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Mar 4, 1998 - Grapefruit juice has been found to significantly increase oral bioavailability of several drugs metabolized by cytochrome P450 3A4 (P450...
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Chem. Res. Toxicol. 1998, 11, 252-259

Articles Inactivation of Cytochrome P450 3A4 by Bergamottin, a Component of Grapefruit Juice Kan He,† Krishna R. Iyer,‡ Roger N. Hayes,‡ Michael W. Sinz,‡ Thomas F. Woolf,‡ and Paul F. Hollenberg*,† Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, and Department of Pharmacokinetics and Drug Metabolism, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, Michigan 48105 Received October 27, 1997

Grapefruit juice has been found to significantly increase oral bioavailability of several drugs metabolized by cytochrome P450 3A4 (P450 3A4) through inhibiting the enzymatic activity and decreasing the content of intestinal P450 3A4. HPLC/MS/MS and HPLC/UV analyses of ethyl acetate extracts from grapefruit juice revealed the presence of several furanocoumarins of which bergamottin (BG) is the major one. BG was shown to inactivate P450 3A4 in a reconstituted system consisting of purified P450 3A4, NADPH-cytochrome P450 reductase, cytochrome b5, and phospholipids. Inactivation was time- and concentration-dependent and required metabolism of BG. The loss of catalytic activity exhibited pseudo-first-order kinetics. The values of kinactivation and KI calculated from the inactivation studies were 0.3 min-1 and 7.7 µM, respectively. While approximately 70% of the erythromycin N-demethylation activity was lost during incubation with BG in the reconstituted system, P450 3A4 retained more than 90% of the heme as determined either by UV-visible spectroscopy or by HPLC. However, approximately 50% of the apoP450 in the BG-inactivated P450 3A4 incubation mixture could not be recovered from a reverse-phase HPLC column when compared with the -NADPH control. The mechanism of the inactivation appears to involve modification of the apoP450 in the active site of the enzyme instead of heme adduct formation or heme fragmentation. These results indicate that BG, the primary furanocoumarin extracted from grapefruit juice, is a mechanismbased inactivator of P450 3A4. BG was also found to inhibit the activities of P450s 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 in human liver microsomes.

Introduction Oral coadministration of grapefruit juice has been demonstrated to significantly increase the oral bioavailability of several clinically used drugs including dihydropyridines (1, 2), cyclosporine A (3), midazolam (4), triazolam (5), terfenadine (6), and ethinylestradiol (7). Since all of these drugs are metabolized primarily by cytochrome P450 3A4 (P450 3A4), the predominant intestinal and hepatic P450 enzymes (8, 9), it has been suggested that the grapefruit juice effect may be due to the inhibition of P450 3A4 activity. More recently, grapefruit juice has been shown to dramatically decrease the immunoreactive P450 3A4 content in enterocytes of human intestines with no change in the content of P450 3A4 mRNA (10). These results suggest that the degradation of P450 3A4 protein may be accelerated by ingestion of grapefruit juice (10). Because suicide inac* Corresponding author address: Department of Pharmacology, University of Michigan, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0632. Tel: 313-764-8166. Fax: 313-763-4450. E-mail: [email protected]. † University of Michigan. ‡ Parke-Davis Pharmaceutical Research.

tivation of rat P450 3A could accelerate degradation of the apoP450 (11), mechanism-based inactivation of P450 3A4 has been suggested to be involved in grapefruit juice effects. To identify the principal components in grapefruit juice responsible for increasing the bioavailability of some drugs, a number of flavonoids found in grapefruit juice, such as naringenin, naringin, quercetin, and kaemferol, have been chosen as possible candidates because they have been shown to competitively inhibit P450 3A4 activity in vitro (12, 13). However, oral administration of these flavonoids does not reproduce the grapefruit juice effects (14, 15). Recently, HPLC purification of a methylene chloride extract of grapefruit juice led to the identification of 6′,7′-dihydroxybergamottin as a component of grapefruit juice which caused inhibition of testosterone 6β-hydroxylase in liver microsomes from dexamethasone-induced rats (16). We have shown that 6′,7′-dihydroxybergamottin causes mechanism-based inactivation of P450 3A4 (17, 18). Since several furanocoumarins have been reported to inactivate P450s (19,

