60
Chem. Res. Toxicol. 1992, 5 , 60-66
Hepatic Microsomal Metabolism of the Anthelmintic Benzimidazole Fenbendazole: Enhanced Inhibition of Cytochrome P450 Reactions by Oxidized Metabolites of the Drug Michael Murray,* Alison M. Hudson, and Violette Yassa Liver Research Group, Department of Medicine, University of Sydney, Westmead Hospital, Westmead, NSW 2145, Australia Received June 14, 1991
Potentiation of the anthelmintic action of benzimidazole carbamates, such as fenbendazole has been noted during concurrent [methyl 5(6)-(phenylthio)-lH-benzimidazol-2-ylcarbamate], administration of benzimidazoles that possess no intrinsic anthelmintic activity. This study investigated the possibility that inhibition of P450 enzymes by fenbendazole and its metabolites could play a role in the potentiation phenomenon. Fenbendazole underwent P450-mediated oxidation in microsomes from untreated rat liver to the sulfoxide and (4’-hydroxypheny1)thio metabolites [2.92 and 2.87 nmol/ (mg of proteimh)]. Pretreatment of rats with phenobarbital or dexamethasone enhanced sulfoxidation by 1.9- and 2.9-fold, respectively. 4’-Hydroxylation was increased slightly (by 28%) by phenobarbital and decreased slightly (by 41%) by dexamethasone. Induction also promoted further metabolism of the sulfoxide to fenbendazole sulfone. Immunoinhibition and chemical inhibition studies suggested that P450 3A proteins and the flavin-containing monooxygenase are involved in sulfoxide and sulfone formation whereas 4’hydroxylation involved the P450s 2Cll,2C6, and 2B1, depending on the type of induction. In untreated rat liver, the sulfoxide and (4’-hydroxyphenyl)thio metabolites of fenbendazole were relatively potent inhibitors of P450-mediated androstenedione 16a-, 16&, and 6P-hydroxylation (IC5ovalues of 42, 36, and 74 pM, respectively); 7a-hydroxylase activity was uninhibited. In contrast, fenbendazole and its sulfone metabolite were not inhibitors of these reactions. Mixed-function oxidase activities in phenobarbital-induced rat hepatic microsomes were refractory to inhibition by most compounds, but P450 1Al mediated activities in microsomes from flnaphthoflavone-induced rat liver were quite susceptible to inhibition by fenbendazole sulfoxide. Studies with two analogous sulfoxides yielded similar findings. Since it is unlikely that the increased hydrophilicity of the sulfoxides could be responsible for enhanced inhibition of P450 1A1, it is possible that the relatively rigid pyramidal conformation of the sulfoxide could facilitate the coordination of aromatic substituents within the P450 1 A l active site. These findings c o n f i i that fenbendazole undergoes hepatic oxidation to produce metabolites with greater inhibitory potency against P450 reactions. Thus it is possible that such metabolites, which may have diminished anthelmintic activity, may potentiate the action of fenbendazole in vivo by virtue of their anti-P450 action.
Introduction The benzimidazole carbamate (BZC)l class of anthelmintic agents has a broad-spectrum activity against nematode, cestode, and trematode species (2), as well as anticancer ( 3 ) and antifungal (4) properties. This wide therapeutic profile is thought to be due to the ability of BZCs to bind to the cytoskeletal protein tubulin (5). In sheep, BZCs with potent mammalian tubulin binding capacity have been shown to potentiate the in vivo kinetics and anthelmintic efficacy of coadministered BZCs of low potency (6). This phenomenon has been attributed to a direct effect on the hepatic tubular network. However, many benzimidazoles are themselves quite effective inhibitors of the hepatic microsomal mixed-function oxidase (MFO) system (7-11). The inhibitory capacity of benzimidazoles is due to their interaction with cytochrome P450 (P450), the terminal oxidase of the MFO system. Inhibition of P450-mediated reactions is an established mechanism underlying drug potentiation (12,13) and insecticide synergism (14).
* Address correspondence to this author at the Department of Medicine, Westmead Hospital, Westmead, NSW 2145, Australia. 0893-228X/92/2705-0060$03.00/0
The present investigation addressed the activity of fenbendazole and several metabolites (15,16) as inhibitors of specific microsomal P450 reactions in rat liver. Since certain metabolites were found to be more potent P450 inhibitors than the parent drug, studies were conducted to investigate the potential significance of this phenomenon in potentiation of BZC action.
