Abstract
The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite,trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen ortrans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 ± 20 pmol/40 min/0.2 nmol P450 using 18 μM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 ± 0.8 and 3.1 ± 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 μM tamoxifen). These two forms also catalyzed 4′-hydroxylation (13.0 ± 1.9 and 1.4 ± 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 μM tamoxifen; 0.51 ± 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 μM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifentrans-cis isomerization but CYP2B6 and CYP2C19 also contributed.
The nonsteroidal antiestrogen tamoxifen is currently the most widely used chemotherapeutic agent for the adjuvant treatment of breast cancer. It may act, at least in part, as a prodrug, by conversion to the primary metabolite, trans-4-hydroxytamoxifen, which has enhanced binding affinity and potency compared with the parent compound (Katzenellenbogen et al., 1984). Tamoxifen has also been shown to halve the risk of breast cancer in women with high susceptibility to the disease (Fisher et al., 1998). However, its prophylactic benefit is compromised by a small but statistically significant increase in the risk of developing endometrial cancer (International Agency for Research on Cancer, 1996).
The P4501-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite,trans-4-hydroxytamoxifen. Flavin-containing monooxygenases appear to be responsible for N-oxide formation. Tamoxifen has been shown to be metabolized by human P450s toN-desmethyl, 4-hydroxy, and α-hydroxy metabolites. Previous studies have pointed to CYP3A4 being the major catalyst ofN-demethylation (Jacolot et al., 1991; Crewe et al., 1997) with potential contributions by CYP1A1 and 1A2 (Simon et al., 1993). Studies in one of our laboratories (Crewe et al., 1997) and others (Dehal and Kupfer, 1997) have suggested that CYP2D6 plays a predominant role in the 4-hydroxylation of tamoxifen, with contributions from CYP2C9 and 3A4.
In addition, secondary metabolites resulting from further metabolism of these derivatives may be formed by phase I processes. P450-catalyzed isomerization of trans-4-hydroxytamoxifen has been reported (Williams et al., 1994), resulting in the conversion of a potent antiestrogen (trans-4-hydroxytamoxifen) into a weak estrogen agonist (cis-4-hydroxytamoxifen). Clinical resistance to tamoxifen therapy has been associated with decreased tumor concentrations of tamoxifen and an increase in the cis/transratio of 4-hydroxytamoxifen (Osborne et al., 1992). Biotransformation of tamoxifen by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of the drug, with respect to both the removal of parent drug and the generation of therapeutic or toxic metabolites.
The objectives of the current study were to determine which human P450 forms might participate in the extrahepatic metabolism of tamoxifen to its N-desmethyl, 4-hydroxy, and 4′-hydroxy metabolites and in the isomerization of trans-4-hydroxytamoxifen (Fig.1). In addition, since it is metabolized via several alternative pathways, tamoxifen may serve as a useful probe drug for examining the regioselectivity of different P450 enzymes. Accordingly, the ability of closely related forms to metabolize tamoxifen via its primary routes was studied.
Materials and Methods
Chemicals and Drugs.
The metabolite standards for HPLC analyses were kindly provided by Dr. I. N. H. White (Medical Research Council, Toxicology Unit, University of Leicester, UK; racemic 4-hydroxytamoxifen,N-desmethyltamoxifen, N,N-didesmethyltamoxifen, tamoxifen N-oxide) and Dr. P. Jank (Klinge Pharma GmbH, