Introduction

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N-[2(R)-Hydroxy-1(S)-indanyl]-5-[2(S)-(1,1-dimethylethylaminocarbonyl)-4-[(furo[2,3-b]pyridin-5-yl)methyl]piperazin-1-yl]-4(S)-hydroxy-2(R)-phenylmethylpentanamide (L-754,394)¹, is a potent and highly selective inhibitor of HIV-1 protease (Fig. 1). Previous in vivo and in vitro studies in rats showed that L-754,394 is a potent suicide inhibitor of rat liver microsomal cytochrome P-450 (P-450) enzymes (Chiba et al., 1995; Lin et al., 1995; Sahalisahly et al., 1996). There have been several publications documenting the effect of chronic treatment by HIV protease inhibitors, which resulted in in vitro, time-dependent P-450 inhibition and alterations in P-450 catalytic functions in vivo (Kumar et al., 1996; Grubb et al., 1997). Although in vitro studies indicated that L-754,394 destroys both CYP3A2- and 2C11-mediated activities in a time-dependent manner in rat liver microsomes, the long-term effect on the expression of the two male-predominant isoforms by this suicide inhibitor was unknown. The purpose of this paper is to examine how chronic dosing of L-754,394 would affect the regulation of the P-450s it destroys. The consequences of this chronic treatment on the catalytic function, expression, and mRNA levels of the major P-450 isoforms in male adult rats are investigated.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Chemical structure of HIV protease inhibitor L-754,394.

	Materials and Methods

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Chemicals. L-754,394 was synthesized at Merck Research Laboratories (West Point, PA). Hydroxylated metabolites of testosterone (2, 2, 6, 7, 16, and 16-hydroxytestosterone) were obtained from Steraloids (Wilton, NH). Testosterone, tolbutamide, thyroxine (T₄), and triiodothyronine (T₃) were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were of analytical grade.

Animal Treatments and Microsome Preparation. Adult male Sprague-Dawley rats (Harlan-Sprague-Dawley, Inc., Frederick, MD) were 10 to 12 weeks old at the beginning of treatment and weighed 230 to 250 g. The sulfate salt of L-754,394, dissolved in 0.05 M citric acid, was administered p.o. with a gavage needle every morning at a dose of either 1, 5, or 20 mg/kg/day for 7 consecutive days. Rats were sacrificed on day 2 (i.e., 24 h after the first dose, but before the second dose), day 4, day 6, and day 8. Blood was collected and serum samples were stored at 20°C until use. Livers were quickly excised, perfused with ice-cold 1.15% KCl (w/v), and homogenized for the preparation of hepatic microsomes by differential ultracentrifugation (Omura and Sato, 1964). Microsomes were washed and the final pellets were resuspended in 0.15 M Tris-HCl buffer (pH 7.4) and stored at 70°C until use. Microsomal protein was measured by the method of Lowry et al. (1951) with BSA as the standard. Quantitation of P-450 content was carried out according to the method of Omura and Sato (1964).

Antibodies. Polyclonal anti-rat CYP 2C11 and 3A1/2 goat antiserum, both obtained from Gentest Corp. (Woburn, MA), were used for immunoblotting studies. For inhibition studies, preliminary experiments revealed that polyclonal anti-rat CYP 2C11 goat antiserum (Gentest Corp., Woburn, MA) and anti-rat CYP 3A1 rabbit antibody (Human Biologics, Inc., Phoenix, AZ) showed a specific inhibitory effect on 2-/16-hydroxylase and 2-/6-hydroxylase activities of testosterone, respectively, in rat liver microsomes.

Immunoinhibition Study. Immunoinhibition of testosterone 2- and 16-hydroxylase activities by anti-CYP 2C11 goat antiserum (Gentest Corp., Woburn, MA) and that of 2- and 6-hydroxylase activities by anti-CYP 3A1 rabbit antibody (Human Biologics, Inc., Phoenix, AZ) were estimated as described herein after a 30-min preincubation at room temperature. A sufficient amount of anti-CYP 2C11 antiserum (60 µl/mg microsomal protein) or anti-CYP 3A1 antibody (10 mg/nmol P-450) for maximum inhibition, which had been determined in preliminary experiments, was added to the microsomes and the reaction was initiated by the addition of an NADPH-generating-system as described herein. The same amount of control goat serum or preimmune rabbit IgG was added to control samples.