Materials and Methods Chemicals. Authentic samples of fenbendazole (I) and fenbendazole sulfoxide (oxfendazole;11) were obtained from Hoechst (Australia) and Syntex Agribusiness (Australia), respectively. Fenbendazole sulfone (111) was prepared from I1 by hydrogen peroxide oxidation in glacial acetic acid in quantitative yield; m p >300 “C [lit. m p >320 “C (191.4’-Hydroxyfenbendazole (IV) Abbreviations: M,, maximal absorbance change; BZC, benzimidazole carbamate; BNF, 8-naphthoflavone;ECOD, 7-ethoxycoumarin 0-deethylase; EROD, 7-ethylresorufin 0-deethylase; FMO, flavin-containing monooxygenase;IgG, immunoglobulin G; K,,spectral dissociation constant; log,, P, logarithm (base 10) of the n-octanollwater partition coefficient;MFO, mixed-functionoxidase; P450, cytochrome P450 [the recommended nomenclature ( I ) is used throughout this paper]; P450 reductase, NADPH-cytochrome P450 reductase; PB, phenobarbital; PROD, 7-pentylresorufin 0-depentylase.
1992 American Chemical Society
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 61
Fenbendazole Metabolism a n d P450 Inhibition was synthesized by modification of the method of Averkin et al. ( 1 7 ) involving the reaction of 5-chloro-2-nitroaniline with 4mercaptophenol (yield 75%; mp 216 OC). Subsequent oxidations to the sulfoxide (V; mp 256 OC) and sulfone (VI; mp 299 OC) were accomplished with 1.2 and 4 equiv of hydrogen peroxide in glacial acetic acid. 2-Amino-5(6)-(phenylthio)bemimidazole(VII) was synthesized by a slight modification of the method of Allan et al. (18)involving alkaline hydrolysis of I; mp 238 OC [lit. mp 145 OC (19)l. The amines (VI11 and IX) were prepared similarly; mp(VI1) 217 OC, mp(1X) 263 "C [lit. mp 220 OC (19)]. Yields from carbamate hydrolyses ranged from 75% to 90% of the theoretical. Purity was assessed by thin-layer chromatography and highpressure liquid h m a t u g r a p h y , and by comparison with literature melting points where available; melting points were determined in a Mettler FP61 instrument. All spectroscopic data ('H NMR and CH,-CI/MS) were consistent with the assigned structures. Biochemicals were obtained from Sigma Chemical Co. (St. Louis, MO), and other reagents and chemicals were analytical reagent grade. Chromatographic resins for P450 purification were obtained from Pharmacia Pty. Ltd. (North Ryde, NSW, Australia), and DEAE-Affi-Gel Blue for IgG isolation was from Bio-Rad Laboratories (Richmond, CA). Animals. Male Wistar rata (approximately 250 g) were used in this study. Some animals received either phenobarbital (PB; 100 mg/kg/day ip for 3 days), dexamethasone (400 mg/kg day ip for 3 days), or 8-naphthoflavone (BNF; 40 mg/kg/day ip for 3 days); rata were sacrificed 24 h after the final injection. Washed hepatic microsomes were prepared by conventional procedures (20),and protein was determined by the method of Lowry et al. (21) with bovine serum albumin as standard. Purification of Microsomal Enzymes and Preparation of IgG Fractions. The purifications of P450 2Cll and P450 2B1 from untreated and PB-induced adult male rat liver have been described previously (22,23).P450 2C6 was isolated from PBinduced male rat liver essentially as described by Waxman and Walsh (24)except that DEAE-Sephacel (Pharmacia) was used in place of Whatman DE52 and CM-Trisacryl M (LKB, Bromma, Sweden) waa used in place of CM-Sepharose. P450 3A1 isolated from PB-induced male rat liver (2.5) and rabbit anti-rat P450 IgG were generously supplied by Dr. Anders Astrom (University of Stockholm,Stockholm,Sweden). The specific contents of all P450 preparations were in the range 9-14 nmol/mg of protein. NADPH-cytochrome P450 reductase (P450 reductase) was purified by the method of Yasukochi and Masters (26). Antisera to P455Os 2Cll,2B1, and 2C6 and P450 reductase were raised in female New Zealand rabbits by a standard protocol of three inoculations over a 4-week period (22). Animals were bled via an ear vein 2 weeks after the final inoculation and a t fortnightly intervals for a total of three bleeds. The IgG against each P450 was then isolated by ammonium sulfate precipitation and chromatography on DEAE-Affi-Gel Blue (Bio-Rad); the IgG eluted from this column in the unbound fraction. Immunoinhibition of MFO activities by the various IgGs was assessed. As documented previously (22),anti-P450 2Cll preferentially inhibited androstenedione 16a-hydroxylation from control male rat liver; Westem immunoblot analysis also indicated that this IgG recognized an antigen present in male, but not female, rat hepatic microsomes. Anti-P450 2B1 effectively inhibited androstenedione 160-hydroxylation and 7-pentylresorufin 