N-Aralkylated derivatives of 1-aminobenzotriazole as

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N-aralkylated derivatives, BBT and N-a-methylbenzyl- 1 - aminobenzotriazole (aMB), in hepatic microsomes of untreated, PB-induced, and @-NF-induced ...
N-Aralkylated derivatives of 1-aminobenzotriazole as isozyme-selective, mechanism-based inhibitors of guinea pig hepatic cytochrome P-450 dependent monooxygenase activity KIMBEWLEY J. W O O ~ WAND O ~JOHN R. BENDI Department of Phamccplsgy and Toxicology, University of Western Ontario, London, Octt., 66~

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Received November 6, 1989 W o o ~ w o m ,K. J., and BEND, J. R. 1990. N-AralkylateQ derivatives of 1-arr;ainobenzotriazole as isozyme-selective, mechanism-based inhibitors of guinea pig hepatic cytochrome P-458 dependent monooxygenase activity. Can. 3. Physisl. PhamacoB. 68: 1278- 1285. The mechanism-based inactivation of hepatic cytochrome P 4 5 8 by the suicide inhibitor 1-aminokmotriazole and two of its derivatives, N-benzyl- 1-aminobemotrimole and N-a-methylbenzyl- 1-aminobemotriazole, was investigated in microsomes from untreated, phenobarbihl-induced, and b-naphthoflavone-inducedguinea pigs. Microsomd '7'3-ethsxyresomfin0-deethylase, 7-pentoxyressmfin 0-dedkylase, and bemphetamine N-Bemethylase activities, and cytochrome P-450 content were determined following incubation with 1-aminoknzotriazole and its analogues. The lass of hepatic cytochrome P-450 content and msnooxygemse activity was dependent on inhibitor concentration and required NADPH. N-Benzyl-1-arninobemtriazole and N-a-methylbemyl-1-aminobemotrimole were more potent inhibitors of monssxygenase activity than the parent compound in microsames from untreated and phenobarbital-induced guinea pigs. In rnicrssomes from phenobarbital-induced guinea pigs, N-a-methylbemyl-1-aminobemotriazole(10 pM) was highly selective for the inactivation of the major eytochrome P-450 isozyme catalyzing '7-pentoxyresomfin 0-dmlkylation (the guinea pig ortholog of P-450KB1) compared with those isozymes catalyzing 7-ethoxyresomfin 0-deethylation or bemphetamine N-deanethylation (88 f 3 % loss of activity vs. 35 f 1B and 13 7 96, respectively). N-Bemyl- 1-aminobemotriazole was also selective for the inactivation of 7-pentoxyresomfin 0-dealblase activity, but to a lesser degree (56 f 6 vs. 3 1 f 8 and 21 f 8 % ,respectively). In hepatic Imicrosomes from untreated guinea pigs, the two N-substituted analogues were selective for the inhibition of 7-pentoxyresomfin 0-dealblation compmd with bemphetamine N-demethylation, but not 7-ethoxyresomfi 0-deethylation. The spectrally assayed loss of cytochmme P 4 5 8 caused by 1-aminobemotriazoie paralleled the inhibition sf enzyme activity in all three treatment groups; however, the loss of eytochrsme P-450 caused by N-bemyl- 1-aminsbemotrimole and N-a-methyHbemyl- I -aminobemotriazole was never greater than 45% even when msnooxygemse activity was virtually 180% inhibited. In general, N-bemyl-1-aminobemotriazole and N-a-methylbemyl-1-aphlinobenzo&.iaolewere more potent inhibitors of cytochmme P-450-dependent monosxygenase activity in hepatic microsomes from untreated compared with induced guinea pigs (for example, 100 pM N-bemyl-1-aminsbenzotriazoleinhibited 93 f 3, 81 1, and 61 7 % of the '7-ethoxyresomfin 0-deethylase activity in hepatic microsomes from untreated, phenobarbital-indaaced, and 0 - m p h t h o a v o n e - d u d guinea pigs, respectively). These latter data are consistent with the facile inactivation of guinea pig P-450IA1 but not P450IA2 by N-bemyl- 1-aminobemotriazole. Key words: cytochrome P-450, guinea pig, isozyme selective, suicide inhibitors.