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Inactivation of P450 3A4 by Bergamottin

20), we hypothesized that bergamottin (BG),1 the parent compound of 6′,7′-dihydroxybergamottin, might also be an inactivator of P450 3A4. The results presented here demonstrate that BG is a component in grapefruit juice responsible for the mechanism-based inactivation of human P450 3A4.

Materials and Methods Caution: BG is photoactive and may be carcinogenic in the presence of UV light. It should be considered hazardous and handled carefully. Chemicals. NADPH, L-R-dilauroyl- and L-R-dioleyl-snglycero-3-phosphocholines, phosphatidylserine, catalase, GSH, δ-aminolevulinic acid hydrochloride, testosterone, 6β- and 11βhydroxytestosterone, phenacetin, acetaminophen, 3-acetamidophenol, chlorzoxazone, coumarin, tolbutamide, and R-naphthoflavone were purchased from Sigma Chemical Co. (St. Louis, MO). 7-Hydroxycoumarin (umbelliferone) was obtained from Aldrich (Milwaukee, WI). 4-(Hydroxymethyl)tolbutamide, 6-hydroxychlorzoxazone, 4′-hydroxymephenytoin, racemic bufurolol, and 1′-hydroxybufurolol were obtained from Gentest Corp. (Woburn, MA). Isopropyl β-D-thiogalactoside was purchased from Calbiochem Corp. (La Jolla, CA). (S)-Mephenytoin was a gift from Dr. W. F. Trager (University of Washington, Seattle, WA). BG was purchased from Indofine Chemical Co., Inc. (Somerville, NJ). HPLC/MS/MS Identification of BG and Its Derivatives in Grapefruit Juice. Grapefruit juice or orange juice was made by hand-squeezing halved pink Florida grapefruits or oranges, respectively. The juice was extracted with ethyl acetate, and the dried extract was dissolved in the HPLC buffer for subsequent analysis. HPLC/MS identification of the components was performed by using a Quattro II triple quadrupole mass spectrometer (Micromass, Manchester, U.K.). Sample introduction and ionization were by electrospray ionization (ESI) in the positive ion mode (cone voltage of 30 V). Scan data were acquired under the control of the Micromass Masslynx NT data system (version 2.22). The components of grapefruit juice were separated by HPLC on a C8 column (Zorbax XDB, 5 µm, 2.1 × 150 mm; MAC-MOD, Chadds Ford, PA) eluted with 100 mM acetic acid (A) and acetonitrile (B) by a gradient of 30% B for 5 min and then 30-70% B within 25 min at a flow rate of 200 µL/min. Molecular weight determinations were performed by acquiring mass spectra over a mass range of 100-500 amu at a scan rate of 1.0 s/decade. Determinations of molecular structure were performed by acquiring MS/MS product ion scans at a scan rate of 1.0 s/decade. Collision activation was achieved by using argon at an indicated gas cell pressure of 2.0 × 10-3 Torr and collision energy of 20 eV. Expression of P450 3A4 and Purification of the Expressed Enzyme. A 5′-end-modified P450 3A4 cDNA constructed as described by Gillam et al. (21) and inserted into the pCW vector was obtained from Dr. R. W. Estabrook (University of Texas Southwestern Medical Center, Dallas, TX). The P450 3A4-containing vector was transformed into MV1304 cells. Growth of the transformed Escherichia coli was carried out in modified Terrific Broth, and the expression of P450 3A4 was induced by addition of 1 mM isopropyl β-D-thiogalactoside (21). δ-Aminolevulinic acid (0.5 mM) was added to increase heme synthesis. The membrane fraction was prepared from the bacterial cells by sonication after treatment with lysozyme and subsequently isolated from the bacterial cell homogenate by differential centrifugation. P450 3A4 was purified to homogeneity by the method of Gillam et al. (21). 1 Abbreviations: BG, bergamottin; CID, collision-induced dissociation; ES, electrospray; ESI, electrospray ionization; Hepes, N-(2hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid); kinactivation, the maximal rate constant of inactivation; KI, the concentration required for half-maximal inactivation.