0-depentylation in microsomes from PB-induced rat liver (23). Anti-P450 3A was also male-specific and inhibited androstenedione 6b-hydroxylation effectively. This antigen also significantly decreased the rate of androstenedione 7a-hydroxylation, and although this activity is attributed to P450 2A1/2, a previous study documented the same phenomenon (27).Anti-P450 2C6 decreased progesterone 21-hydroxylation in microsomes from untreated rat liver but had no measureable effect on other pathways of progesterone, testosterone, or androstenedione metabolism (not included). Anti-P450 reductase (8 mg of IgG/mg of protein) inhibited 7-ethylresorufin 0-deethylase activity in BNF-induced rat hepatic microsomes by 50%. Microsomal Metabolism of Fenbendazole. Metabolism of fenbendazole was studied in hepatic microsomes from control and induced rats. In these studies the concentrations of microsomal protein and substrate were 0.25 mg/mL and 50 pM, respectively.
W
I
I
0
22 RETENTION TIME, MINUTES
Figure 1. Typical HPLC separation of fenbendazole and its metabolites. Key: sulfone, fenbendazole sulfone; 4-hydroxy, 4'-hydroxyfenbendazole. Oxibendazole is the internal standard. The standard NADPH-generating system (1mM NADP, 5 mM glucose 6-phosphate, and 1 unit of glucose-6-phosphate dehydrogenase in 0.1 M potassium phosphate buffer, pH 7.4) was used to initiate the reactions, which were conducted at 37 OC and then quenched after 30 min by removal to a dry ice/acetone bath. The contents of the flasks were thawed in warm water and loaded immediately onto a Sep-Pak C18 cartridge (Waters Associates, Milford, MA). The internal standard (oxibendazole) was added, and the Sep-Pak was washed sequentially with water and methanol. In immunoinhibition experiments, microsomal fractions were preincubated with the appropriate IgG (8 mg/mg of microsomal protein) for 10 min prior to initation of the reaction with the NADPH-generating system; control incubations included preimmune IgG which did not markedly influence metabolite formation. Incubations were performed in duplicate, and products of fenbendazole metabolism were extracted as described above. Separation of Fenbendazole Metabolites by High-Performance Liquid Chromatography. Fenbendazole and its metabolites were applied to a Beckman Ultrasphere ODS column (5 pm, 25 cm X 4.6 mm i.d., Beckman Instruments Inc., San Ramon, CA) attached to a Waters Associates HPLC system and eluted with a mobile phase of acetonitrile/25 mM ammonium acetate, pH 7.2 (2:3). Under these conditions the retention times of the compounds of importance were as follows: oxfendazole (3.65 min), fenbendazole sulfone (5.55 min), 4'-hydroxyfenbendazole (6.32 min), oxibendazole (6.87 min), and fenbendazole (18.28min). The eluate was monitored at 292 nm, and peak areas were calculated on a Waters 730 Data Module. Figure l shows a typical HPLC separation of the five compounds. Microsomal Steroid Hydroxylase Activity. The metabolism of [4-'*C]androstenedione in hepatic microsomal fractions was conducted as described previously (28). Hydroxyandrostenedione derivatives were separated by thin-layer chromatography and identified under UV light by comigration with authentic standards. Metabolite formation was quantified by 8-counting (ACS 11, Amersham Australia). Inhibitors were incorporated into incubations in dimethylformamide (25 pL); incubations were conducted in duplicate at each inhibitor concentration. Other Microsomal MFO Activities. Direct spectrofluorometric procedures (29) were used for the assay of 7-ethoxycoumarin 0-deethylase (ECOD), 7-ethylresorufin 0-deethylase (EROD), and 7-pentykeSOrufin 0-depentylase (PROD). Inhibitors were included in reaction incubations (final volume 2.0 mL) in 25 pL of dimethylformamide; solvent was added to control incubations. ICN values were calculated from plots of percent inhibition versus loglo [inhibitor]. Each plot was constructed from the average percent inhibition at 4-6 different inhibitor concentrations, each determined in duplicate. Optical Difference Spectroscopy. Optical difference spectra were determined a t 37 "C in Aminco-Chance DW-2a spectrophotometer using 1-cm cuvettes and 1-mL aliquots of microsomal suspensions (1-2 mg/mL in 0.1 M potassium phosphate buffer, pH 7.4). Compounds were added to the sample cuvette in microliter volumes of dimethylformamide, and an equal volume of the solvent was added to the reference cuvette. The resultant difference spectra were recorded between 380 and 500 nm. Double-reciprocal plots of aA (between the peak and trough of
62 Chem. Res. Toxicol.,
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Murray et al.
