at a ‰ow rate of 0.35 mL W min by a linear gradient with the mobile phase, which v) acetonitrile consisted of a mixture of A (10z (v W containing 10 mM ammonium acetate) and B (80z (v W v) acetonitrile containing 10 mM ammonium acetate). The gradient conditions for elution were as follows: 0 to 100z B (0.0–1.0 min); 100z B (1.0–2.0 min); 100 to 0z B (2.0–2.5 min); and 0z B (2.5–6.0 min). In the detection of 1?-hydroxymidazolam, the supernatants of the reaction mixture were injected into a Phenomenex Luna C18 column (2×50 mm; 3 mm, min Phenomenex) and eluted at a ‰ow rate of 0.35 mL W by a linear gradient with the mobile phase, which consisted of a mixture of C (95z 20 mM acetic acid, 5z acetonitrile) and pH adjusted to 4.0 with ammonia W D (5z 20 mM acetic acid, pH adjusted to 4.0 with 95z acetonitrile). The gradient conditions ammonia W for elution were as follows: 0 to 50z D (0.0–2.5 min); 50z D (2.5–3.0 min); 50 to 0z D (3.0–3.5 min); and 0z D (3.5–6.0 min). Data analysis: The kinetic parameters and the IC50 values were calculated from the following curves using WinNonlinTM 4.1 (Pharsight Corporation, Mountain View, CA). Michaelis-Menten plots; v＝

C×Vmax C＋Km

where v is the velocity, C is the concentration of substrate, Vmax is the maximum velocity, and Km is the Michaelis constant. IC50 determination;

Ø

z Control activity＝100A× 1－

I I＋IC50

»

where A is the maximum activity, IC50 is the in‰ection

point on the curve, and I is the concentration of inhibitor. In the data analysis of the time-dependent inhibition assay, the kobs value, which is an initial rate constant of inactivation, was calculated ˆrst by linear regression analysis, and obtained as a slope of the regression line generated by plotting logarithm of the residual activity against the preincubation time. The kinetic parameters, namely kinact and Ki values, were determined as the y-intercept (1 W kinact) and the x-intercept (－1 W Ki), respectively, in the double reciprocal plots of kobs versus inhibitor concentration by linear regression analysis. The kinact and Ki values are the maximal rate constant of inactivation and inhibitor concentration required for half-maximal rate of inactivation, respectively. Results Km values of probe substrates: To obtain the activity speciˆc to each CYP isoform in the inhibition study using HLM, it is recommended to use the probe substrate concentration close to the Km value. Therefore, the Km values in all drug oxidations were determined prior to the inhibition study using HLM prepared in-house, and were as follows; POD, 36 mM; DFOH, 4.9 mM; S-MPOH, 21 mM; BFOH, 11 mM; CZXOH, 110 mM; MDZ1?OH, 0.89 mM; NIFOX, 5.5 mM; and TESOH, 18 mM (Table 1). These results were almost equivalent to the reported values in a two-fold range except NIFOX. The Km for NIFOX was more than 4 times lower than the reported values. EŠect of preincubation on the inhibition by ABT and SKF-525A: Preincubation of ABT for 30 min in the presence of NADPH shifted the IC50 values in inhibiting the probe metabolic reactions to a low concentration (Table 2). The IC50 values of ABT with preincubation were as follows; POD, 21 mM; DFOH, 400 mM; S-MPOH 37 mM; BFOH, 16 mM; CZXOH, 38 mM; MDZ1?OH, 0.71 mM; NIFOX, 0.99 mM; and TESOH,

354

CHIE EMOTO et al. Table 2.

