Introduction

Abstract Introduction Results Discussion References

(+)-S-145¹, is a potent and selective thromboxane A₂ receptor antagonist, and its calcium salt, S-1452, is currently being developed as an effective agent for treating asthma (1, 2). In previous studies on the in vivo metabolism of S-1452, the plasma and urine of animals administered S-1452 were found to contain products in which two (DH-bisnor or bisnor) or four carbons (tetranor) had been removed from the carboxyl side chain, along with hydroxylated metabolites of (+)-S-145 and its carbon-shortened products (3-7). Recently, we reported that chain shortening was catalyzed by the peroxisomal -oxidation system and hydroxylation by NADPH-dependent microsomal monooxygenase systems (8). The hydroxylated metabolites of tetranor at the C-5 and C-6 positions of the bicyclo ring (5-OH- and 6-OH-tetranor) were identified as the major metabolites in the urine of rats and humans (3, 7). In humans, S-1452 was mainly excreted into urine as hydroxylated metabolites, which primarily consisted of 5-OH- and 6-OH-tetranor and their conjugates (7). 6-OH-Tetranor and its glucuronides excreted in urine account for more than half of the total metabolites after oral administration of S-1452 in humans (7). Determination of the pathway for the formation of OH-tetranors revealed that the carboxyl side chain was first -oxidized, followed by hydroxylation of the bicyclo ring (8).

Recently, major P450 enzymes in human liver have been expressed in Escherichia coli at high levels and purified from membranes (9-12). Immunoinhibition studies can be done to study in vitro drug metabolism with human liver microsomes, as well as those prepared from rats.

In the present study, we investigated the hydroxylation of (+)-S-145 and its -oxidized metabolites (bisnor, DH-bisnor, and tetranor) with liver microsomes from rats and humans to identify P450s playing major roles in these reactions.

Materials and Methods

Chemicals. (+)-S-145 and its metabolites (fig. 1) were synthesized in our laboratories (13). HEPES, PB, DEX, and DLPC were purchased from Wako Pure Chemicals (Osaka, Japan). N,O-Bis-(trimethysilyl)-trifluoroacetoamide + 1% trimethylchlorosilane was obtained from Pierce Chemicals Co. (Rockford, IL). N-Nitrosomethyl urea was obtained from Maruwaka Kagaku Co. (Osaka, Japan) and used for generation of diazomethane. DOPC, PS (bovine brain), and PC (bovine liver) were purchased from Sigma Chemical Co (St. Louis, MO). All other chemicals were of the highest quality commercially available.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Metabolic scheme for the formation of hydroxylated (+)-S-145, bisnor, DH-bisnor, and tetranor.

Animals and Preparation of Liver Microsomes. Rat liver microsomes were prepared from male Wistar rats (weighing 200-250 g, 8-9 weeks old) obtained from Japan SLC, Inc. (Shizuoka, Japan). In some experiments, rats were treated by intraperitoneal administration of PB (40 mg/kg/day in saline) or DEX (80 mg/kg/day in sesame oil), once per day for 3 days. Livers were homogenized with 4 volumes of 50 mM Tris-HCl buffer (pH 7.4) containing 0.15 M KCl and then centrifuged at 9,000g for 20 min. Supernatants were collected and centrifuged at 105,000g for 60 min. Pellets were resuspended in 50 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose and 1.0 mM EDTA.

Purification of P450 and Related Enzymes and Preparation of Antibodies. P4502C11 and cytochrome b5 were purified from liver microsomes of untreated male rats, NADPH-P450 reductase from PB-treated male rats, P4502C6 from PB-treated female rats, and P4503A2 from DEX-treated male ratsall isolated as described previously (14-16). The apparent specific contents or specific activity of all enzymes from the rat were as follows: P4502C11, 16 nmol/mg protein; P4502C6, 15 nmol/mg protein; P4503A2, 18 nmol/mg protein; NADPH-P450 reductase, 31 µmol cytochrome c reduced/min/mg protein; and cytochrome b₅, 54 nmol/mg protein.