Table I. Microsomal Metabolism of Fenbendazole in Control and Induced Rat Liver and Effect of IgGs Directed against P450 Enzymes on Metabolite Formationa fenbendazole metabolite, nmol/ (mg of proteinoh) 4‘hydroxy- fenbenoxfenfenbendazole microsomes IgG dazole dazole sulfone ND control preimmune 2.92 2.87 1.43 (49) 2.50 (87) ND anti-P450 3A anti-P450 2Cll 3.10 (106) 1.66 (58) ND anti-P450 2C6 3.08 (105) 1.81 (63) ND 0.41 phenopreimmune 5.50 3.66 barbital anti-P450 2Cll 4.19 (76) 2.58 (70) 0.44 (107) induced anti-P450 2C6 6.34 (115) 3.31 (90) 0.88 (215) anti-P450 2B1 5.90 (107) 2.55 (70) 0.62 (151) 1.10 dexapreimmune 8.54 1.70 4.87 (57) 4.01 (236) 0.59 (54) methasone anti-P450 3A anti-P450 2B1 9.74 (114) 1.10 (65) 2.90 (264) induced ____
~
~~
~~~
aValues are means from duplicate incubations. The average variation from stated mean values was 9.5%. Numbers in parentheses are percentages of activities in the presence of preimmune
Table 11. Effect of Methimazole and Anti-NADPH-P450 Reductase on Microsomal Fenbendazole Metabolism in Untreated Rat Liver“ nmol/ (mg of protein-h) 4‘-hydroxyaddition to incubation oxfendazole fenbendazole none 4.03 f 0.49 4.78 f 0.51 methimazole (1mM) 2.76 f 0.37 (68Ib 4.96 f 0.17 (104)b preimmune IgG 3.61 f 0.39 4.02 f 0.24 (8mg/mg) anti-P450 reductase 2.67 f 0.23 (74)e 2.88 f 0.36 (72)c (8mg/mg) ~
~~
aValue~are mean f SD of three individual incubations. Numbers in parentheses indicate percent of metabolite formation with no addition to incubation and preimmune IgG. 120
E
100
a
80
E0
El
IgG.
the difference spectrum) versus [ligand] were constructed, and the spectral dissociation constants (K,) and maximal absorbance changes (M”) were determined from the x-axis and y-axis intercepts, respectively (IO). P450 was determined according to the method of Omura and Sat0 (30).
8
60
u. 0
ku
40
W 0:
n
20
Results Microsomal Metabolism of Fenbendazole. The anthelmintic agent fenbendazole [methyl 5(6)-(phenylthio)-lH-benzimidazo1-2-ylcarbamate(I)] underwent NADPH-dependent oxidation in hepatic microsomes from untreated male rats to the corresponding sulfoxide (11)and 4’-hydroxy-5(6)-(phenylthio)(IV) metabolites. Rates of metabolite production were relatively slow [2.92 and 2.87 nmol/(mg of protein-h) for I1 and IV, respectively; Table I]. Formation of I1 was induced 1.9- and 2.9-fold by pretreatment of rats with PB and dexamethasone [5.50and 8.54 nmol/ (mg of proteinoh), respectively;Table I] whereas IV production was increased by 28% after PB induction and decreased by 41% by dexamethasone pretreatment. The sulfone I11 was not produced in microsomes from untreated male rats but was formed in PB- and dexamethasone-induced rat liver (Table I). Immunoinhibition studies of metabolite formation from fenbendazole were undertaken with a series of IgG raised in rabbits against specific purified rat P450s (Table I). In control microsomes, formation of the sulfoxide (11) was effectively inhibited by anti-P450 3A and formation of the 4’-hydroxy metabolite (IV) was inhibited by anti-P450 2Cll (by 42%) and by anti-P450 2C6 (by 37%). These data are consistent with the notion that P450 3A2 is involved in fenbendazole sulfoxidation and the P450s 2Cll and 2C6 are both important in 4’-hydroxylation. In hepatic microsomes from induced rats anti-P450 3A inhibited fenbendazole sulfoxidation and anti-P450 2C11 and anti-P450 2B1 both produced approximately 30% inhibition of 4’-hydroxylation. Formation of the sulfone in dexamethasone-induced rat liver was inhibited efficiently (by 46%) by anti-P450 3A. Stimulation of 4’-hydroxylation and sulfone formation was observed in some cases and was most pronounced in induced microsomal fractions. Although the mechanism underlying this phenomenon is unclear, it is possible that inhibition of one pathway of fenbendazole metabolism by a particular anti-P450 IgG increases the availability of the drug to other P450s. Similar effects have been noted in reports from other