Gel Electrophoresis and Western Immunoblotting. SDS electrophoresis was conducted in 10% polyacrylamide gels according to the procedure of Laemmli (1970). Proteins were transferred onto a nitrocellulose membrane after electrophoresis (Towbin et al., 1979). The proteins of CYP 3A1/2 and CYP 2C11 were probed by goat antiserum (Gentest Corp., Woburn, MA) directed against the corresponding isoform(s). The nitrocellulose membrane was washed, and then incubated with anti-goat IgG rabbit antibody (Sigma Chemical Co., St. Louis, MO). After washing the membrane, anti-rabbit IgG goat antibody conjugated with a horseradish peroxidase (Sigma Chemical Co., St. Louis, MO) was applied. The appropriate amount of 3',3-diaminobenzidine was used as a substrate for the quantitation. The density was measured by a chromatoscanner (BioRad GS670; BioRad, Richmond, CA) to determine the relative intensity within the linear condition established by a microsomal standard supplied with antibodies.

Testosterone Hydroxylase Assay. P-450 isoform-selective hydroxylase assays of testosterone were performed using an HPLC method. An incubation mixture (250 µl), containing an NADPH-generating system (20 mM glucose 6-phosphate; 4 I.U./ml glucose 6-phosphate dehydrogenase; 20 mM MgCl₂), 1 mg/ml microsomes, and 500 µM testosterone, was preincubated for 5 min at 37°C. Metabolism was initiated by the addition of NADPH at a final concentration of 1 mM. The reaction was conducted at 37°C for 10 min and stopped by the addition of ice-cold ethyl acetate (1.0 ml). An internal standard (50 µl of 50 µM tolbutamide) then was added to each sample, followed by an additional 1.5 ml ethyl acetate to extract metabolites. The resulting mixture was vortex-mixed and the ethyl acetate layer was separated by centrifugation, followed by an evaporation to dryness under nitrogen. The residue was reconstituted in 150 µl of 20% methanol in water. An HPLC assay was performed on a Supelco LC-18DB column (3 µm, 4.6 mm × 15 cm) with a Spectra-Physics HPLC system (Fremont, CA). The HPLC method involved the following gradient system using 30% methanol in water as mobile phase A and 10% acetonitrile in methanol as mobile phase B: From 0 to 3.5 min, the ratio of mobile phase A to B remained at 83/17%. Mobile phase A was then decreased to 70% at 10 min. Mobile phase A remained at 70% until 25 min, after which it was further decreased to 50% at 30 min. Mobile phase A then was returned to 83% at 35 min. Both mobile phases were adjusted to pH 4.5 with glacial acetic acid and the flow rate was set at 1.0 ml/min. The detector was set at 240 nm. Under these conditions, the retention times were 13, 13.6, 16.2, 18.4, 21.9, 23.4, and 17 min for 6-, 7-, 16-, 16-, 2-, 2-hydroxytestosterone, and internal standard (tolbutamide), respectively. Calibration curves were linear (r = 0.99) for all metabolites up to 10 nmol/injection with a good reproducibility (c.v. values <15%).