Chem. Res. Toxicol., Vol. 11, No. 4, 1998 253 Isolation of NADPH-Cytochrome P450 Reductase and Cytochrome b5. NADPH-cytochrome P450 reductase and cytochrome b5 were purified by the methods described previously from liver microsomes of phenobarbital-treated Long-Evans rats (22, 23). BG-Mediated Inactivation of P450 3A4 in a Reconstituted System. P450 3A4 (0.5 nmol) was reconstituted with 20 µg of a mixture (1:1:1) of L-R-dilauroyl- and L-R-dioleyl-snglycero-3-phosphocholines and phosphatidylserine, 200 µg of cholic acid, 1 nmol of NADPH reductase, 0.5 nmol of cytochrome b5, 500 U of catalase, 2 µmol of GSH, 30 mM MgCl2, 0.5 mM EDTA, and 20% glycerol in a final volume of 1 mL of 50 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (Hepes) buffer (pH 7.5). Reactions with various concentrations of BG were initiated by addition of 1 mM NADPH and stopped by cooling on ice. The incubations were performed at 37 °C for the time periods indicated. At the end of the incubation, 0.2 mL of the incubation mixture was diluted into 0.8 mL of 50 mM Hepes buffer (pH 7.5) containing 20% glycerol and 0.5 mM EDTA. The spectra were recorded between 330 and 700 nm against the diluting buffer as reference on a DW2-OLIS spectrophotometer in the split beam mode. An aliquot of 0.25 mL was used for the determination of the P450 content by the method of Omura and Sato (24). Additional aliquots were taken for determination of erythromycin N-demethylation or testosterone 6β-hydroxylation activity and HPLC analysis. Determination of Erythromycin N-Demethylation and Testosterone 6β-Hydroxylation Activity. An aliquot (0.05 mL) of the incubation mixture was diluted into 0.95 mL of 50 mM Hepes buffer (pH 7.5) containing 1 mM erythromycin or 200 µM testosterone, 500 U of catalase, 2 µmol of GSH, 30 mM MgCl2, 0.5 mM EDTA, and 20% glycerol in a final volume of 1 mL and then incubated for 10 min at 37 °C. For erythromycin N-demethylation, formaldehyde was determined using the Nash reagent followed by fluorimetric detection on a SLM-AMINCO spectrofluorometer (25). 6β-Hydroxytestosterone was determined by HPLC on a C18 column (Microsorb-MV, 5 µm, 4.6 × 15 cm; Rainin, Woburn, MA) eluted isocratically with a mobile phase of 65% methanol at a flow rate of 1 mL/min, and the eluate was monitored by UV detection at 254 nm. HPLC Analysis of BG-Inactivated P450 3A4. After a 10min incubation of P450 3A4 with 50 µM BG in the reconstituted system as described above, 200 µL of the reaction mixture was directly analyzed on a Poros column (R/H, 4.6 × 100 mm, S/N 195; PerSeptive Biosystems, Framingham, MA) as described previously (26). The eluate was monitored by visible-UV detection at 214, 310, and 405 nm simultaneously. Inhibition of the Activities of Human Liver Microsomal P450 Enzymes by BG. Human liver tissues were obtained from the University of Chicago Distribution Center of LTPADS (Liver Transplant Procurement and Distribution Service, University of Minnesota, Minneapolis, MN), Human Biologics Inc. (Phoenix, AZ), and the International Institute for the Advancement of Science (Exton, PA). The liver microsomes were prepared by differential centrifugation. Phenacetin O-deethylation (27), coumarin 7-hydroxylation (28), tolbutamide hydroxylation (29), (S)-mephenytoin 4′-hydroxylation (30), racemic bufurolol 1′-hydroxylation (31), chlorzoxazone 6-hydroxylation (32), and testosterone 6β-hydroxylation (33) were used to determine the activities of P450s 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4, respectively. Pooled human liver microsomes (N ) 6, 0.1-1 mg of protein/mL) were incubated with BG (1, 10, and 100 µM) in the presence of the corresponding probe substrates, 10 µM phenacetin, 4 µM coumarin, 100 µM tolbutamide, 10 µM bufurolol, 40 µM chlorzoxazone, or 50 µM testosterone, in a final volume of 0.5 mL of 0.1 mM phosphate buffer (pH 7.4) at 37 °C for the appropriate time periods. The reactions were initiated by the addition of 1 mM NADPH and stopped by cooling on ice. (S)-Mephenytoin (50 µM) was incubated with pooled human liver microsomes in a final volume of 0.125 mL in the presence of 0.2, 2, and 20 µM BG, respectively. 4′Hydroxymephenytoin concentration was determined using HPLC/