n
16a 160 60 7cy Figure 2. In vitro inhibition of hepatic microsomal androstenedione metabolism by fenbendazole and metabolites. Drugs were included into microsomal incubations a t concentrations of 100 p M . Uninhibited rates of androstenedione metabolite production were as follows: testosterone [2.02 nmol/ (mg of proteimmin)], l6a-hydroxy (1.94), 16P-hydroxy (0.28), 6B-hydroxy (1.75), and 7a-hydroxy (0.35). Closed boxes, fenbendazole; open boxes, fenbendazole sulfone; diagonal hatching, fenbendazole sulfoxide, and dots, 4’-hydroxyfenbendazole.
laboratories in studies of anti-P450 IgGs against steroid hydroxylation (27,31). Further experiments were undertaken to investigate the possible involvement of the flavin-containing monooxygenase (FMO) in microsomal fenbendazole Soxygenation. The data in Table I1 indicate that the FMO inhibitor methimazole (1mM) decreased the rate of sulfoxidation to 32% of control without influencing the rate of 4’-hydroxylation of the anthelmintic. Incubation with anti-P450 reductase decreased the rates of both pathways of oxidation similarly. Thus, these findings are consistent with the participation of P450 and FMO in fenbendazole sulfoxidation. Inhibition of MFO Activity by Fenbendazole and Metabolites. Fenbendazole was essentially nonpotent as an inhibitor of P450-mediated androstenedione hydroxylation in hepatic microsomes from untreated rat liver (Figure 2). Slight inhibition (15%) of P450 2Cll and P450 3A2 dependent steroid 16a- and 6P-hydroxylase activities was produced by I, but its metabolites were somewhat more inhibitory. Thus, 20-37% inhibition of rates of androstenedione 6P-, 16a-, and 16P-hydroxylation was observed with the sulfoxide (11)and sulfone (111)metabolites of fenbendazole (Figure 2). Most interestingly, however, 4’-hydroxyfenbendazole (IV) was a quite potent inhibitor of these three pathways of microsomal androstenedione hydroxylation (Figure 2). ICsovalues were obtained for compound IV, and it was found that formation of the
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 63
Fenbendazole Metabolism and P450 Inhibition
Table 111. Inhibition of Hepatic Microsomal Mixed-Function Oxidase Activities by Fenbendazole and Derivatives '
/ X\
O
~
N
)
-
.
'1 compound I I1 I11 IV V VI VI1 VI11 IX
R NHC02CH3 NHC02CH3 NHCOzCH3 NHCOzCH3 NHC02CH3 NHCOzCH3 NH2 NH2 NH2
X S
SO SO2 S
SO SO2
S SO SO2
Y H H H OH OH OH H H H
mixed-function oxidase activity (ICw, pM) BNP PB" EROD ECOD PROD NIb 1.1 NI 47 64 41% at 100 pM' 68 0.074 NI 5.8 63 NI 52 2.8 65 NI 43% at 100pM 0.42 28 NI NI NDd NI NI UT" ECOD
34 76 59
6.9 0.31 4.6
63 15 48
ECOD NI
6.1 45% at 100 pM 61
97
NI 47
NI NI 29 55 79
Source of microsomal fraction: UT, untreated; BNF, 8-naphthoflavone-induced;PB, phenobarbital-induced male rat liver. NI, not inhibitory (ICw > 200 pM). cPercent inhibition observed at test concentration of 100 pM (when 100 pM < IC, < 200 pM). dND, not determined. a
Table IV. Binding of Fenbendazole and Derivatives to Cytochrome P450 in Hepatic Microsomes from Untreated and BNF-Induced Rats" binding parameter untreated liver BNF-induced liver AA,,, absorbance AA,,, absorbance K,,pM units/nmol of P450 type Ks,pM unita/nmol of P450 compound typeb I ND' ND I1 RI 21 0.98 X RI 0.73 2.38 X I11 mixed RI 6.5 0.94 X 160 2.43 X ND IV RI 51 1.38 X RI V RI 0.98 1.42 X VI ND RI 7.2 0.71 X 12 0.94 X ND VI1 I VI11 mixed RI 1.3 2.06 X IX mixed RI 32 0.76 X lo-' ~
a Values are means of at least two estimates from individual titrations, each employing at least five substrate concentrations. type: RI, reverse type I; I, type I; and mixed, mixed type I/reverse type I. 'ND, no detectable spectral change.