RNA Quantitation by Slot-Blotting Analysis. Gene-specific oligonucleotide probes for CYP 2C11, CYP 3A2, and -tubulin, as presented elsewhere (LeBlanc et al., 1992), were synthesized on a Cyclone DNA synthesizer (Biosearch Inc., San Rafael, CA). Oligonucleotides were 5'-end labeled with [-³²P]ATP by T4 polynucleotide kinase and purified on a NENSORB 20 cartridge (DuPont-NEN, Boston, MA). Total RNA was prepared from portions of freshly frozen liver by RNeasy kit (Qiagen, Chatsworth, CA). The samples of 10 µg of total RNA were denatured by heating for 15 min at 65°C with equal volume of sample treatment buffer (67% formamide, 8.6% formaldehyde, 1.33X SSC) and then diluted with 20X SSC (3 M NaCl, 300 mM sodium citrate) to 0.5, 1, 2, and 6 µg. The diluted RNA from individual livers was loaded onto a GeneScreen membrane using a slot-blot apparatus (BioRad Bio-Dot SF Apparatus; BioRad) and immobilized by UV cross-linking. Membranes were hybridized according to the multiple-sandwich filter paper hybridization method (Wu et al., 1995) in 10 ml of hybridization buffer containing 1 pmol of 5'-end labeled probe. The hybridization conditions for each probe were conducted as described (LeBlanc et al., 1992). Membranes were washed at room temperature with 2X SSC for 5 min twice, at 45°C with a mixture of 2X SSC and 1% SDS for 30 min twice, and lastly at room temperature with a mixture of 0.1X SSC and 1% SDS for 30 min twice. The washed membranes were thoroughly drained and loaded onto a PhosphorImager plate (Molecular Dynamics, Sunnyvale, CA) to quantify the intensities of the bands.

Hormone Measurements. Total serum estradiol, testosterone, T₄, and T₃ were separately measured by solid phase radioimmunoassay using commercially available kits (Diagnostic Products Corporation, Los Angeles, CA).

Statistics. Differences between means from control (untreated) and treated microsomal studies were detected using Student's t test with results presented as mean ± S.D.

	Results

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General Effect of L-754,394. A significant increase in liver weight (as a percentage of body weight) was observed over the 7-day period in rats treated with L-754,394. The liver weights (normalized by body weight) in animals dosed with 1, 5, and 20 mg/kg L-754,394 on day 8 were 3.71 ± 0.28, 3.77 ± 0.27, and 4.83 ± 0.17 (mean ± S.D., n = 4-6), respectively. The values for the three doses were significantly (p < .01) larger than those of control rats (3.22 ± 0.05, mean ± S.D., n = 4). There were no significant changes in body weight during the treatment.

Effect of L-754,394 on Testosterone Hydroxylase Activities. Testosterone metabolism has been widely accepted as a marker to measure P-450 isoform activities. Testosterone is hydroxylated at the 2/6 and 2/16 positions mainly by CYP 3A1/2 and CYP 2C11, respectively, and these activities were measured in rat liver microsomes during the treatment with L-754,394. Testosterone 2- and 6-hydroxylase activities were elevated approximately 2-fold by treatment with 20 mg/kg L-754,394; both activities reached maximum values on day 2, declining thereafter to basal levels by day 8 (Fig. 2, top). In contrast, 2- and 16-hydroxylase activities of testosterone showed a marked decrease to 40% of control values on days 2 and 4 during the same treatment (Fig. 2, bottom). The reduced activities, however, returned to basal levels afterward and exceeded control values by 20 to 40% on day 8. Notably, as shown in Fig. 2, the changes in CYP3A and 2C11 activities mirrored each other over the course of treatment. Lower doses of L-754,394 (1 and 5 mg/kg) had little or no effect on these hydroxylase activities (data not shown). Additionally, CYP2A1-mediated 7- and CYP2B1/2-mediated 16-hydroxylase activities of testosterone showed increasing, then decreasing, trends with 20 mg/kg L-754,394 (Fig. 3); the activities increased to 200% of control values on day 6, followed by a decrease to control levels on day 8. 

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Comparison of marker catalytic activities and immunodetected P-450s in microsomes from untreated rats and rats treated with 20 mg/kg L-754,394 over the 7-day period. Both 2- (filled columns)/6 (open columns)-hydroxylase activities and immunodetected CYP 3A protein () are plotted in the top panel. Similarly, 2- (filled columns)/16 (open columns)-hydroxylase activities and immunodetected CYP 2C11 protein () are shown in the bottom panel. Data for 2-hydroxylase activities were multiplied by a factor of 10 for the sake of comparison. Significant differences between data from control and treated microsomes are shown by asterisks (*p < .05, **p < .01).