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Figure 1. Representative reverse-phase HPLC profiles of ethyl acetate extracts of grapefruit juice. The grapefruit juice components were separated on a Zorbax XDB C8 column eluted isocratically with 100 mM acetic acid (A) and acetonitrile (B) using a gradient of 30% B for 5 min and then 30-70% B within 25 min. The eluate was monitored by UV detection at 310 nm (panel A). ES-MS ion chromatograms are shown for the protonated molecules of BG (MH+ of m/z 339; panel B), monooxygenated BG isomers (MH+ of m/z 355; panel C), and dihydroxybergamottin isomers (MH+ of m/z 373; panel D).

He et al.

Figure 2. Typical ES-MS/MS spectrum obtained for BG (MH+: m/z 339). The fragment ion of m/z 203 corresponds to the 5-hydroxypsoralen moiety which subsequently fragments to product ions of m/z 175 and 159 by loss of CO and CO2 groups, respectively. The fragment ion of m/z 137 corresponds to the remaining geran side chain.

MS/MS. Experimental controls consisted of the complete incubation mixture without the addition of BG.

Results Identification of BG and Its Derivatives in Grapefruit Juice. Several components of the ethyl acetate extract of grapefruit juice were separated by HPLC (Figure 1) under the conditions described in Materials and Methods. Electrospray (ES)-MS/MS analysis of the components having absorption at 310 nm revealed that the peak eluted at 26 min (Figure 1) was BG. HPLC retention time and the product ion spectrum (Figure 2) were identical to that of the authentic standard. The predominant fragment ion at m/z 203 corresponds to the 5-hydroxypsoralen moiety which subsequently fragments to give ions of m/z 175 and 159 by loss of CO and CO2, respectively. The fragment ion at m/z 137 corresponds to the remaining side chain. As shown in Figure 1, there appear to be a number of monooxgenated BG products in grapefruit juice. Two of the major ones were further characterized by ES-MS/MS (Figure 3). Similarly, two major dihydroxylated BGs in grapefruit juice were also characterized by ES-MS/MS (Figure 4). BG appeared to be the predominant furanocoumarin in the ethyl acetate extract of grapefruit juice by HPLC/UV determination with a concentration ranging from 12 to 23 µM in grapefruit juice, depending on the

Figure 3. Typical ES-MS/MS spectra of two major monooxygenated BG isomers (MH+: m/z 355). Panel A: MS/MS spectrum of the isomer eluting at 13.3 min. Panel B: MS/MS spectrum of the isomer eluting at 24.1 min (37%). The indicated sites of oxidation are based on the CID behavior of the precursor ion of m/z 355; viz., the product ion of m/z 203 corresponds to the intact 5-hydroxypsoralen moiety, and thus oxidation of the isoprene chain is indicated. Specific sites of oxidation cannot be determined from the MS/MS data.