Microsomal Incubations. (+)-S-145 or -oxidized metabolites (50 µM) were incubated at 37°C for 10 min with 250 µg (rat) or 200 µg protein (human) of liver microsomes and 2 mM NADPH in 0.5 ml of incubation buffer consisting of 120 mM KCl, 4.7 mM NaCl, 1.2 mM KH₂PO₄, 7.5 mM MgSO₄, 25 mM NaHCO₃, and 10 mM HEPES (pH 7.4). Reactions were stopped by adding an equal volume of acetone. After centrifugation at 2,000g for 10 min, supernatants were collected, and subsequently 0.25 M phosphate buffer (pH 2.3) and ethyl acetate were added to extract the metabolites. Products were determined by gas chromatography as described previously (7, 8).

Immunoinhibition Studies. Each anti-P450 IgG (2.5, 5, or 10 mg protein/nmol P450) was preincubated with microsomes for 30 min at room temperature before starting the reactions. Anti-P4503A1/2 (5 mg protein/nmol P450) and P4502C11 IgGs (2.5 mg protein/nmol P450) significantly and selectively inhibited testosterone 6-hydroxylation (>80%) and 16-hydroxylation (>80%) activities by male rat liver microsomes, respectively. Furthermore, anti-P4502C6 IgG (5 mg protein/nmol P450) did not affect testosterone 16-hydroxylation activity. Anti-P4502C6 and P4502C11 antibodies did not affect testosterone 6-hydroxylation activity under the conditions described previously. Anti-human P4501A2, P4502C9/10, P4502E1, and P4503A4 IgGs (10 mg protein/nmol P450), respectively, inhibited 7-ethoxyresorufin O-deethylation (>65%), tolbutamide methylhydroxylation (>95%), aniline hydroxylation (>80%), and testosterone 6-hydroxylation activities (>90%) by human liver microsomes (results not shown).

Reconstitution of Recombinant Human P450 Enzymes. Hydroxylation activities toward (+)-S-145, bisnor, and tetranor by recombinant human P450 were also determined in reconstituted systems. A standard incubation system contained 100 pmol of purified P450, 200 pmol of rat liver NADPH-P450 reductase, 100 pmol of rat liver cytochrome b₅, and 30 µg of phospholipid mixture (DLPC:PS = 1:1, w/w for P4501A2, P4502C10, and P4502E1; DOPC:PS:PC = 1:1:1, w/w/w and 200 µg sodium cholate for P4503A4) in 0.5 ml of 50 mM Tris-HCl buffer (pH 7.4) containing 150 mM KCl and 10 mM MgCl₂. Metabolites that formed after incubation at 37°C for 15 min were measured as described previously.

Other Assays. Protein concentrations were estimated using a bicinchoninic acid protein assay kit (Pierce Chemical Co.), with bovine serum albumin as standard. 7-Ethoxyresorufin O-deethylation (18), tolbutamide methylhydroxylation (19), aniline hydroxylation (20), and testosterone hydroxylation (21) activities were determined with substrate concentrations as follows: 7-ethoxyresorufin, 5 µM; tolbutamide, 2.5 mM; aniline, 2 mM; and testosterone, 0.5 mM.

Statistical Analysis. Significant differences were determined by Student's t test. A value of p < 0.05 was considered to be statistically significant.

	Results

Abstract Introduction Results Discussion References

Effects of P450 Inducers on Hydroxylation of (+)-S-145 and Its -oxidized Metabolites by Rat Liver Microsomes. PB treatment increased all hydroxylation activities toward (+)-S-145 and its -oxidized metabolites by rat liver microsomes (table 1). 5-Hydroxylation activities were affected more than those of 6-hydroxylation. DEX exhibited more induction of hydroxylation activities for (+)-S-145 and its -oxidized metabolites, except the 6-hydroxylation of (+)-S-145. On the contrary, -naphthoflavone and clofibric acid did not increase the rates of these reactions (data not shown).

Immunoinhibition Studies with Rat Liver Microsomes. Inhibitory effects of antibodies against P4503A1/2, P4502C6, and P4502C11 on hydroxylation activities toward (+)-S-145 and its -oxidized metabolites by untreated rat liver microsomes are summarized in table 2. Anti-P4503A1/2 IgG completely inhibited 5-hydroxylation of bisnor and 6-hydroxylation of tetranor. This IgG also inhibited 5-hydroxylation of (+)-S-145 and DH-bisnor and 6-hydroxylation of bisnor by ~50%. Anti-P4502C6 IgG inhibited 6-hydroxylation of (+)-S-145, bisnor, and DH-bisnor and also slightly affected 5-hydroxylation of bisnor and 6-hydroxylation of tetranor. Anti-P4502C11 IgG inhibited 5-hydroxylation of DH-bisnor and 6-hydroxylation of bisnor and weakly inhibited 5-hydroxylation of tetranor and 6-hydroxylation of DH-bisnor and tetranor. Only tetranor 5-hydroxylation activity was not strongly inhibited by any anti-P450 IgG used in these experiments.