IC50 values of ABT and SKF-525A in inhibiting the CYP-dependent drug oxidations with and without preincubation IC50 (mM)

Probe reactions

POD DFOH S-MPOH BFOH CZXOH MDZ1?OH NIFOX TESOH

Substrate concentration (mM)

40 5 20 10 80 1 5 20

ABT Preincubation Without

With

340±31 860±150 110±9 120±11 690±170 11±0.9 3.3±0.4 0.58±0.07

21±1 400±52 37±2 16±1 38±5 0.71±0.04 0.99±0.06 0.70±0.11

SKF-525A Ratioa) 16 2.2 3.0 7.5 18 15 3.3 0.83

Preincubation Without

With

1100±170 9.3±0.8 3.1±0.2 0.030±0.009 950±130 6.2±0.5 16±1 2.9±0.3

320±31 9.3±0.6 6.0±0.5 0.14±0.03 720±59 2.6±0.6 10±2 4.0±0.9

Ratio 3.4 1.0 0.52 0.21 1.3 2.4 1.6 0.73

The inhibitory eŠects were investigated with and without preincubation for 30 min in the presence of NADPH. Data are the mean±S.E. (nÆ3). a): The ratio of IC50 without preincubation to IC50 with preincubation.

Fig. 1. Inhibitory eŠects of ABT and SKF-525A on BFOH. Preincubation of HLM with ABT or SKF-525A was conducted for 30 min in the presence of NADPH (: preincubation conducted, : preincubation not conducted). After preincubation, bufuralol at 10 mM was added to the reaction mixture. Data points with bars represent the mean± S.E. (n＝3).

0.70 mM. ABT exhibited the strongest inhibitory eŠect on the CYP3A-dependent drug oxidations (MDZ1?OH, NIFOX, and TESOH) and the weakest eŠect on the DFOH with and without preincubation. A remarkable IC50 shift (À10 times) by preincubation was observed on the POD, CZXOH, and MDZ1?OH. On the other hand, the IC50 shift of ABT was not recognized on the TESOH. SKF-525A had a wide range of IC50 values in inhibiting the probe drug oxidations by HLM as well as ABT (Table 2). SKF-525A potently inhibited the BFOH, but weakly inhibited the POD and the CZXOH with and without preincubation. However, the preincubation of SKF-525A did not cause a remarkable IC50 shift as the preincubation of ABT did. On the other hand, in BFOH, the inhibitory eŠect of SKF-525A was decreased

by preincubation (Fig. 1). The IC50 value of SKF-525A with preincubation was 5 times higher than that without preincubation on BFOH. Time-dependent inhibition by ABT: As mentioned above, a remarkable IC50 shift by the preincubation was observed in the inhibition of the POD, CZXOH, and MDZ1?OH by ABT. Therefore, the time-dependent inhibition assay was conducted on these drug oxidations, preincubating varying concentrations of ABT with HLM for varying times. POD, CZXOH, and MDZ1?OH in HLM were inactivated by ABT in a timeand concentration-dependent manner in the presence of NADPH (Fig. 2). The initial linear phase in the plot of logarithm of the residual activity against the preincubation time was observed up to 10 min for POD, 30 min for CZXOH, and 5 min for MDZ1?OH. The Ki and kinact

In vitro Inhibitory EŠect of 1-Aminobenzotriazole on Drug Oxidations

355

Fig. 2. Time- and concentration-dependent inhibition of on POD (A), CZXOH (B), and MDZ1?OH (C) by ABT. HLM was preincubated with ABT in the presence of NADPH. After preincubation, each substrate was added to the reaction mixture and the metabolite was determined. Phenacetine, chlorzoxazone, and midazolam concentrations were set at 40 mM, 80 mM, and 1.0 mM, respectively. ABT concentrations were as follows: POD (A), %, 0.0 mM, &, 4.7 mM, , 9.4 mM, , 19 mM, #, 38 mM, $, 75 mM, , 150 mM, and , 300 mM; CZXOH (B), , 0.0 mM, , 4.7 mM, #, 9.4 mM, $, 19 mM, , 38 mM, and , 75 mM; MDZ1?OH (C), , 0.0 mM, , 0.63 mM, #, 1.3 mM, $, 5.0 mM, , 10 mM, and , 20 mM. Data points represent the mean of duplicate determinations.