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W o o m ~ o mK. ~ J., and BEND, J. R. 1990. Wr-Aralkgrlated derivatives of 1-aminobemotrizole as isozyme-selective, mechanism-based inhibitors of guinea pig hepatic cytochrorne P-450 dependent monooxygenase activity. Can. J. Physiol. Phamacol. 68 : 1278-1285. &'inactivation du cytochrome P-450 hkpatique par l'inhibiteur 1-aminobemotrizole et deux de ses dtrivts, N-bemyi-laphlinobemotriazole et N-a-mCBgaylbenzyl-1-aminobemotrizole, a t t t examinte &ins des microsomes de cobayes induits de B-maphthoflavone et de ph6nobarbita.I ainsi que dans des rmicrosomes de cobayes now trait&. La teneur en cytschrome B-458 et les activitCs de bemphCtaamine N-dCmCthylase, 7-~ntoxyrCsomfine-0-d6sdkylaseet 7-Cthoxyrtssmfine 0-dttthylase microsomiales ont CtC dCtednCes aprbs une incubation avee 1-aminoknzotriazole et ses analogues. La perte de teneur en cytoehrome P 4 5 0 hCpatique et d9activitdmonooxygCnasique a CtC fonction de la concentration des inhibiteurs et a n6cessitC le NADPH. N-Bemyl- 1-aminobemotriazo1e et N-a-mkthylbeql-l -aminobenzotrimole ont CtC de plus puissants inhibiteurs d'activitt de rnonooxygtnase que le csmposC mkre dans les microsomes des cobayes induits de phknobarbital et dans ceux des cobayes non traitCs. Dans les microsomes des cobayes induits de phknobarbital, N-a-mCthylbenzyl-1-aminobemotriazole (10 pM) a CtC trks sklectif pour I'inactivation du principal isszyme du cytschrome P-450 catalysant la '7-pentoxyrCsomfine 0-dCsdkylation (ortholope du P-45OIIB1 du cobaye) comparativement aux isozymes catdysant la '74thoxyrCssmfine 0-dktthylation ou la benzphktamine N-dCrnCthylation (perte d'activitk de 88 f 3 % contre 35 f 11 et 13 f 7 %, respectivement). N-Bemyl-1-amimbemotriazolea aussi CtC stlectif pour l'inactivation de l'activitk de 7-pentoxyrCsomfine-Odtsalkylase, m i s ?i un degrt moindre (56 f 6 % contre 3 1 f 8 et 21 f 8% , respectivement). Dans les microsomes hkpatiques des csbayes non trait&, les deux analogues N-substitats ont CtC stlectifs pour l'inhibition de la 7-pentoxyresomfine O46salkylation comparativemesat ?i celle de la bemphCtamine N-dCmCthylation, mais non pour celle de la '7-CthoxyrCsomfine 0-dCtthylation. L'analyse spectrde a indiquC que la perte de cytschrome P-450 provoquCe par 1-aminobemotriazole suivait l'inhibition de 19activitC emymatique dans les trois groupes; toutefois, la perte de cytschrome P-450 prgavoquke par N-benzyl- 1-aminobemotriazole et N-a-mtthylbemyl- l -araainobenzotriazole n9ajamais Ctt supCrieure 2 45 % , meme lorsque l'activitC de monooxygCnase a Ctk inhibte de prbs de 100%. En gCnCral, N-bemyl-1-aminobemotrimoleet N-a-methylbemyl-1-a~nobemotriazoleont CtC de plus puissants inhibiteurs de 19activitCde monooxygtnase ddpendante du cytochrome 'Author to whom all correspondence should be addressed. ABBREVI~IONS: P-450, cytochrome P-458; ABT, 1-aminobemotriazole; PB, sodium phenobarbital; BBT, N-knzyl-1-aminobemotriazole; PAH, polycyclic aromatic hydrocarbon; 0-NF, 0-naphthoflavone; aMB, N-a-mehylbemyl-1-aminobemotrimsle; ERF, 7-ethoxyresomfin 0-deethylase; P W , 7-pentoxyresomfin 0-dealblase; BND, benzphetamine N-demethylase. Printed In Canada / Imprid au Canada