Hydroxylation of (+)-S-145 and Its -oxidized Metabolites by Human Liver Microsomes and Recombinant Human P450s in Reconstituted Systems. Because DH-bisnor was not detected as a -oxidized metabolite of S-1452 in early clinical studies, we examined the formation of hydroxylated metabolites of (+)-S-145, bisnor, and tetranor by human liver microsomes (table 3). 6-Hydroxylation was more prominent than 5-hydroxylation of (+)-S-145 and bisnor, whereas 5-hydroxylation was faster than 6-hydroxylation in the case of tetranor.

Immunoinhibition Studies with Human Liver Microsomes. The effects of antibodies raised against four recombinant human P450s (P4501A2, P4502C9/10, P4502E1, and P4503A4) on the hydroxylation of (+)-S-145, bisnor, and tetranor were investigated (table 4). Anti-P4501A2 IgG only slightly inhibited tetranor 6-hydroxylation, whereas anti-P4502E1 IgG did not inhibit any catalytic activities. Anti-P4502C9/10 IgG strongly inhibited tetranor 5-hydroxylation and slightly inhibited (+)-S-145 5- and 6-hydroxylation and tetranor 6-hydroxylation. Anti-P4503A4 IgG strongly inhibited (+)-S-145 and bisnor 5-hydroxylation, and tetranor 6-hydroxylation, and slightly inhibited tetranor 5-hydroxylation and bisnor 6-hydroxylation.

	Discussion

Abstract Introduction Results Discussion References

In the metabolism of (+)-S-145, 5-OH- and 6-OH-tetranor and their conjugates are the major metabolites eliminated in urine and feces (3, 7), and hydroxylation on the bicyclo ring has as significant a role as -oxidation of the carboxyl side chain in rats and humans. Hydroxylation activities toward (+)-S-145 and its -oxidized metabolites were previously shown to be dependent on NADPH and liver microsomes (8), and P450 enzymes were suggested to be involved in these reactions.

Treatment of rats with some P450 inducers increased the hydroxylation activities toward (+)-S-145 and its -oxidized metabolites by rat liver microsomes (table 1). DEX, a P4503A inducer, extensively induced (+)-S-145 5-hydroxylation and bisnor, DH-bisnor, and tetranor 5- and 6-hydroxylation, suggesting that P4503A enzymes may have significant roles in these reactions. Anti-P4503A1/2 IgG inhibited (+)-S-145, bisnor and DH-bisnor 5-hydroxylation, and bisnor and tetranor 6-hydroxylation by liver microsomes prepared from untreated rats (table 2). P4503A2, a constitutive form of P450 (15), seems to have certain roles in these reactions in untreated rats. In liver microsomes of DEX-treated rats, anti-P4503A1/2 IgG significantly inhibited all of the activities induced by DEX treatment (results not shown), which indicated that P4503A1, a DEX-inducible form (22, 23), shares a major role in these reactions with P4503A2 in DEX-treated rat liver microsomes. In the case of tetranor 5-hydroxylation, it was not clear which P450 enzyme mainly catalyzes this reaction in untreated rat liver microsomes, but P4503A1 may have a significant role in DEX-treated rats, as judged by the 31-fold increase in activity on DEX treatment (table 1) and significant inhibition by anti-P4503A1/2 IgG (results not shown).

PB treatment also increased these hydroxylation activities in rat liver microsomes, but to a lesser extent than DEX (table 1); thus suggesting that the contribution of P4502B subfamily enzymes may be minor. (+)-S-145 6-hydroxylation activity was not induced by DEX treatment, but was induced slightly by PB treatment. This result indicated that a constitutive and PB-inducible P450 form, but not P4503A2, was responsible for this reaction. Similar results were observed for progesterone 21-hydroxylation and sulfamethoxazole N-hydroxylation in the rat, and the P450 form responsible for these reactions was identified as P4502C6 (24, 25), which is known to be a constitutive and somewhat PB-inducible form (26). As expected, antibodies raised against P4502C6 strongly inhibited (+)-S-145 6-hydroxylation, in addition to bisnor and DH-bisnor 6-hydroxylation (table 2).

