Drug Metabolism and Disposition Fast Forward
First published on January 27, 2006; DOI: 10.1124/dmd.105.008060
0090-9556/06/3404-702-708$20.00
DMD 34:702-708, 2006
INVESTIGATION OF DRUG-DRUG INTERACTION POTENTIAL OF BORTEZOMIB IN VIVO IN FEMALE SPRAGUE-DAWLEY RATS AND IN VITRO IN HUMAN LIVER MICROSOMES
Chuang Lu,
Richard Gallegos,
Ping Li,
Cindy Q. Xia,
Sandeepraj Pusalkar,
Vinita Uttamsingh,
Darrell Nix,
Gerald T. Miwa, and
Liang-Shang Gan
Drug Metabolism and Pharmacokinetics, Drug Safety and Disposition, Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts
(Received October 28, 2005;
Accepted January 25, 2006)
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Abstract
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Bortezomib (Velcade, PS-341), a dipeptidyl boronic acid, is a first-in-class proteasome inhibitor approved in 2003 for the treatment of multiple myeloma. In a preclinical toxicology study, bortezomib-treated rats resulted in liver enlargement (35%). Ex vivo analyses of the liver samples showed an 18% decrease in cytochrome P450 (P450) content, a 60% increase in palmitoyl coenzyme A ß-oxidation activity, and a 41 and 23% decrease in CYP3A protein expression and activity, respectively. Furthermore, liver samples of bortezomib-treated rats had little change in CYP2B and CYP4A protein levels and activities. To address the likelihood of clinical drug-drug interactions, the P450 inhibition potential of bortezomib and its major deboronated metabolites M1 and M2 and their dealkylated metabolites M3 and M4 was evaluated in human liver microsomes for the major P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4/5. Bortezomib, M1, and M2 were found to be mild inhibitors of CYP2C19 (IC50 
18.0, 10.0, and 13.2 µM, respectively), and M1 was also a mild inhibitor of CYP2C9 (IC50 11.5 µM). However, bortezomib, M1, M2, M3, and M4 did not inhibit other P450s (IC50 values > 30 µM). There also was no time-dependent inhibition of CYP3A4/5 by bortezomib or its major metabolites. Based on these results, no major P450-mediated clinical drug-drug interactions are anticipated for bortezomib or its major metabolites. To our knowledge, this is the first report on P450-mediated drug-drug interaction potential of proteasome inhibitors or boronic acid containing therapeutics.
The approval of bortezomib (Velcade, PS-341) by the U.S. Food and Drug Administration for treatment of multiple myeloma made it the first drug in a new class of medicines called proteasome inhibitors. The proteasome is an enzyme complex found in the nucleus and cytoplasm of all cells in the body. It degrades intracellular proteins, such as I
B and p53, through the ubiquitin proteasome pathway (Ciechanover, 1994
) and regulates cell growth, apoptosis, and cell adhesion. In tumor cells, the blockage of degradation of IB by proteasome inhibitors makes the inflammatory nuclear factor B remain in an inactive form, thus enhancing tumor cell apoptosis (Palombella et al., 1998; Berenson et al., 2001; Garg and Aggarwal, 2002). In addition, proteasome inhibitors block the degradation of the tumor suppressor protein p53. When cells undergo radiation or chemotherapy, the p53 expressed in normal cells allows the arrest of cell proliferation and permits the repair of damaged DNA. In contrast, tumor cells express mutated forms of p53, which hinders cell cycle arrest and the ability to repair damaged DNA (Kuerbitz et al., 1992). Thus proteasome inhibitors help normal cells to recover from DNA damage while allowing tumor cells to undergo apoptosis (Adams, 2001; Chauhan et al., 2005).