grapefruit samples. HPLC/MS analysis of the ethyl acetate extracts of orange juice indicated that there is

Inactivation of P450 3A4 by Bergamottin

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Figure 4. Typical ES-MS/MS spectra of two major dihydroxylated BG isomers (MH+: m/z 373). Panel A: MS/MS spectrum of the isomer eluted at 12.9 min. Panel B: MS/MS spectrum of the isomer eluted at 15.9 min. The indicated sites of oxidation are based on the CID behavior; viz., the ion of m/z 203 (panel A) corresponds to the intact 5-hydroxypsoralen moiety, and thus oxidation of the isoprene chain is indicated. Specific sites of oxidation cannot be determined from the MS/MS data. Table 1. BG-Mediated Inactivation of P450 3A4 in a Reconstituted Systema

BG-/NADPH+ BG+/NADPHBG+/NADPH+

P450 (nmol/mL)

erythromycin N-demethylation (nmol/min/nmol)

0.44 0.47 0.27

7.6 7.5 2.2

a P450 3A4 (0.5 nmol/mL) was incubated with 50 µM BG in a reconstituted system at 37 °C for 15 min as described in Materials and Methods. An aliquot (0.05 mL) of incubation mixture was diluted into 0.95 mL of 50 mM Hepes buffer (pH 7.5) for the determination of erythromycin N-demethylation activity. The results are reported as the average of two experiments.

no detectable BG in orange juice (data not shown). In addition, it was found that BG binds to a C18 column so tightly that it could not be eluted from the column with 60% methanol (data not shown), conditions previously used to identify 6′,7′-dihydroxybergamottin in grapefruit juice (16). Inactivation of P450 3A4. As shown in Table 1, incubation of P450 3A4 with BG in the reconstituted system resulted in approximately 70% loss of erythromycin N-demethylation activity in 15 min. Moreover, the P450 contents as measured by the reduced-CO spectrum decreased by approximately 40% after incubation of P450 3A4 with BG in the presence of NADPH for 15 min (Table 1). There was no emergence of a peak at 420 nm or any other absorption peaks replacing that at 450 nm in the range from 400 to 500 nm in the CO-reduced P450 difference spectrum (Figure 5). The maximum absorption of the absolute spectrum of the BG-inactivated P450 3A4 was shifted about 2 nm to a longer wavelength (425 nm) when compared to P450 3A4 in the presence of

NADPH but without BG added. The observation that there was no significant decrease in the absorbance of Soret peak of the modified P450 indicated that there was no heme destruction. Similar results were observed when the -NADPH/+BG sample was used as reference. HPLC Analysis of BG-Inactivated P450 3A4. As shown in Figure 6, the amount of apoP450 3A4 was selectively decreased by about 50% when the sample containing P450 3A4 inactivated by BG was analyzed by reverse-phase HPLC on a Poros column. Nearly 100% of the reductase and cytochrome b5 protein were recovered from the column when compared with the -NADPH controls. Approximately 90% of the heme was recovered from the sample containing BG-inactivated P450 3A4, which was in agreement with the results obtained from the spectral analysis. No modified heme peak could be detected using these HPLC conditions. Time- and Concentration-Dependent Inactivation of P450 by BG. As shown in Figure 7, BGmediated inactivation of P450 3A4 in a reconstituted system was time- and concentration-dependent and required metabolism of BG. The inactivation exhibited pseudo-first-order kinetics. Linear regression analysis of the data in Figure 7 was used to determine the initial rate constants of inactivation (kobs). Double-reciprocal plots of the values of kobs and BG concentrations gave a maximal rate constant (kinactivation) for inactivation of 0.3 min-1 and a concentration of inactivator required for halfmaximal inactivation (KI) of 7.7 µM (34). A concentration-dependent inhibition was also observed for the sample without preincubation. Effect of r-Naphthoflavone on BG-Mediated Inactivation. R-Naphthoflavone has been reported to stimulate the metabolism of several substrates by P450 3A4 (35). Therefore, it was decided to assess whether it would increase the formation of the reactive metabolite of BG and subsequently enhance inactivation of P450 3A4. R-Naphthoflavone did not change the potency of BG-mediated inactivation of P450 3A4. The testosterone 6β-hydroxylation activity of P450 3A4 was inactivated by about 55% when 2 µM BG and R-naphthoflavone were simultaneously incubated in the reconstituted mixture at final concentrations of R-naphthoflavone ranging from 6 to 50 µM (data not shown). Higher concentrations of R-naphthoflavone were not used because of solubility problems. R-Naphthoflavone does not exhibit any stimulatory or cooperative effect on the 6β-hydroxylation of testosterone by P450 3A4 (35). Inhibition of Human Liver Microsomal P450 Enzymes by BG. As shown in Figure 8, the P450 2A6, 2C9, 2D6, 2E1, and 3A4 activities in human liver microsomes were inhibited by more than 50% by 10 µM BG. Approximately 92% of P450 1A2 activity was inhibited by 1 µM BG. Approximately 71% and 100% of P450 2C19 activity was inhibited by 2 and 20 µM BG, respectively.