(+)-S-145, bisnor, and DH-bisnor 5-hydroxylation and tetranor 6-hydroxylation were mainly catalyzed by P4503A2, and (+)-S-145 and DH-bisnor 6-hydroxylation were mainly catalyzed by P4502C6 in untreated rat liver microsomes. Both P4503A2 and P4502C6 were also partially involved in bisnor 6-hydroxylation. Furthermore, P4502C11, the major form of P450 in male rat liver microsomes (14, 22), was partially involved in DH-bisnor 5-hydroxylation and bisnor 6-hydroxylation (table 2).

(+)-S-145 and its -oxidized metabolites were also hydroxylated by human liver microsomes (table 3). When the balance of 5- and 6-hydroxylation was compared with that observed in vivo, discrepancy was found only in the case of tetranor hydroxylation (i.e. 5-hydroxylation predominated over 6-hydroxylation in vitro, but 6-OH-tetranor is predominantly excreted in vivo) (7). Kinetic analysis (conducted in vitro) showed that K_M and Vmax values of tetranor hydroxylation by human liver microsomes (pooled from five subjects) were 116 µM and 333 pmol/min/mg protein for 5-hydroxylation, and 66 µM and 65 pmol/min/mg protein for 6-hydroxylation, respectively (results not shown). Thus, Vmax/K_M value for 5-hydroxylation (2.9 µl/min/mg protein) of tetranor was higher than that for 6-hydroxylation (1.0 µl/min/mg protein) of tetranor, suggesting that tetranor 5-hydroxylation might be expected to predominate over to 6-hydroxylation in vivo. Further investigations will be required to clarify this apparent discrepancy between in vitro and in vivo findings.

P4502C9/10 and P4503A4 oxidized (+)-S-145 and its -oxidized metabolites in reconstituted systems (table 3). P4502C9/10 catalyzed (+)-S-145 6-hydroxylation and tetranor 5-hydroxylation (table 3), and these activities were inhibited by anti-P4502C9/10 IgG in human liver microsomes (table 4). Purified P4503A4 catalyzed (+)-S-145 and bisnor 5-hydroxylation and tetranor 6-hydroxylation (table 3), and these activities were inhibited by anti-P4503A4 IgG in human liver microsomes (table 4). These results clearly show that P4502C9/10 and P4503A4 have significant roles in these reactions (table 5). Since >60% of the total excreted metabolites in humans are 6-OH-tetranor and its glucuronides (7), it is noteworthy that P4503A4, which is important in the metabolism of a wide range of clinically important compounds (27), was mainly involved in this reaction of S-1452.

On the other hand, antibodies raised against these two P450 forms only slightly inhibited bisnor 6-hydroxylation (table 4). Reconstituted P4503A4 showed catalytic activity, whereas reconstituted P4502C9/10 showed none (table 3). The possibility remains that other P450 forms might be responsible for this reaction, although omeprazole (50 µM) failed to indicate the involvement of P4502C19 and anti-P4502D6 sera did not affect the activity (results not shown). At this time, we hypothesize that bisnor 6-hydroxylation is catalyzed by P4503A and P4502C enzymes, as in the rat (table 5).

Comparison of the P450 forms involved in each hydroxylation pathway demonstrated that similar P450 subfamily enzymes play major roles in rats and humans (table 5). Although we could not identify the P450 forms responsible for tetranor 5-hydroxylation in untreated rats, the similarity of P450 forms in the oxidative metabolism of S-1452 between rats and humans may suggest that some P4502C enzymes might be involved in this reaction.

Similarity between rats and humans was also observed in the switching of regioselectivity at the bicyclo ring. Except for tetranor, P4503A forms primarily play major roles in 5-hydroxylation reactions and P4502C forms prefer 6-hydroxylation reactions, both in rats and humans. On the other hand, in the hydroxylation of tetranor, each P450 form switched the position of hydroxylation with respect to each other. Selectivity might be affected by the length of the carboxyl side chain. It is of interest that such a small difference between tetranor and other drugs might alter orientation of the substrate.