Bortezomib, a dipeptidyl boronic acid (Fig. 1; Wu et al., 2000), is a potent, selective, and reversible inhibitor of the proteasome in mammalian cells (Adams, 2001; Richardson et al., 2003; Chauhan et al., 2005). Bortezomib is approved for intravenous administration of 1.3 mg/m2 and has a maximum plasma concentration (Cmax) of 150 nM. In multiple myeloma patients, bortezomib has a mean elimination half-life ranging from 9 to 15 h. The boronic acid group was found to be essential for activity, and the metabolites are not pharmacologically active. Like many other cancer drugs, bortezomib is a cytotoxic agent with a narrow therapeutic index. Thus, it is important to understand the metabolism of bortezomib and the consequences of inhibition or induction of that metabolism. Furthermore, for cancer patients on multiple drug therapy, adverse effects may arise if bortezomib affects the enzymes metabolizing these coadministered drugs. Therefore, understanding the enzymes that affect the metabolism of bortezomib and the enzymes that are affected by bortezomib would help to predict possible drug-drug interactions.
Bortezomib was found to be primarily metabolized by CYP3A4 and CYP2C19 (Pekol et al., 2005; Uttamsingh et al., 2005). In this study, the effect of bortezomib on P450 enzymes was evaluated ex vivo in Sprague-Dawley rats. Rats were selected as the ex vivo model because liver enlargement was observed in preclinical toxicology studies in rats but not in other species, such as monkeys. The microsomal activities and protein levels of major inducible P450s (2B1/2, 3A1/2, and 4A1/3) and peroxisomal palmitoyl coenzyme A (CoA) oxidation activity in rat liver were determined. The P450 inhibitory potential of bortezomib and its major metabolites M1, M2, M3, and M4 on the major P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4/5 as well as the time-dependent CYP3A4/5 inhibition potential were evaluated in human liver microsomes.
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Materials and Methods
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Bortezomib was obtained from the Process Chemistry Department at Millennium Pharmaceuticals, Inc. Major bortezomib metabolites M1, M2, M3, and M4 (Fig. 1) were synthesized by Albany Molecular Research (Albany, NY). Testosterone, lauric acid, 12-hydroxylauric acid, 10-hydroxydecanoic acid, 16ß-hydroxytestosterone, 6ß-hydroxytestosterone, 6ß-hydroxydianabol, sucrose, imidazole, ß-nicotinamide adenine dinucleotide, flavin adenine dinucleotide, Triton X-100, Tween 20, dithiothreitol, coenzyme A, palmitoyl CoA, sodium dithionite, midazolam, NADPH, phenacetin, tolbutamide, dextromethorphan, furafylline, sulfaphenazole, quinidine, ketoconazole, troleandomycin, and horseradish peroxidase-conjugated rat anti-rabbit and anti-goat polyclonal secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO). S-Mephenytoin was purchased from Biomol, Inc. (Plymouth Meeting, PA). Human liver microsomes, naive rat liver microsomes, phenobarbital-induced or dexamethasone-induced rat liver microsomes, and rabbit anti-rat CYP3A polyclonal antibody were purchased from Xenotech, LLC (Lenesa, KS). Goat anti-rat CYP2B and CYP4A polyclonal antibodies and rat CYP2B1 and CYP3A1 Supersomes were purchased from BD Gentest (Woburn, MA). [14C]Palmitoyl CoA and ECL plus Western blotting detection reagents were purchased from Amersham (Piscataway, NJ). NuPage Novex Bis-Tris gels and an electrophoresis reagent kit were purchased from Invitrogen (Carlsbad, CA).
Female Sprague-Dawley rats (8 weeks old with body weights 200 g) were dosed intravenously with 0.15 mg/kg bortezomib or the vehicle control (mannitol saline, 1.5 ml) on days 1, 4, 8, and 12 at Charles River Laboratories (Worcester, MA). This single dose was the highest dose that could be administered to rats without causing any adverse effects, such as neuropathy. Twenty-four hours after the last dose, the animals were sacrificed, and livers were removed, frozen in liquid nitrogen, and stored at –80°C. Microsome- and peroxisome-enriched fractions were prepared in-house following published protocols (Small et al., 1985; Guengerich, 1989). In the peroxisome preparation, the liver homogenates were first centrifuged at 6000g for 10 min to remove mitochondria and other heavy cellular fragments. Then a 30-min centrifugation at 30,000g was applied to collect the peroxisome-enriched fractions. Microsome- and peroxisome-enriched fractions prepared from the livers of female rats treated with 200 mg/kg clofibric acid in 2.5 ml of Mazola oil in-house for 4 days were used as positive controls in the CYP4A1 and peroxisomal activity assays, respectively. The total P450 content in microsomes was determined using the method described by Omura and Sato (1964). Protein concentrations in microsomes or peroxisomal fractions were determined using the BCA Protein Assay kit (Pierce, Rockford, IL).