Discussion Our present results indicate that BG is the furanocoumarin found in the highest concentration in the ethyl acetate extract of grapefruit juice and that it is responsible for the mechanism-based inactivation of P450 3A4. Several monooxygenated or dihydroxylated BG derivatives were also identified in grapefruit juice. The content of dihydroxybergamottins, one of which has previously

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He et al.

Figure 5. Reduced-carbon monoxide difference spectra (left panel) and UV-visible spectra (right panel) of the reconstituted P450 3A4 reaction mixture incubated with BG in the presence of NADPH (s), with BG in the absence of NADPH (‚‚‚), and without BG in the presence of NADPH (- - -), respectively. P450 3A4 (0.5 nmol/mL) was incubated with 50 µM BG in a reconstituted system at 37 °C for 15 min as described in Materials and Methods. Aliquots of 0.25 or 0.2 mL of the incubation mixtures were diluted into 1.75 or 0.8 mL of 50 mM Hepes buffer (pH 7.5) containing 20% glycerol and 0.5 mM EDTA, and the reduced-carbon monoxide P450 difference and UV-visible spectra were recorded respectively as described in Materials and Methods.

Figure 6. HPLC profiles of the P450 3A4 reconstituted system after incubation with 50 µM BG in the presence (s) or absence of NADPH (‚‚‚). The eluate was monitored at 214 and 405 nm (inset). Peaks A, B, C, and D represent short P450-NADPH reductase, P450-NADPH reductase, cytochrome b5, and P450 3A4, respectively. The heme group was eluted at 5.5 min (inset).

been isolated and demonstrated to inhibit testosterone 6β-hydroxylase in rat liver microsomes (16), was determined to be less than 20% of the content of BG in grapefruit juice based on relative UV extinction. Most of the BG derivatives in grapefruit juice contain the intact furanocoumarin group which is presumed to be responsible for the inactivation of P450s. The inactivation of P450 3A4 by grapefruit juice may involve BG as well as several of its derivatives although the specificity and potency for inactivation could differ significantly between these components. BG and its mono- and dihydroxylated derivatives were not detected in orange juice, which is consistent with reports that orange juice does not produce inhibitory effects on intestinal drug metabolism like those seen with grapefruit juice (1). The content of BG and its derivatives may vary significantly among different preparations of grapefruit juice which could account for the discrepancy reported concerning the grapefruit juice effect (36). The inactivation of P450 3A4 activity was time- and concentration-dependent and required metabolism of BG.