epimeric 16-alcohols of the steroid was most susceptible
to inhibition by IV ( E m sof 42 and 36 pM were obtained against 16a- and 16-hydroxylase activities, respectively). An ICw of 74 r M was estimated against androstenedione 6&hydroxylation, but the 7a-hydroxylase pathway was essentially refractory to inhibition by IV so that an IC50 value could not be determined. Other MFO activities in control and induced rat liver were assessed for their susceptibility to inhibition by I and its analogues. ECOD activity in untreated rat hepatic microsomes was uninhibited by the parent drug (IC50> 200 pM; Table 111). Again it was noted that several metabolites of the anthelmintic agent were more potent inhibitors of ECOD than I. The data in Table I11 indicate that the sulfoxide (11)and 4'-hydroxy (IV) metabolites are more potent than the parent (I) as inhibitors of ECOD activity from untreated rat liver microsomes; the sulfone (111)was uninhibitory. Examination of the effectiveness of these agents against P450 activities in induced rat liver yielded different results (Table 111). Some of the nonpotent inhibitors of P450s in untreated rat liver were more potent against MFO activities from BNF-induced rat liver. Thus, IC50sin the range 0.074-5.8 pM were obtained for I and metabolites II-IV. It also emerged from this study that compounds I-IV were relatively inactive as inhibitors of P450 activities in microsomes from PB-induced rat liver. PROD activity, which is considered to be a useful catalytic marker for P450 2B1 (32),was not inhibited effectively by any of the fenbendazole metabolites, although the parent
Spectral
drug exhibited an ICso of 47 pM against the activity. Similarly, ECOD activity catalyzed by the same hepatic microsomal fractions was refractory to inhibition by the fenbendazole analogues. Only the putative 2-decarbamylated metabolite, 2-amino-5(6)-(phenylthio)benzimidazole (VII), exhibited notable activity against either PROD or ECOD from PB-induced rat liver (Table 111). Further analogues of I were tested as MFO inhibitors in the present study. In vitro studies in microsomal fractions indicated that these compounds are not hepatic metabolites of I. However, some of them have been identified after in vivo administration to certain species. Thus, it is possible that species differences exist for the hepatic P450-dependent conversion of I to oxidized metabolites, but it is more likely that they reflect extrahepatic metabolism, especially along the 2-decarbamylation pathway. From Table I11 it appears that the 2-amino analogues (VII-IX) are, in general, somewhat more potent than the corresponding carbamates (1-111). Interestingly, the further oxidized (putative) metabolites-the 4'-hydroxy sulfoxide (V) and the 4'-hydroxy sulfone (VI)-were less inhibitory toward MFO activities in untreated and PBinduced rat liver than the primary oxidized metabolites. Binding Interactions of Benzimidazoles with Oxidized Microsomal P450. As indicated in Table IV, only four of the nine fenbendazole derivatives generated quantifiable difference spectra in control rat liver microsomes. Fenbendazole itself elicited no binding spectrum, but the sulfoxide, (4'-hydroxyphenyl)thio, and 4'-
64 Chem. Res. Toxicol., Vol. 5, No. I, 1992
Murray et al. 0
a
b
C
Figure 3. Conformations of fenbendazole (a) and its sulfoxide (b) and sulfone (c) metabolites.