Testosterone And Lauric Acid Oxidation Assays. Rat liver microsomes (0.25 mg/ml), in triplicate, were prewarmed for 5 min at 37°C in 0.1 M potassium phosphate buffer, pH 7.4. The microsomes were then incubated with testosterone (200 µM) or lauric acid (100 µM) with 2 mM NADPH and 3 mM MgCl2 for 30 min at 37°C. The reactions were stopped by the addition of equal volume of acetonitrile containing 1 µM internal standards (6ß-hydroxydianabol and 10-hydroxydecanoic acid for the testosterone and lauric acid assays, respectively). After placing the sample on ice for 30 min, the precipitated proteins were removed via a 10-min centrifugation at 3000g. Metabolites 16ß- and 6ß-hydroxytestosterone and 12-hydroxylauric acid were analyzed using an Applied Biosystems API 4000 LC/MS/MS system (Applied Biosystems, Foster City, CA) and a Phenomenex Synergi C18 column (75 x 4.6 mm; Phenomenex, Torrance, CA). Metabolite separation was achieved with a gradient consisting of 0.1% formic acid/water (mobile phase A) and 0.1% formic acid/acetonitrile (mobile phase B) at a flow rate of 1.0 ml/min; specifically, 5% of mobile phase B was applied for 0.5 min after injection and increased linearly to 95% B from 0.5 to 3.5 min. Mobile phase B was held at 90% from 3.5 to 3.6 min, and the column was re-equilibrated to 5% B from 3.6 to 5.0 min. Positive and negative ion spray modes were applied to 6ß-hydroxytestosterone, 16ß-hydroxytestosterone, and 12-hydroxylauric acid, respectively. The following precursor/product ion pairs were monitored: 305.3: 269.1 (6ß-hydroxytestosterone), 305.3:97.1 (16ß-hydroxytestosterone), 215.2: 169.0 (12-hydroxylauric acid), 187.1:140.9 (10-hydroxydecanoic), and 317.0: 281.0 (6ß-hydroxydianabol). Enzyme catalytic activities were calculated based on external standard curves of the analytes prepared in the same matrix.
[14C]Palmitoyl CoA Oxidation Assay. Palmitoyl CoA ß-oxidation is an assay widely used to determine peroxisomal activity. Palmitoyl CoA is an acid-insoluble substrate; however, its metabolite, acetyl CoA, is acid-soluble. By incubating a [14C]-labeled palmitoyl CoA with peroxisomes and then stopping the reaction with acid, the metabolite formation rate can be measured by counting the radioactivity in the supernatants. Reaction mixtures were prepared with 50 mM Tris-HCl, pH 8.0, 0.2 mM ß-nicotinamide adenine dinucleotide, 0.01 mM flavin adenine dinucleotide, 0.0075% bovine serum albumin, 0.01% Triton X-100, 1 mM dithiothreitol, 0.1 mM CoA, 0.01 mM palmitoyl CoA, and 20 nCi/ml [14C]palmitoyl CoA. Aliquots (10 µl) of peroxisomes (0.25 mg/ml in sucrose imidazole buffer) were incubated in triplicate with 500 µl of the reaction mixture solution for 10 min in a 37°C water bath. The reactions were stopped by adding 250 µl of ice-cold 6% perchloric acid, and the samples were kept on ice for 1 h before being centrifuged at 14,000g for 10 min. Supernatants (500 µl) were transferred into scintillation vials containing 5 ml of Ultima Flo M cocktail (Perkin Elmer, Boston, MA), and the total radioactivity was counted using a Beckman LS6500 scintillation counter (Beckman Coulter, Inc., Fullerton, CA). An identical control set of samples was incubated on ice (Lazarow, 1981). Specific activity (dpm per picomoles) of [14C]palmitoyl CoA was determined by counting 100 µl of the reaction mixture. Because of the limitation of our laboratory setup, for the concern of safety, potassium cyanide, an inhibitor of residual mitochondrial activity, was not used in this experiment. Thus, minor contribution of mitochondria to the palmitoyl CoA oxidation activity is possible. The palmitoyl CoA oxidation activity was calculated using the following formula: Activity (pmol/min/mg protein) = (dpm of incubation sample–dpm of control samples) x 1.5/incubation time (min)/protein amount (mg)/specific activity of reaction mixture (dpm/pmol).