P-450 &ins les microsomes hkpatiques des cobayes non trait& que dans ceux des cobayes induits (par exemple, 188 yM de

N-knzyl-1-aminobenzotriazoleont inhibC de 93 f 3, 81 f 1 et 61 f 7% l'activit6 de 7-Cthoxyr6sorufine 0-dCCthylase dans

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les microsomes des cobayes non traitCs et des cobayes induits de ph6nobarbital et de 8-naphthoflavone, respectivement. Ces deniers r6sultats confirment l'inactivation facile du P-450M1 mais non du P-458M2 par N-knzyl-1-aminobenzotriazole chez le cobaye. Msa ekJs : cytochrome P-450, cobaye, s6lectivitC isoenzymatique, inactivateurs. [Traduit par la revue]

Introduction The cytochrome P-450 (P-450) dependent monooxygenase system is important in the metabolism of both exogenous and endogenous compounds, and the products of these oxidations can have equal, greater, or less biological activity or toxicity than the parent compound. The delineation of the role played by P-450 in the metabolism of various substrates is complicated by the large number of P-450 isozymes, which often have overlapping substrate specificities (Black and Coon 1986). One method of determining the relative catalytic contribution of various P-458 isozymes is the use of isozymeselective suicide substrates (Ortiz de Montellano and Correia 1983), also called mechanism-based inhibitors (Wando 1984). Several of these are known, including derivatives of progesterone and pregnenolone (Halpert et al. 1989a, 1989b), acetylenic fatty acids (Ortiz de Montellano and Reich 1984), derivatives of chloramphenicol (Miller and Halpert 1987), secobarbital (Lunetta et al. 1989), and 1-ethynylpyrene (Gan et al. 1984). 1-Mnoknzotriazole (ABT) is a suicide substrate for a number of P-458 isozymes in lung (Mathews et al. 1985), liver (Ortiz de Montellano and Mathews 1981; Ortiz de Montellano et al. 1981), and plants (Reichhart et al. 1982). The enzymatic oxidation of ABT to the reactive intermediate, benzyne, leads to the inactivation of P-450 via alkylation of the prosthetic heme moiety as demonstrated by the isolation of an N,N1-bridged porphyrin adduct following incubation of hepatic microsomes from phenobarbital (PB) treated rats with ABT (Ortiz de Montellano and Mathews 1981; Ortiz de Montellano et al. 1984). The same heme adduct was isolated from livers of PB-induced rats treated with N-benzyl-l-aminobenzotriazole (BBT; Mathews and Bend 1986). N-Akylated analogues of ABT have been synthesized and shown to be potent suicide inhibitors of rat hepatic P-458 (Ortiz de Montellano et al. 1984). The N-arakylated derivatives studied here were previously shown to be very potent and isozyme-selective inhibitors of rabbit pulmonary P-450 (Mathews and Bend 1986). Hepatic microsomes contain numerous isozymes of P-458 (Nebert et al. 1989), and the relative monooxygenase activity of different classes of P-450 isozymes or individual isozymes can be determined through the use of isozyme selective substrates. 7-Ethoxyresorufln is a substrate selective for those isozymes induced by polycyclic aromatic hydrocarbons (PAH) such as P-naphthoflavone (o-NF), while 7-pentoxyresomfin is selective for isozymes induced by PB (Burke and Mayer 1983; Burke et al. 1985; Lubet et al. 1985). The N-demethylation of benzphetamine is also catalyzed by isozymes inducible by PB treatment in mammalian liver (Serabjit-Singh et al. 1983). The primary objective of this study was to determine the potency and isozyme selectivity of ABT and two of its N-aralkylated derivatives, BBT and N-a-methylbenzyl- 1aminobenzotriazole (aMB), in hepatic microsomes of untreated, PB-induced, and @-NF-inducedguinea pigs. The

two N-arakylated derivatives synthesized earlier (Mathews and Bend 1986) are potent inactivators of pulmonary P-450 in vivo in the rabbit (J. M. Mathews and J. W. Bend, unpublished results) and have not been characterized for their potency or isozyme selectivity in detail in liver of any species. A similar study has been completed with guinea pig pulmonary microsomes and will be published elsewhere (Woodcroft et al. 1990).