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Testosterone 7- and 16-hydroxylase activities in microsomes from untreated rats and rats treated with 20 mg/kg L-754,394 over the 7-day period. Rats were sacrificed on day 2 (24 h after the first dose, but before the second dose), day 4, day 6, and day 8. Control microsomes were obtained from untreated rats. Data represent the mean ± S.D. of determinations obtained from four to five rats. Values are expressed relative to those of the control. Control values were: 0.0689 ± 0.0069 nmol/min/mg protein for testosterone 7-hydroxylase activity and 0.00854 ± 0.00146 nmol/min/mg protein for testosterone 16-hydroxylase activity. Significant differences between data from control and treated microsomes are shown by asterisks (**p < .01).

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Testosterone hydroxylase activities in the presence (open column) or absence (shaded column) of anti-rat CYP 2C11 antiserum for the inhibition of 2- and 16-hydroxylation (left) and of anti-rat CYP 3A1 antibody for the inhibition of 2- and 6-hydroxylation (right). A sufficient amount of anti-CYP 2C11 antiserum (60 µl/mg microsomal protein) or anti-CYP 3A1 antibody (10 mg/nmol P-450) for maximum inhibition was preincubated with microsomes before testosterone metabolism, whereas the same amount of control goat serum or preimmune rabbit IgG was added to control samples.

Immunoquantitation of CYP3A1/2 and CYP2C11. To examine whether altered protein levels were the cause for the changes in the catalytic activities observed during treatment, immunoquantitation experiments using antibodies for CYP 3A or CYP 2C11 were conducted. As shown in Fig. 2, the protein levels for CYP 3A and CYP 2C11 were consistent with the respective catalytic activities. The content of CYP 3A showed an upward (to 180% of control)-and-downward (to basal level) alteration with a 20-mg/kg dose of L-754,394 (Fig. 2). For CYP 2C11, the same dose of L-754,394 decreased the CYP 2C11 content (to 50% of control) during the first 4 days, followed by a rebound to exceed the basal level on day 8 (Fig. 2).

Slot-Blot Analysis for mRNA Levels Encoding CYP3A2 and CYP2C11. In addition, slot-blot analyses were carried out to determine the effect of chronic treatment with L-754,394 on the level of mRNA encoding CYP 3A2 and CYP 2C11 (Fig. 5). Although there was a large interindividual variation among animals, mRNA levels for CYP3A2 and CYP2C11 showed the same trends as the protein expression of the respective isoform. The integrity of the sample preparation was not a factor for the observed variation as shown by the relatively consistent mRNA levels of -tubulin, a constitutively expressed gene. These results suggest that the observed alteration is due to an effect at the pretranslational level.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Alteration in liver mRNA expression during the treatment with 20 mg/kg L-754,394. Quantitation was carried out by slot-blot analysis using CYP3A2 and CYP2C11 gene-specific oligonucleotides. Rat -tubulin mRNA levels obtained from the same livers are also shown to indicate consistency of RNA loading. Each line represents the mean of data obtained from three to four rats.

Serum Levels of Steroid and Thyroid Hormones. To examine the mechanism responsible for the effect of L-754,394 on P-450 regulation, we have measured serum levels of steroid hormones (estradiol and testosterone) and thyroid hormones (T₄ and T₃) in the rats treated with L-754,394 (Table 1). Estradiol was not detectable before and after treatment and no discernible change was observed for testosterone. In contrast, thyroid hormones showed a marked decrease on days 2 and 4, followed by a return to basal levels on day 8. 

	Discussion

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It has been demonstrated that the half-lives of rat P-450 de novo syntheses are relatively short (12-20 h) (Shiraki and Guengerich, 1984). Therefore, any chemical reagent, such as L-754,394, which directly affects P-450 synthesis and/or degradation, may alter the steady-state level. Chronic dosing of L-754,394 indeed resulted in an immediate response in the regulation of major P-450 isoforms. Interestingly, although in vitro, both CYP 2C11 and CYP 3A were destroyed by L-754,394, chronic dosing resulted in a striking and surprising response that was isoform-dependent; mirror-image profiles were observed for the catalytic activities of the two predominantly male isoforms, CYP 3A and CYP 2C11 (Fig. 2). These distinct catalytic profiles were confirmed by Western immunoblotting (Fig. 2) and immunoinhibition (Fig. 4) studies. Furthermore, slot-blot mRNA analysis using cDNA oligo-probes specific for the constitutive CYP 3A2 and CYP 2C11 revealed that L-754,394 is likely acting at a pretranslational step in P-450 regulation resulting in mRNA profiles (Fig. 5) paralleling the respective isoform protein contents and catalytic activities (Fig. 2).