Table 3. The apparent inactivation constants for ABT of each drug oxidation by HLM Probe reactions POD CZXOH MDZ1?OH

Kia) (mM)

Kinactb) (W min)

Ki Kinact W

9.70 6.4 0.85

0.14 0.069 0.12

0.014 0.011 0.14

The apparent inactivation constants were calculated by the KitzWilson double reciprocal plot between ABT concentration and Kobs, which is the initial rate constant of the enzyme inactivation. a): Inhibitor concentration required for half-maximal rate of inactivation. b): Maximal rate constant of inactivation.

values of ABT were 9.7 mM and 0.14 min－1 for POD, respectively, 6.4 mM and 0.069 min－1 for CXZOH, and 0.85 mM and 0.12 min－1 for MDZ1?OH (Table 3). Ratio of kinact to Ki value for MDZ1?OH was about 10 times higher than those ratios for POD and CZXOH. Discussion CYP enzymes play an important role in the drug oxidation, which is one of the major clearance processes in the body.3) Therefore, in the drug development process, it is useful to obtain the information on the contribution of CYP enzymes to the overall metabolism of drug candidate compounds. ABT is known as a suicide substrate for CYP isoforms and widely used as a non-speciˆc CYP inhibitor. It is believed that benzyne, which was produced from ABT, forms an N,N-bridged

benzyne-protoporphyrin IX adduct in rats.6,7) However, selectivity of ABT in inhibiting the drug oxidations catalyzed by various CYP isoforms in HLM has not been fully investigated. Therefore, we investigated the inhibition of CYP enzymes by ABT with and without preincubation using conventional CYP substrates and HLM prepared in-house. ABT had a wide range of IC50 values in inhibiting the drug oxidations in HLM, 0.58–860 mM without preincubation and 0.70–400 mM with preincubation. ABT had the strongest inhibitory eŠect on the CYP3A-dependent drug oxidations, namely MDZ1?OH, NIFOX, and TESOH and the weakest eŠect on the DFOH with and without preincubation. The inhibitory eŠects of ABT using HLM in the present study are consistent with our previous results using human recombinant CYP enzymes.5) Balani et al. also investigated the inhibitory eŠects of ABT on the oxidations of ‰uorescence substrates by recombinant CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 after preincubation for 30 min in the presence of NADPH, and reported that the eŠect is highest in inhibiting the activity of CYP3A4.8) Therefore, our present and previous results with HLM and recombinant CYP enzymes using the probe substrates in combination with their results using the ‰uorescence substrates demonstrated that ABT shows the highest inhibition on the activity of CYP3A4 among the human CYP isoforms. A remarkable IC50 shift (À10 times) by preincubation with ABT was observed in the inhibition of POD, Ki value of ABT, CZXPH, and MDZ1?OH. The kinact W which indicates the inactivation potency, was 0.014 for

356

CHIE EMOTO et al.