Methods Animk pretreatment Male or female Wartley guinea pigs (250-300 g) were used. Animals were treated intraperitoneally with 80 mg/kg PB (2% in saline) or 80 mg/kg 6-NF (2% in corn oil) daily for 4 days. Animals were sacrificed 24 h following the last injection by asphyxiation with CO,. All animals were allowed free access to food (Purina guinea pig chow) and water throughout the treatment period. In vitro imdivatisn sf hepatic P-$50 Liver microsomes were prepared as previously described (Bend et ak. 1972). Incubation mixtures contained hepatic m i c r o s o d protein (14 - 16 mg), NADPH (1 d;no NADPH in controls), and various concentrations of inhibitor (no inhibitor in controls) in 0.1 M potassium phosphate buffer, pH 7.4. The total incubation volume was 2 mL. Inhibitors were dissolved in methanol, added to the incubation vessel, and the methanol was removed under a gentle stream of nitrogen at room temperature prior to the addition of other incubation components. After a 45-fin incubation at 37 'C, the mixtures were cooled on ice and centrifuged for 10 min at 4 12 160 x g (Beckman TL- 100 ultracentrifuge; TLA 1MI.3 rotor). Subsequently, the microsand pellets were washed by resuspension and resedimentation to remove excess inhibitor. The microsomal pellets were resuspended and stored at -80°C. Specific P450 content and 7-ethoxyresomfin 0-deethylase (ERF), 7-pentoxyresomfin 0-dealblase (PRF), and benzphetamine N-demethylase @ND) activities were subsequently determined in thawed samples within 14 days of freezing. Control experiments showed there is no loss of monooxygenase activities or P-450 content under these conditions.

Enzyme assays Specific P-450 content was determined from the dithionite difference spectrum of carbon monoxide saturated microsomes using a Bechan DU-65 spectrophotometer ( E = 100 mM cm-$ Esaabrook et al. 1972). N-Demethyhtion of D-benzphetarmine (2 ELM) was measured by assaying the amount of formaldehyde produced in 15 min at 37 "C (Nash 1953). E W activity was determined by the method of Burke and Mayer (1974) with some modifications. The reaction mixture consisted of microsomal suspension (approximately 13 yg protein/m%), 1 pM 7-ethoxyresorufin (Molecular Probes Inc., Eugene, OR) in DMSO (5 pL), in 0.1 M potassium phosphate buffer, pH 7.8. The reaction volume was 2 mL. A baseline was recorded for a few minutes at an excitation wavelength of 550 nm and an emission wavelength of 585 on a Perkin- Elmer fluorescence spectrometer (model LS-SB), and the reaction was started by the addition of 108 pM NADPH (Sigma Chemical Co., St. Louis, MO). The reaction was run at 37°C and the rate of formation of resorufin was calculated by comparison with known amounts (15 pmol) of resorufin (Molecular Probes Inc., Eugene, OR) added in DMSO (5 y%) to the reaction mixture. The rate of 0-dealblation of 7-pentoxyresomfin

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(Molecula Probes Inc.) was determined by the method of Lubet et al. (1985) with 4 pM substrate and approximately 50 pglmL rnicrosornal protein in 0.1 M potassium phosphate buffer, pH 7.8. The reaction was started by the addition of 168 pM NADPN and was

68, 1990

TABLE 1. Cornpison of the effects s f equimslar amounts (100 yM) of ABT, BBT, and aMB on the B-450 monooxygenase system of hepatic rnicrosomes from untreated, 6-NF-induced, and PB-induced guinea pigs

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incubated at 37°C. Protein concentrations were determined by the method sf Lowry et &el. (1951) using bovine serum albumin as the standard.