Immunoblotting (Western Blot) Assays for CYP2B1/2, 3A1/2, and 4A1/3 Proteins. Liver microsomes from vehicle- and bortezomib-treated rats, P450-induced rats, naive rats, and Supersomes were diluted in deionized water to 0.5 mg/ml, 0.05 mg/ml, and 10 pmol/ml, respectively. The diluted proteins (100 µl) were incubated with 20 µl of NuPage reducing agent (10x), 50 µl of NuPage SDS sample buffer (4x), and 30 µl of deionized water for 10 min at 70°C and then kept on ice for at least 5 min. Aliquots (20 µl) of the reduced proteins were loaded on NuPage Novex Bis-Tris gels. Electrophoresis was applied at a 60-V constant for 4 h. The proteins were then transferred from the gels to polyvinylidene difluoride membranes by applying a 25-V constant for 1.5 h. The membranes were blocked in 5% nonfat dry milk/PBST for 1 h before being incubated with primary antibodies (1:1000 diluted in 5% nonfat dry milk/PBST) overnight at 4°C. After washing three times in PBST, the membranes were incubated with the secondary antibodies for 1 h, followed by three washes in PBST and once in Tris-buffered saline to remove the excess second antibodies. ECL plus Western blotting detection reagents were then used to detect proteins, and the chemiluminescent signals were captured on Kodak Biomax films (Rochester, NY). Images were quantitatively analyzed using the NIH Image software (National Institutes of Health, Bethesda, MD).
Microsomal P450 Inhibition Assay. Serial dilutions of bortezomib, M1, M2, M3, and M4 solutions (triplicate in 0.1 M potassium phosphate buffer, pH 7.4) were prepared in a 96-well plate. Pooled human liver microsomes (0.5 mg/ml) and P450 substrates (30 µM phenacetin, 150 µM tolbutamide, 100 µM S-mephenytoin, 8 µM dextromethorphan, 50 µM testosterone, and 5 µM midazolam) were added, and the samples were preincubated for 5 min at 37°C. The reactions were initiated by the addition of prewarmed NADPH (2 mM) and MgCl2 (3 mM) solution. After 15-min incubation at 37°C, the reactions were quenched with 100 µl of acetonitrile containing 1 µM antipyrine (internal standard). The samples were centrifuged at 800g for 10 min, and the supernatants were transferred to another 96-well plate for LC/MS/MS analyses of the metabolite formation of the probe substrates. The selective CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 inhibitors furafylline (0–10 µM), sulfaphenazole (0–10 µM), omeprazole (0–100 µM), quinidine (0–10 µM), and ketoconazole (0–10 µM), respectively, were included in the experiments as positive controls. The IC50 values were determined using 12-point inhibition curves for bortezomib, M1, M2, M3, and M4 (0, 0.000508, 0.00152, 0.00457, 0.0137, 0.0412, 0.123, 0.370, 1.11, 3.33, 10, and 30 µM). Data were analyzed using Prism software (GraphPad, San Diego, CA).