POD in this assay. The kinact and Ki values of furafylline, another known mechanism-based inactivator of CYP1A2, were 0.87 min－1 and 23 mM, respectively, and the kinact W Ki value was 0.038.18) For MDZ1?OH, on the other hand, the kinact W Ki value of ABT (0.14) was much higher than that for POD. Diltiazem, erythromycin, ritonavir, troleandomycin, and verapamil, all known as a typical mechanism-based inactivator for CYP3A, Ki values of 0.055, 0.0017, 1.11, 0.83, showed the kinact W and 0.053, respectively.19) According to the available data, ABT seems to be a comparable inactivator for CYP1A2 to furafylline and a moderate inactivator for CYP3A4. SKF-525A inhibited the tested 8 probe drug oxidations with a wide range of the IC50 values. SKF-525A potently inhibited the BFOH, but weakly inhibited POD and CZXOH. From the IC50 values without preincubation in this study, SKF-525A as a CYP inhibitor was classiˆed into three classes: potent inhibitor for BFOH by CYP2D6 (IC50º1.0 mM); moderate inhibitor for DFOH by CYP2C9, S-MEOH by CYP2C19, and MDZ1?OH, NIFOX, and TESOH by CYP3A (1.0 mMºIC50º20 mM); and relatively weak inhibitor for POD by CYP1A2, CZXOH by CYP2E1 (IC50À20 mM). Ono et al. reported that the inhibitory eŠects of SKF525A diŠer among CYP-dependent drug oxidations in HLM, in a rank order for the inhibition at 300 mM as CYP1A2ºCYP2E1ºCYP2CºCYP3AºCYP2C19º CYP2D6.20) These results were equivalent with those in this study. In addition to the HLM studies, SKF-525A inhibited the drug oxidations by recombinant CYP enzymes in cDNA-expressed HepG2 cell lysate and bacurovirus-infected insect cells in the same manner as it did in HLM.5,20) The inhibitory eŠect of SKF-525A on BFOH in HLM was decreased by preincubation in the presence of NADPH, suggesting that SKF-525A is metabolized in HLM. In fact, it has been reported that SKF-525A is metabolized to a mono-deethylated form and minor metabolites in rat and swine liver microsomes.21) The inhibitory eŠects of the metabolites are normally smaller than those of the parent compound as reported in several drugs such as azelastine, amiodarone, and troglitazone.22–24) Therefore, it would be acceptable that the preincubation decreased the concentration of SKF525A leading to a decreased inhibition. As described above, ABT and SKF-525A showed a wide range of IC50 values in inhibiting the drug oxidations in HLM with and without preincubation, and their inhibitory eŠects were increased by preincubation in some probe reactions. Therefore, we should be careful in interpreting the results of the inhibition study using single CYP inhibitor for estimation of the contribution of CYP enzymes to the overall metabolism of the compounds in question.

Acknowledgements: We thank Ms. Kimberly Berls for reviewing this manuscript. References 1)

2)

3)

4)

5)

6)

7)

8)

9)

10)

11)

12)

Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I. C. and Nebert, D. W.: P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics, 6: 1–42 (1996). Krishna, D. R. and Klotz, U.: Extrahepatic metabolism of drugs in humans. Clin. Pharmacokinet., 26: 144–160 (1994). Spatzenegger, M. and Jaeger, W.: Clinical importance of hepatic cytochrome P450 in drug metabolism. Drug Metab. Rev., 27: 397–417 (1995). Mugford, C. A., Mortillo, M., Mico, B. A. and TarloŠ, J. B.: 1-Aminobenzotriazole-induced destruction of hepatic and renal cytochromes P450 in male SpragueDawley rats. Fundam. Appl. Toxicol., 19: 43–49 (1992). Emoto, C., Murase, S., Sawada, Y., Jones, B. C. and Iwsaki, K.: In vitro inhibitory eŠect of 1-aminobenzotriazole on drug oxidations catalyzed by human cytochrome P450 enzymes: a comparison with SKF525A and ketoconazole. Drug Metabol. Pharmacokinet., 18: 287–295 (2003). Ortiz de Montellano, P. R. and Mathews, J. M.: Autocatalytic alkylation of the cytochrome P-450 prosthetic haem group by 1-aminobenzotriazole. Biochem. J., 195: 761–764 (1981). Ortiz de Montellano, P. R., Mathews, J. M. and Langry, K. C.: Autocatalytic inactivation of cytochrome P-450 and chloroperoxidase by 1-aminobenzotriazole and other aryne precursors. Tetrahedron, 40: 511–519 (1984). Balani, S. K., Zhu, T., Yang, T. J., Liu, Z., He, B. and Lee, F. W.: EŠective dosing regimen of 1-aminobenzotriazole for inhibition of antipyrine clearance in rats, dogs, and monkeys. Drug Metab. Dispos., 30: 1059–1062 (2002). Balani, S. K., Li, P., Nguyen, J., Cardoza, K., Zeng, H., Mu, D. X., Wu, J. T., Gan, L. S. and Lee, F. W.: EŠective dosing regimen of 1-aminobenzotriazole for inhibition of antipyrine clearance in Guinea pigs and mice using serial sampling. Drug Metab. Dispos., 32: 1092–1095 (2004). Mathews, J. M., Dostal, L. A. and Bend, J. R.: Inactivation of rabbit pulmonary cytochrome P-450 in microsomes and isolated perfused lungs by the suicide substrate 1-aminobenzotriazole. J. Pharmacol. Exp. Ther., 235: 186–190 (1985). Woodcroft, K. J. and Bend, J. R.: N-aralkylated derivatives of 1-aminobenzotriazole as isozyme-selective, mechanism-based inhibitors of guinea pig hepatic cytochrome P-450 dependent monooxygenase activity. Can. J. Physiol. Pharmacol., 68: 1278–1285 (1990). Ren, S., Yang, J. S., Kalhorn, T. F. and Slattery, J. T.: Oxidation of cyclophosphamide to 4-hydroxycyclophosphamide and deschloroethylcyclophosphamide in human liver microsomes. Cancer Res., 57: 4229–4235