A number of physiological and nutritional factors, as well as in vivo treatment with foreign chemicals, have been documented to affect the relative abundance of male-dominant constitutive P-450 isoforms in rats. One of the most important factors that may inversely regulate CYP3A (CYP2A1 and CYP2B1/2) and CYP2C11 appears to be the thyroid hormone. T₄ and its potent metabolite, T₃, have been demonstrated to suppress the expressions and/or catalytic functions of CYP 3A (Waxman et al., 1990), CYP2A1 (Arlotto and Parkinson, 1989), and CYP2B1/2 (Yamazoe et al., 1989). Furthermore, the full expression of CYP2C11 requires a pulsatile secretion of growth hormone (Mode et al., 1989; Shapiro et al., 1989; Waxman et al., 1991). Thyroid hormone is also known to be a potent regulator for growth hormone secretion at the pituitary in rats (Korytko and Cuttler, 1997). Therefore, a circulating thyroid hormone could also be a positive regulator for the CYP2C11 expression level by maintaining the growth hormone secretion. We speculate that the inversely altered expressions of CYP 3A and 2C11 by L-754,394 is due to its effect on the homeostasis of thyroid and possibly growth hormones. Although the changes of thyroid hormone levels were small (~35%), a significant decrease was observed in the serum levels of both T₄ and T₃ 2 and 4 days after the onset of treatment with a 20-mg/kg dose of L-754,394 (Table 1). This decrease in thyroid hormone levels may partially relieve its suppressive effect on CYP 3A, resulting in an increase in CYP 3A, as well as a decrease in the expression of CYP 2C11 which could be a consequence of the decreased growth hormone secretion secondary to the reduced thyroid hormone at the pituitary. A positive correlation was reported between an individual CYP2C11 activity and the plasma T₄ concentration in cyclophosphamide-treated rats (Kraner et al., 1996). Recently, it was also demonstrated that the reduction of thyroid hormone level in the plasma was responsible for the alterations in CYP2C11 (30% decrease) and 3A2 (30% increase) after the administration of retinol to rats (Badger et al., 1998).

Steroid hormones, such as testosterone and estradiol, are among such factors that play a major role in regulating the gender-specific expression of P-450 isoforms (Dannan et al., 1986; Waxman, 1988). Circulating testosterone is known to be required for maintaining the full expressions of CYP2C11 and CYP3A in adult male rats (Gustafsson and Stenberg, 1974; Waxman et al., 1985). Thus, decrease of this androgen level results in the suppression of both constitutive, male-predominant CYP 2C11- and 3A-catalyzed metabolism. Unlike the anticancer compounds that caused decreased expression of both isoforms (LeBlanc and Waxman, 1988, 1990), L-754,394 treatment resulted in an increase in CYP 3A and simultaneous decrease in CYP 2C11 expressions. Examination of serum testosterone levels showed no discernible trends and serum estradiol levels remained below the detection limit (Table 1). These observations precluded the possibility that perturbation of these steroids is involved in the P-450 modulation by L-754,394.

In summary, the protein contents of two predominantly male P-450 isoforms and their associated catalytic activities were found to be inversely affected by L-754,394 during chronic treatment in rats. Unlike in vitro, in which L-754,394 destroys both CYP 3A and CYP 2C11, chronic treatment affected the isoforms in opposite directions. Analyses of CYP 2C11 and CYP 3A2 mRNA indicated that L-754,394 is acting at a pretranslational step in P-450 regulation. Although this effect likely involves the perturbation of the thyroid hormone homeostasis, the mechanism remains to be examined.