hydroxyphenyl sulfoxide metabolites (11, IV, and V, respectively) generated reverse type I or mixed-type spectral changes. This spectral type has not yet been explained fully, but stabilization of the protein conformation by an interaction between the P450 heme iron and a weak ligand from an amino acid residue within the protein has been proposed (33). It appears from the present study that oxidation of the 5(6)-(phenylthio)substituent facilitates the reverse type I interaction. Compound VI1 produced a type I difference spectrum in untreated rat hepatic microsomes, despite the presence of a 2-amino substituent. The interaction was of quite high affinity (K, = 12 pM) but low capacity (AAmax= 0.94 X absorbance units/nmol of P450). The sulfoxides 11, V, and VI11 were potent inhibitors of MFO activities from BNF-induced rat liver. All three elicited reverse type I difference spectra of high affinity (K, values 0.73,0.98, and 1.3 pM, respectively; Table IV) and exhibited AAmm values that were 2- to 3-fold larger than those produced in these microsomes by other fenbendazole analogues. Thus, the binding experiments yielded data that were consistent with the findings from inhibition experiments. The conformation of the diaryl sulfoxide (Figure 3) appears to be clearly superior to the diaryl sulfide or diaryl sulfone configurations for interaction with P450 in BNF microsomes.
Discussion The potency of BZC metabolites against P450-dependent microsomal oxidation was approximately an order of magnitude lower than that required for the inhibition of microtubule formation by the parent drugs in vitro (34). From the present data it is possible that in vivo potentiation of BZC action could contain a component related to inhibition of microsomal oxidation by BZC metabolites. The relative importance of MFO inhibition to potentiation of anthelmintic activity would be expected to be species-dependent and dependent upon the actual BZC used in therapy. However, a mechanism of this type may well account for previous reports of drug synergism produced by non-carbamate benzimidazole agents, such as thiabendazole, that possess a low intrinsic capacity for microtubule inhibition (35-37). Many studies have emphasized the importance of lipophilicity to effective MFO inhibition potency (7,8,38), but in the case of benzimidazoles, the steric nature of substituents is also important ( 8 , l l ) . The present series of compounds is not extremely lipophilic [log P for benzimidazole = 1.38 (39)j, and the phenylthio substituent would add 2.32 log units to the overall hydrophobicity of fenbendazole (39). The methyl carbamate group makes a negative contribution to hydrophobicity (a = -0.39), and the product of carbamate hydrolysis is less hydrophobic still [a(NHZ)= -1.23 ( 3 9 ) ] . Inclusion of a phenolic hydroxyl group in the 4’-position of the 5(6)-substituent of fenbendazole, or oxidation of the thioether to sulfoxide or sulfone, would decrease lipophilicity further but is clearly associated with enhanced capacity for inhibition of P450 activity. Since the major P450 enzyme in BNF microsomes, 1A1, is active in the metabolic oxidation of highly
lipophilic polycyclic aromatic hydrocarbons, it is unlikely that enhancement of hydrophilic character could be the physicochemical factor responsible for increased inhibition of this enzyme by metabolites of I. Indeed, studies with other benzimidazoles have noted that it is the steric nature of the derivative and not its hydrophobic character that is the principal determinant of potency against P450 1 A l activities (9, 10, 38). The geometries of the phenylthio, phenyl sulfoxide, and phenyl sulfone moieties are an important consideration (Figure 3). In the phenylthio group the sulfur has two lone pairs of electrons that could be available for binding to the P450 heme. However, rotation about the carbon-sulfur bonds of the phenylthio substituent is likely and would be expected to hinder the approach of these lone pairs to the P450 heme. In contrast, the configuration of the sulfoxides 11, V, and VI11 is pyramidal, with the lone electron pair coordinated away from the three groups attached to that atom (the benzimidazolyl nucleus, phenyl ring, and sulfoxide oxygen). This is a more rigid structure, and any interaction between the sulfur lone pair and the heme iron would be less impeded by rotation about u bonds. Finally, in the sulfone, which has a tetrahedral configuration (40),rotation about the sulfur atom might be expected, but the only available lone electron pairs are those on the sulfone oxygen atoms, and these are probably not sufficiently nucleophilic to interact with the P450 heme. This is one explanation that could account for the diminished potency of the sulfones compared to those of the analogous sulfoxides. Similar observations have been made previously with a series of benzimidazoles with extremely hydrophobic, but highly flexible, 2-n-alkyl substituents (38). From these considerationsit seems unlikely that lipophilicity is the principal determinant of inhibitory potency. Instead, the electronic configuration and the steric nature of the bonding in the substituents of these compounds may be more important. Whereas the carbamate group in the 2-position of fenbendazole appears to prevent the interaction of the drug with ferric P450, the