CYP3A4/5 Time-Dependent Assay. In 96-well plates, bortezomib, M1, M2, M3, and M4 (0, 1, 10, and 30 µM final concentrations) were preincubated with 2.5 mg/ml human liver microsomal protein in the presence or absence of 2 mM NADPH/3 mM MgCl2 in 100 µl of 0.1 M potassium phosphate buffer, pH 7.4. The reactions (often referred as preincubation) were initiated with the addition of microsomes. The residual activities after 0-, 15-, and 30-min preincubation were measured in secondary incubations by transferring 15 µlof the incubation mixture solutions into another 96-well plate containing 135 µl of testosterone and NADPH/MgCl2 in 0.1 M potassium phosphate buffer, pH 7.4. The final concentrations of testosterone, NADPH, and MgCl2 were 200 µM, 2 mM, and 3 mM, respectively. After a 10-min incubation at 37°C, reactions were terminated by the addition of 100 µl of acetonitrile containing 1 µM antipyrine (internal standard) and chilled at 4°C for 30 min. The samples were centrifuged at 800g for 10 min. The supernatants were analyzed using LC/MS/MS for the 6ß-hydroxytestosterone formation. Troleandomycin (10 µM) was included as a positive control. The results were processed following a method described by Kitz and Wilson (1962).
Statistical Analysis. Data were processed using Microsoft Excel (Microsoft, Redwood, WA) to calculate means and standard deviations. Student's t test with 95% confidence (p < 0.05) was performed on data from different treatment groups to determine any significant differences between the means of the treatment groups.
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Results
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Rat Ex Vivo Liver Enzyme Characterization Total P450 content in rat liver microsomes. Body weights, liver weights, and P450 contents were measured. Results in Table 1 show that bortezomib-treated rats had an average liver weight of 13.8 g compared with the vehicle control group (10.2 g). This represented a 35% increase in liver weight, whereas the body weights were barely changed (291 versus 302 g in the bortezomib-treated rats). On the other hand, microsomes prepared from bortezomib-treated rats had an average total P450 content of 0.503 nmol/mg protein compared with 0.614 nmol/mg protein for the vehicle control group, resulting in an 18% decrease in P450 content.
CYP2B1/2, CYP3A1/2, and CYP4A1/3 activities and protein expression levels in rat liver microsomes. Testosterone 16ß-hydroxylation, 6ß-hydroxylation, and lauric acid 12-hydroxylation are mediated by CYP2B1/2, CYP3A1/2, and CYP4A1/3 in rats, respectively. The rates of formation of these metabolites were measured to evaluate the effect of bortezomib on rat P450s. Results from this study showed little difference in CYP2B1/2 activities between the control and the bortezomib-treated rats (97.5 ± 9.5 versus 96.9 ± 12.8 pmol/min/mg protein; Table 1). In addition, quantitative image analysis of CYP2B1/2 Western blots (Table 1 and Fig. 2) showed that the microsomes from the vehicle control group had an average band intensity of 1.49 ± 0.08 compared with 1.23 ± 0.16 of the bortezomib-treated group. This 17% decrease in CYP2B1/2 protein expression level was statistically insignificant (p < 0.05). Regarding CYP3A1/2, whereas the vehicle control rats had an average activity of 217 ± 92 pmol/min/mg protein, the bortezomib-treated rats showed an average activity of 167 ± 65 pmol/min/mg protein (Table 1). This represented a 23% decrease in CYP3A1/2 activity, but it was found not statistically significant (p < 0.05). The Western blot analysis (Table 1 and Fig. 2) showed that the control group had an average CYP3A1/2 band intensity of 1.64 ± 0.04 compared with that of 0.97 ± 0.34 in the bortezomib-treated group. This was a 41% decrease in 3A1/2 protein expression level, which could be attributed to the bortezomib treatment in rats. The CYP4A1/3 activity in the vehicle control rats was 1286 ± 236 pmol/min/mg protein, whereas the bortezomib-treated rats had an average specific activity of 1418 ± 379 pmol/min/mg protein (Table 1). In the Western blots (Table 1 and Fig. 2), the vehicle control rats showed an average CYP4A1/3 band intensity of 2.24 ± 0.58 and the bortezomib-treated rats showed an average band intensity of 1.93 ± 0.38. This 10% increase in CYP4A1/3 activity and 14% decrease in the CYP4A1/3 protein expression in bortezomib-treated rats was statistically insignificant (Student's t test, p < 0.05).