In vitro Inhibitory EŠect of 1-Aminobenzotriazole on Drug Oxidations

13)

14)

15)

16)

17)

18)

19)

20)

21)

22)

23)

24)

(1997). Bhamidipati, R., Mujeeb, S., Dravid, P. V., Khan, A. A., Singh, S. K., Rao, Y. K., Mullangi, R. and Srinivas, N. R.: Pre-clinical assessment of DRF 4367, a novel COX-2 inhibitor: Evaluation of pharmacokinetics, absolute oral bioavailability and metabolism in mice and comparative inter-species in vitro metabolism. Xenobiotica, 35: 253–271 (2005). Prakash, C., Kamel, A., Cui, D., Whalen, R. D., Miceli, J. J. and Tweedie, D.: Identiˆcation of the major human liver cytochrome P450 isoform(s) responsible for the formation of the primary metabolites of ziprasidone and prediction of possible drug interactions. Br. J. Clin. Pharmacol., 49: 35S-42S (2000). Huang, Z. and Waxman, D. J.: Modulation of cyclophosphamide-based cytochrome P450 gene therapy using liver P450 inhibitors. Cancer Gene Ther., 8: 450–458 (2001). Kenworthy, K. E., Bloomer, J. C., Clarke, S. E. and Houston, J. B.: CYP3A4 drug interactions: correlation of 10 in vitro probe substrates. Br. J. Clin. Pharmacol., 48: 716–727 (1999). Helin-Tanninen, M., Naaranlahti, T., Kontra, K. and Ojanen, T.: Enteral suspension of nifedipine for neonates. Part 2. Stability of an extemporaneously compounded nifedipine suspension. J. Clin. Pharm. Ther., 26: 59–66 (2001). Kunze, K. L. and Trager, W. F.: Isoform-selective mechanism-based inhibition of human cytochrome P450 1A2 by furafylline. Chem. Res. Toxicol., 6: 649–656 (1993). Zhou, S., Yung Chan. S., Cher Goh, B., Chan, E., Duan, W., Huang, M. and McLeod, H. L.: Mechanismbased inhibition of cytochrome P450 3A4 by therapeutic drugs. Clin. Pharmacokinet., 44: 279–304 (2005). Ono, S., Hatanaka, T., Hotta, H., Satoh, T., Gonzalez, F. J., and Tsutsui, M.: Speciˆcity of substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes. Xenobiotica, 26: 681–693 (1996). Barber, P. J. and Wilson, B. J.: Microsomal conversion of SKF 525-A and SKF 8742-A. Biochem. Pharmacol., 29: 2577–2582 (1980). Nakajima, M., Ohyama, K., Nakamura, S., Shimada, N., Yamazaki, H. and Yokoi, T.: Inhibitory eŠects of azelastine and its metabolites on drug oxidation catalyzed by human cytochrome P-450 enzymes. Drug Metab. Dispos., 27: 792–797 (1999). Ohyama, K., Nakajima, M., Suzuki, M., Shimada, N., Yamazaki, H. and Yokoi, T.: Inhibitory eŠects of amiodarone and its N-deethylated metabolite on human cytochrome P450 activities: prediction of in vivo drug interactions. Br. J. Clin. Pharmacol., 49: 244–253 (2000). Yamazaki, H., Suzuki, M., Tane, K., Shimada, N.,