Figure 7. Time- and concentration-dependent inhibition of testosterone 6β-hydroxylation activity of P450 3A4 by BG in a reconstituted system. The experimental details are described in Materials and Methods. In brief, P450 3A4 was first incubated with 0 µM (0), 5 µM (]), 10 µM (O), 25 µM (4), 50 µM (3), and 100 µM (×) BG in the reconstituted system for 0, 2, 5, and 10 min at 37 °C, respectively. The incubation mixture was then diluted 20-fold with 50 mM Hepes buffer (pH 7.5) containing 200 µM testosterone and the appropriate components and incubated for 10 min at 37 °C. 6β-Hydroxytestosterone was determined by HPLC.

These results suggest that BG is a mechanism-based inactivator of P450 3A4 (32). Several other furanocoumarins have previously been reported to cause mechanism-based inactivation of P450s, e.g., corandrin (18) and 8-methoxypsoralen (17, 35). The furan ring has been suggested to be the group responsible for the inactivation of P450 1A based on studies of a series of naturally occurring coumarins (20). Some other furan-containing compounds have also been shown to cause inactivation of P450. One example is the furanopyridine L-754,394, a HIV protease inhibitor shown to cause mechanismbased inactivation of P450 3A4 by forming a chemically reactive epoxide on the furan ring (38, 39). BG-mediated mechanism-based inactivation of P450 3A4 is presumed to follow a similar mechanism in which the furan ring is activated to a reactive intermediate which covalently

Inactivation of P450 3A4 by Bergamottin

Figure 8. Inhibition of the activities of P450s 2A6, 2C9, 2D6, 2E1, and 3A4 in human liver microsomes by BG. The experimental details are described in Materials and Methods. Briefly, human liver microsomes were incubated with BG (1, 10, and 100 µM) in the presence of the appropriate substrates of P450 enzymes in 0.1 mM phosphate buffer at 37 °C for the appropriate time period, respectively. The activities of the P450 enzymes were determined using the methods described in Materials and Methods. The results are reported as the average of three experiments.

modifies a critical moiety in the active site of the enzyme. Characterization of the reactive metabolite(s) is currently in progress. The inactivation appears to occur at the active site because it was not inhibited by the addition of 2 or 3 mM GSH to the incubation system (data no shown). The values of kinactivation and KI for the BGmediated inactivation of P450 3A4 are 0.3 min-1 and 7.7 µM. The value of the KI indicates that BG is a potent inactivator of P450 3A4. The values of kinactivation and KI for two other inactivators are 0.4 min-1 and 46 µM for gestodene (40) and 1.6 min-1 and 7.5 µM for L-754,394 (38), respectively. In addition, BG was found to be more potent than 6′,7′-dihydroxybergamottin, whose KI was determined to be 59 µM for the inactivation of P450 3A4 in the reconstituted system (18). The kinactivation was 0.16 min-1 (18). BG also appears to be a competitive inhibitor of P450 3A4. This is consistent with the observation that BG is readily metabolized to several hydroxylated metabolites by P450 3A4 in the reconstituted system (data not shown). Since P450 3A4 activity has been reported to be stimulated by R-naphthoflavone (35), we investigated the possibility that R-naphthoflavone may have a synergistic effect on the generation of the reactive metabolite of BG which would then lead to an increase in the rate of inactivation. Testosterone 6β-hydroxylation was chosen to measure P450 3A4 activity remaining in this experiment since this reaction has shown no stimulation or cooperativity with R-naphthoflavone (35). Our results indicated that R-naphthoflavone had no effect on BGmediated inactivation of P450 3A4. However, it is not clear whether R-naphthoflavone stimulates the cytochrome P450 3A4-catalyzed hydroxylation of BG. The mechanism of BG-mediated inactivation of P450 3A4 was also explored in the present study. Heme adduct formation is well-documented to be a mechanism for the inactivation of P450 by terminal olefins and acetylenes (41). Heme adduct formation in a reconstituted system was identified and characterized by visible spectroscopy, HPLC, and mass spectrometry (42, 43). The visible spectrum of the BG-inactivated P450 3A4 showed