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FIG. 2. Electrophoresis and immunoblot (Western blot) of P450 proteins in control and bortezomib-treated female Sprague-Dawley rats. In all three gels, lanes 1, 2, and 3: vehicle control rat microsomes (5 µg); lanes 4, 5, and 6: bortezomib-treated rat microsomes (5 µg); and lanes 7 and 8: Xenotech female and male rat microsomes (5 µg). In CYP2B gel, lane 9: phenobarbital-treated rat microsomes (0.5 µg); lane 10: Gentest CYP2B1 Supersomes (0.1 pmol). In CYP3A gel, lane 9: dexamethasone-treated rat microsomes (0.5 µg); lane 10: Gentest CYP3A1 Supersomes (0.1 pmol). In CYP4A gel, lane 9: clofibric acid-treated rat microsomes (0.5 µg).
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Fatty acid ß-oxidation activity in rat liver peroxisomal fractions. Results from this study showed that vehicle control rats had an average peroxisomal activity of 1.57 ± 0.43 nmol/min/mg protein, whereas the bortezomib-treated rats had an average activity of 2.57 ± 1.04 nmol/min/mg protein (Table 1), representing a 60% increase in peroxisomal activity in the bortezomib-treated rats. Female rats treated in-house with clofibric acid, the prototypical CYP4A and peroxisomal acyl-CoA oxidase inducer, were included in this study as a positive control. An average peroxisomal activity of 21.7 nmol/min/mg protein was observed in the clofibric acid-treated rats compared with the oil-diet vehicle control rats, which had a specific activity of 2.42 nmol/min/mg protein—a 9-fold increase. It has been suggested that rats treated with an oil diet have slightly higher (50% increase) basal peroxisomal activity compared with rats fed a standard laboratory diet (Huber et al., 1997). In our study, it was indeed the oil-diet control rats that had higher peroxisomal activity than the chow-diet control rats.
P450 Inhibition Studies in Human Liver Microsomes. The P450 inhibition potential of bortezomib and its major metabolites M1–M4 was studied in human liver microsomes using drug-like probe substrates. The results presented in Table 2 and Figs. 3, 4, 5 indicated that bortezomib was a mild inhibitor of CYP2C19 (IC50 18 µM), its deboronated metabolite M1 was a mild inhibitor of CYP2C9 and CYP2C19 (IC50 11.5 and 10.0 µM, respectively), and another deboronated metabolite M2 was a mild inhibitor of CYP2C19 (IC50 13.2 µM). Furthermore, bortezomib, M1, and M2 showed little inhibition of the other P450 isozymes (IC50 values >30 µM). In addition, the secondary dealkylation metabolites M3 and M4 did not inhibit P450 isozymes 1A2, 2C9, 2C19, 2D6, or 3A4/5 (IC50 values >30 µM). IC50 values of the known selective P450 inhibitors are also presented in Table 2 for comparison. Because the CYP3A substrates used in this study (testosterone and midazolam) could not distinguish the CYP3A4 from the CYP3A5, the CYP3A activities in human liver microsomes were expressed as CYP3A4/5, although CYP3A4 is the major CYP3A isoform in liver.
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TABLE 2 Inhibition of P450 activities by bortezomib, major metabolites M1–M4, and known P450 inhibitors in human liver microsomes
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FIG. 5. Selective inhibition of CYP3A4/5 substrate testosterone metabolism (not midazolam) by bortezomib metabolites M1 and M2.
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In the time-dependent inhibition study, the percentage of CYP3A4/5 activities remaining in various preincubation groups (in the presence of different concentrations of bortezomib or its metabolites) were plotted (in log scale) versus the preincubation time. A negative slope of the plot (Kobs) would usually indicate enzyme inactivation. If enzymes became inactivated during preincubation, the slope (Kobs) would also increase as the test compound concentration increased. Plotting of our data resulted in a set of flat regression lines (slopes 0), suggesting that there was no time-dependent or concentration-dependent loss of CYP3A4/5 activities. This indicated that bortezomib and its metabolites M1, M2, M3, and M4 were unlikely to cause time-dependent inactivation of CYP3A4/5 (data not shown).