decarbamylated derivatives are able to generate type I and/or mixed type I/reverse type I spectra. In the side chain oxidized metabolites with the carbamate intact (11-VI) it is possible that the 4’hydroxyphenyl or the sulfoxide groups may act as weak nucleophilic ligands at the P450 heme iron. The present study again emphasises that benzimidazoles are effective MFO inhibitors. However, the principal finding is that the metabolites of the anthelmintics as well as the parent drugs themselves are likely to be associated with imparied oxidation capacity. There have been numerous reports of P450 inhibition produced by metabolites of drugs and other foreign compounds. For example, the ethylenic and acetylenic steroids are relatively noninhibitory toward P450 but are converted to destructive species during mixed-function oxidation (41-43). Dihydropyridines related to nifedipine also inactivate specific P45Os (44,451, and thionosulfur agents, such as parathion, elicit P450 destruction (46) and heme-protein adduct formation (47) during the P450-mediated covalent attachment of sulfur. Alkylamines (48-50) and (methy1enedioxy)phenylsynergists (2423,511are also relatively inert chemicals that are activated to metabolite intermediates that form quasi-covalent complexes with the P450 heme. Such processes of P450 inactivation and metabolite intermediate complexation are essentially irreversible and are associated with prolonged inhibition of the MFO system. The observation that fenbendazole metabolites are more inhibitory than the parent drug toward MFO
Fenbendazole Metabolism and P450 Inhibition
activities must, however, be dissociated from the effects of inactivation or complexation of P450. The fenbendazole metabolites are more inhibitory than the parent drug, but no evidence for irreversible inhibition of P450 was found when fenbendazole was preincubated with hepatic microsomes (not shown). One feature that distinguishes the fenbendazole derivatives from irreversible inhibitors is that the P450s involved in the formation of a particular metabolite were not preferentially susceptible to inhibition. That is, these inhibitory metabolites apparently leave the active site of the P450 that produces them. Thus, the pharmacokinetic consequences from inhibition of P450 by fenbendazole metabolites are likely to be short term and less specific than those due to mechanism-based inhibitors. Like mechanism-based inhibition, it would be expected that the significance of drug interactions due to fenbendazole metabolism would be dependent on the activity of the P450(s)involved in the process.
Acknowledgment. This work was supported by a grant from the National Health and Medical Research Council of Australia. We are grateful to Dr. E. Lacey, McMaster Laboratory, CSIRO, for generously providing some of the compounds used in this study.
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Quantitative Structure-Activity Analysis of Acetylcholinesterase Inhibition by Oxono and Thiono Analogues of Organophosphorus Compounds Donald M. Maxwell* and Karen M. Brecht United States Army Medical Research, Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425 Received June 27, 1991
A comparison of the bimolecular rate constants (hi) for inhibition of electric eel acetylcholinesterase (AChE) by the oxono (i.e., P=O) and thiono (i.e., P=S) analogues of parathion, methylparathion, leptophos, fonofos, sarin, and soman revealed that the oxono/thiono ratios of ki values varied from 14 for soman to 1240 for parathion. Analysis of the relative importance of the dissociation equilibrium constant and the phosphorylation rate constant in producing this variation in kivalues indicated that the oxono analogues had phosphorylation rate constant values that varied in a narrow range from 8- to 14-fold greater than their thiono counterparts, while the oxono/thiono ratios for dissociation constants varied widely from 1for soman to 82 for fonofos. The lower affmities of thiono analogues for AChE probably resulted from differences in the hydrophobic binding of oxono and thiono analogues to the active site of AChE, inasmuch as the hydrophobicities (i.e., octanol/water partition coefficients) of thiono organophosphorus compounds were much greater than the hydrophobicities of their oxono analogues. Quantitative structure-activity analysis indicated that the hydrophobic effects of oxono and thiono moieties correlated with log ki for AChE inhibition to a greater extent (r2 = 0.79) than their electronic effects (r2I0.48). These observations suggest that the differences in hydrophobicity of oxono and thiono analogues of organophosphorus compounds may be as important as their electronic differences in determining their effectiveness as AChE inhibitors. Introduction The poor reactivity of phosphothioates and phosphonothioaks for acetylchoheskrase (AChE)’in to their oxono analogues has been well documented (1-3).
* Author to whom correspondence should be addressed.
The low reactivity of thiono organophosphorus compounds for mammalian AChE provides a safety factor for agricultural of pesticides, such as or while the rapid metabolic oxidation of these thiono (i-e., Abbreviations: AChE, acetylcholinesterase;Pr’, isopropyl; Pin, pinacolyl.
This article not subject to U.S. Copyright. Published 1992 by the American Chemical Society