25)

26)

27)

28)

29)

30)

31)

32)

33)

357

Nakajima, M. and Yokoi, T.: In vitro inhibitory eŠects of troglitazone and its metabolites on drug oxidation activities of human cytochrome P450 enzymes: comparison with pioglitazone and rosiglitazone. Xenobiotica, 30: 61–70 (2000). Wang, R. W., Newton, D. J., Liu, N., Atkins, W. M. and Lu. A. Y.: Human cytochrome P-450 3A4: in vitro drug-drug interaction patterns are substrate-dependent. Drug Metab. Dispos., 28: 360–366 (2000). Yamazaki, H., Inoue, K., Chiba, K., Ozawa, N., Kawai, T., Suzuki, Y., Goldstein, J. A., Guengerich, F. P. and Shimada, T.: Comparative studies on the catalytic roles of cytochrome P450 2C9 and its Cys- and Leu-variants in the oxidation of warfarin, ‰urbiprofen, and diclofenac by human liver microsomes. Biochem. Pharmacol., 56: 243–251 (1998). Masimirembwa, C. M., Otter, C., Berg, M., Jonsson, M., Leidvik, B., Jonsson, E., Johansson, T., Backman, A., Edlund, A. and Andersson, T. B.: Heterologous expression and kinetic characterization of human cytochromes P-450: validation of a pharmaceutical tool for drug metabolism research. Drug Metab. Dispos., 27: 1117–1122 (1999). Kudo, S., Umehara, K., Hosokawa, M., Miyamoto, G., Chiba, K. and Satoh, T.: Phenacetin deacetylase activity in human liver microsomes: distribution, kinetics, and chemical inhibition and stimulation. J. Pharmacol. Exp. Ther., 294: 80–88 (2000). Patki, K. C., Von Moltke, L. L. and Greenblatt, D. J.: In vitro metabolism of midazolam, triazolam, nifedipine, and testosterone by human liver microsomes and recombinant cytochromes P450: role of CYP3A4 and CYP3A5. Drug Metab. Dispos., 31: 938–944 (2003). Hickman, D., Wang, J. P., Wang, Y. and Unadkat, J. D.: Evaluation of the selectivity of in vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab. Dispos., 26: 207–215 (1998). Nakajima, M., Tane, K., Nakamura, S., Shimada, N., Yamazaki, H. and Yokoi, T.: Evaluation of approach to predict the contribution of multiple cytochrome P450s in drug metabolism using relative activity factor: eŠects of the diŠerences in expression levels of NADPHcytochrome P450 reductase and cytochrome b(5) in the expression system and the diŠerences in the marker activities. J. Pharm. Sci., 91: 952–963 (2002). Shaw, P. M., Hosea, N. A., Thompson, D. V., Lenius, J. M. and Guengerich, F. P.: Reconstitution premixes for assays using puriˆed recombinant human cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5. Arch. Biochem. Biophys., 348: 107–115 (1997). Ito, K., Ogihara, K., Kanamitsu, S. and Itoh, T.: Prediction of the in vivo interaction between midazolam and macrolides based on in vitro studies using human liver microsomes. Drug Metab. Dispos., 31: 945–954 (2003).