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Discussion
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CYP2B1/2, 3A1/2, and 4A1/3 are the major inducible P450 isozymes in rat liver. Peroxisomal acyl-CoA oxidase (ß-oxidation of fatty acids) is also an inducible enzyme, which often attributes to liver enlargement. In our preclinical in vivo toxicity study, an increase in liver weight (35%) after a repeat dose of bortezomib was observed in both male and female rats. Thus, the primary goal for this study was to investigate whether the liver enlargement was caused by the induction of CYP2B1/2, 3A1/2, 4A1/3, and/or peroxisomal acyl-CoA oxidase. Female rats were chosen because their lower hepatic CYP3A expression makes them a more sensitive gender for studying P450 induction, especially for CYP3A. Compared with the vehicle control group, the bortezomib-treated rats showed no changes in CYP2B1/2 and 4A1/3 activities and protein levels. However, a 23% decrease in CYP3A1/2 activity was observed, but it was not statistically significant. This could be due to the variability of the data and the small size of the data set. A decrease in the P450 content and the CYP3A1/2 protein expression and an increase in the palmitoyl CoA ß-oxidation activity were also observed in bortezomib-treated rats. If these changes were normalized to the total liver weight by assuming that the yields of microsomes or peroxisomes (milligram of protein per gram of liver) were similar across all of the treatment groups, the changes in the total P450 content would become insignificant, whereas the increase of palmitoyl CoA ß-oxidation activity would become apparent. Because the microsomal content may vary upon drug treatment in rats (Carlile et al., 1999) and the recovery of microsome preparation was not tracked in this study, our data were not expressed in a per-liver basis. The increase in the palmitoyl CoA ß-oxidation activity may correlate with the increased liver weight in this study. However, compared with the 9-fold induction observed with the positive control compound clofibric acid, the 60% increase by bortezomib is mild. The increase in liver weight may also be attributed to the accumulation of ubiquitinated protein in hepatocytes, because hypertrophy not hyperplasia was observed in these liver samples.
It is known that proteasomes mediate degradation of some P450 isoforms, including 1A2, 2E1, 3A, and 4A, but not 1A1, 2B1/2, or NADPH reductase (Roberts, 1997; Correia et al., 2005). In our study, the CYP2B1/2 activity and the protein expression level in rats were indeed not affected by the proteasome inhibitor bortezomib. CYP1A and 2E were not included in the present study because they are relatively minor enzymes compared with the CYP2B and 3A in rats. It is interesting to note that the CYP4A1/3 activities and protein expression levels in our study were unchanged, whereas Roberts (1997) showed that proteasome inhibitors could enhance CYP4A degradation in rat hepatocyte cultures. Studies by Zangar et al. (2003) also showed that rat hepatocytes treated with proteasome inhibitors resulted in a decrease of CYP3A protein and mRNA levels. However, this is contrary to the CYP3A-ubiqutin-proteasome pathway, thus suggesting that CYP3A may be degraded by multiple pathways or proteasomes could be responsible for the degradation of proteins that suppress CYP3A expression. In our study, we also observed a 41% decrease in CYP3A protein expression and a 23% decrease in CYP3A activity, although the latter is not statistically significant. Although a change in bortezomib exposure has not been observed in clinical trials or preclinical studies, the effect of bortezomib on P450 activities, mRNA levels, transcriptional factors and protein expression, and ubiquitination of P450s is being conducted in human cultured hepatocytes to investigate whether the ubiquitination pathway could mediate P450 degradation and de novo synthesis in humans.