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Results Treatment with B-NF or PB resulted in a marked and selective increase of hepatic microsomal monooxygenase activities relative to those of untreated guinea pigs. For example, P-NF administration increased ERF activity sixfold, PRF activity approximately twofold, P-450 content by 1.Cfold, and had no effect on BND activity. In PB-treated guinea pigs, PRF activity was increased 10-fold and BND and ERF activity and P-450 content all about twofold (Table I). The effects of equimolar concentrations (100 yM) of ABT, BBT, and aMB as suicide inhibitors were initially compared in hepatic microsomes isolated from untreated, P-NF-induced, and PB-induced guinea pigs (Table 1). The concentration of inhibitors selected was based on preliminary experiments which showed that 100 yM BBT inactivated approximately 80% of hepatic ~ c r o s o mERF l and PRF in control animals. The inhibition described here was dependent on incubation with NADPH, i.e., mechanism-based or suicidal inhibition. The purpose of these initial experiments was to compare the ability of the various inhibitors to inactivate P-450 isozyme selective monooxygenase activities in liver of control with induced animals in vitro. All three compounds inactivated at least half of the ERF, PRF, and BND activity in hepatic microsomes from untreated guinea pigs under conditions where approximately 50% $37-53 %) of the spectrally assayed P-450 was lost. However, there was some evidence for differences in potency and isozyme selectivity. BBT and aMB inhibited more of the ERF and PRF activity in these Hnicrosomes than did ABT, and they also inhibited more ERF and PRF activity than BND activity (Table 1). At 100 pM, d l three inhibitors were less potent inactivators of the P-450 monooxygenase system in hepatic microsomes isolated from 0-NF-induced guinea pigs (vs. control). For example, ERF, PRF, and BND activities were only inhibited by 46 -6076 and considerably less P-450 was destroyed (8 and 12% for BBT and aMB, respectively) in P-NF-treated animals. BBT was selective for the inactivation of PRF vs. BND activity (64 vs. 43 %) but ABT and aMB were not. There were no marked differences in the inhibition of PRF vs. ERF activities with ABT, BBT, or aMB (degree of inhibition, PRFIERF = 6.8, I .O, and 0.9, respectively) in P-NF-induced guinea pigs (Table 1). The data obtained in PB-induced guinea pigs were of interest for several reasons. First, virtually all of the PRF activity was inhibited by 100 yM BBT and aMB ( >90%), whereas this concentration of ABT inhibited less than half of the PRF activity in these hepatic microsomes. Also, ABT inhibited more ERF than PRF activity, whereas the reverse was observed with BBT and aMB. The amount of spectrally assayed P-458 destroyed by the three compounds in microsomes of PB-induced guinea pigs was very similar (29 -33 %). Finally, BBT and aMB were significantly more effective as inhibitors of hepatic PRF and ERF than of BND activity after PB treatment (Table 1).

% loss from control values

Inhibitor

ABT BBT ahdB 6-NF induceds ABT BBT a ~ B PB induced

ABT BBT aMB

ERF activity

PWF activity

BND activity

-450 content

$Of$" 93f3" 91f3"

66f7 $6f3' 88f4"

49f 11 68f 7 70f 6

53f4 37f 5 38f 9

52f6" 61f7C@ 45f4"

42f10e Mf8C@ 39fSe

41f11 43fY 49f le

35f6' 8f 3" 12f 2"

58f 3 81fl" 76f4"

40f 3" 93f1" 96f 1"

NDg 50f3e 49f 3"

33f 1' 32f 1 29f 3

n C o n t d (100%) vdms for untreated microsomes were 126 i 22 p m d min-' . mgs' protein (ERF), 11.0 f 2.5 p o l min-' mg-' protein (PW), 4.55 & 0.53 mo1 . min-' . mg-' protein (BND), and 0.77 f 0.05 m o l P-4501mg protein (P-450). Vdues are m a n s f SEM, n = 5 -6. b~elativepercent loss of enzyme activity or P-450 content vs. control following incubation with inhibitor for 45 anin at 37'C. Vdues are m a n s f SBM, pa = 4-6. rSignifkmtly greater than loss s f BND activity ( p < 0.05 by unpaired Student's %-test). dControl (BOO%) vdues for j3-NF-induced microsomes were 792 f 151 pmol . rnin-' . ang-' protein (EWF), 25.9 f 4.0 pmol . rnin-' mg-' protein (PWF), 5.05 f 0.40 m o l - mh-' . rng - protein (BND), and 1.07 f 0.08 m o l P-450Img protein (P-450). Values are m a n s f SBM, m = 4-6. eSi@ifi~mt8yless than loss of rnonooxygemse activity in urntreated microsornes (p