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no sign of heme adduct formation which was expected at 445 nm. The UV-visible absorption spectrum of the BG-inactivated P450 3A4 also showed that the heme content did not decrease significantly even though the sample lost 70% of the erythromycin N-demethylation activity. This result appears to exclude an alternative mechanism seen with several mechanism-based inactivators which cause heme fragmentation that leads to covalent binding of the heme fragments to the apoprotein (41, 44). However, the reduced-CO difference spectrum of P450 3A4 decreased by approximately 40% following inactivation by BG in a reconstituted system. Because there was no evidence for heme destruction or heme adduct formation, it is possible that the BG-derived species may be positioned close to the heme moiety as a result of covalent binding to an active site amino acid residue in such a way that it interferes with the interaction of CO with ferrous heme. Studies on the inactivation of P450 by another furanocoumarin, 8-methoxypsoralen, suggested that covalent binding of 8-methoxypsoralen to apoP450 in the active site might account for the loss of the reduced-CO spectrum of P450 (19, 37). HPLC analysis of BG-inactivated P450 3A4 provided indirect evidence suggesting that BG may inactivate the P450 by modifying the apoprotein. The finding that following inactivation some of the modified apoP450 cannot pass through a reverse-phase HPLC column seems to be a common feature for P450 enzymes inactivated by modification of the apoprotein (26, 42). It might be expected that some hydrophobic active site amino acid residues of the inactivated P450 may be exposed so that they bind tightly to the reverse-phase medium as a result of conformational changes induced by covalent binding. Covalent modification of apoP450 was also reported previously to be a mechanism for the inactivation of P450s by other furanocoumarins (19, 20, 37). Therefore, BG-mediated inactivation of P450 3A4 appears to be due to modification of the apoprotein, as has been observed with 2-ethynylnaphthalene and 9-ethynylphenanthrene for the mechanism-based inactivation of P450 2B1 and 2B4 (45, 46). The mechanism of the inactivation and the identification of the modified residue(s) are currently under investigation. Although the inhibition or inactivation of P450 3A4 by BG and its derivatives is important for understanding the effect of grapefruit juice on the bioavailability of several clinically used drugs which are known to be extensively metabolized by intestinal P450 3A4, we felt that it was important to determine if BG also inhibits the activities of the other human P450s. BG was shown to inhibit the activities of several human P450s including 1A2, 2A6, 2C9, 2C19, 2D6, and 2E1. At this time, the mechanism of inhibition of these enzymes by BG is not clear. BG appears to inhibit P450 1A2 activity more potently than the other P450s. It has been suggested that BG is also a mechanism-based inactivator of P450 1A2 (20). Because the grapefruit juice effect appears to be manifested primarily at the level of intestines, the contribution of each P450 enzyme to the effect may depend on its expression level in intestines. P450 3A4 is the most abundant intestinal P450 enzyme, whereas the other P450s, such as members of the 1A, 2A, 2C, 2D, and 2E families, are poorly expressed in intestines (9, 47). We suspect that BG and its derivatives may be poorly absorbed or extensively metabolized in the gut so

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that they have little chance to inactivate or inhibit liver P450s. In conclusion, BG has been shown to be the furanocoumarin found in the highest concentration in the ethyl acetate extract of grapefruit juice and a potent mechanism-based inactivator of P450 3A4. The finding that grapefruit juice contains several other BG derivatives with an intact furan group suggests that BG and its derivatives may all contribute to the grapefruit juice effect. The mechanism of BG-mediated inactivation of P450 3A4 appears to involve modification of apoprotein rather than either modification of the heme or heme fragmentation. The activities of P450s 1A2, 2A6, 2C9, 2C19, 2D6, and 2E1 in human liver microsomes were also inhibited by BG. Additional studies are planned to explore the in vivo effects of BG on drug bioavailability as well as on hepatic P450 enzymes.

Acknowledgment. We would like to thank Ms. HsiaLien Lin for preparation of cytochrome P450 reductase and cytochrome b5. This work was supported by CA16954 (P.F.H.).

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