CYP1A2, 2C9, 2C19, 2D6, and 3A4 are the five major P450 isozymes in the human liver responsible for metabolism of >90% of drugs (Kwon, 2001). Understanding the P450 inhibition potential of a new chemical entity to these five major P450s helps in predicting possible drug-drug interactions in clinical trials (Bjornsson et al., 2003). To our knowledge, the P450 inhibition potential has not been reported for any boronic acid-containing molecules or proteasome inhibitors. In this study, up to 30 µM bortezomib and its major metabolites were tested. These concentrations are much higher compared with their plasma Cmax, which is approximately 150 nM (unpublished data). Besides being a mild inhibitor of CYP2C19, bortezomib also inhibited CYP3A4/5 and 2C9 to a lesser extent (approximately 20% at 30 µM; Fig. 3). One of its deboronated metabolites (M1) was a mild inhibitor of both CYP2C9 and CYP2C19, but its diastereoisomer (M2) was only a mild inhibitor of CYP2C19 and showed little inhibition toward CYP2C9. M1 and M2 also showed mild inhibition of CYP3A4/5 as determined by the testosterone 6ß-hydroxylation (approximately 40% at 30 µM), whereas midazolam 1'-hydroxylation was unaffected (Fig. 5). It is generally acknowledged that CYP3A4 is an enzyme with multiple active sites (Kenworthy et al., 1999), and midazolam and testosterone are substrates that bind selectively to two different sites of CYP3A4/5 enzymes (Tucker et al., 2001). Therefore, M1 and M2 could interact with one CYP3A4/5 site but not the other. In metabolite profiling and P450-phenotyping studies, bortezomib was found to be primarily metabolized by CYP3A4/5 and 2C19 (Pekol et al., 2005; Uttamsingh et al., 2005). This explains why bortezomib, a substrate of CYP3A4/5 and 2C19, is also a mild reversible inhibitor of these two enzymes. The secondary dealkylated metabolites M3 and M4 did not inhibit any of the five P450s, suggesting that the boron containing chain of bortezomib is responsible for the mild P450 inhibition.
Reactive metabolites are often generated by P450-mediated bioactivation of drugs. These reactive metabolites could covalently bind to microsomes or form a metabolite intermediate complex to inactivate microsomal activity and thus result in an increase in exposure of that compound. Bortezomib was found not to cause exposure change in our preclinical animal species; thus, in the present study, time-dependent inhibition was focused on CYP3A4/5—the primary P450 metabolizing bortezomib as well as over 50% of drugs on the market (Kwon, 2001; Uttamsingh et al., 2005). In our study, the observation of no time-dependent inhibition of CYP3A4/5 by bortezomib and its major metabolites suggests that there were no reactive metabolites being generated that would interact with CYP3A4/5. Consistent with that, bortezomib was found not to cause exposure change in preclinical species.
In conclusion, there was no change in microsomal CYP2B1/2- and 4A1/3-specific activities and protein expression in bortezomib-treated rats; however, there were slight decreases in the CYP3A1/2 protein expression (possibly activity) as well as in the total P450 content. A mild liver weight increase was observed in this study which may be attributed to ubiquitin/protein accumulation in hepatocytes. The liver weight increase seemed to have direct correlation with the increase of peroxisomal activity. In human liver, microsome bortezomib and its deboronated metabolites M1 and M2 were mild inhibitors of CYP2C19, whereas the dealkylated metabolites M3 and M4 did not show any inhibition to P450s. Inhibition of CYP3A4/5 by bortezomib was apparent only at concentrations above 30 µM, which is much higher than the average plasma maximum concentration (Cmax) observed in humans (150 nM). No time-dependent inhibition of CYP3A4/5 by bortezomib or its major metabolites M1, M2, M3, and M4 was observed in human liver microsomes. Based on these results, no major bortezomib- or its metabolite-mediated clinical drug-drug interactions are anticipated.
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Acknowledgments
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We thank Kym Cardoza of the department of Comparative Medicine for treatment of rats with clofibric acid and harvest the liver samples. We thank Sue Colson of Drug Safety and Disposition for proofreading this manuscript.
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Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.105.008060.
ABBREVIATIONS: P450, cytochrome P450; CoA, coenzyme A; LC/MS/MS, liquid chromatography/tandem mass spectrometry; PBST, phosphate-buffered saline with 0.1% Tween 20.
Address correspondence to: Chuang Lu, Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139. E-mail: chuang.lu{at}mpi.com
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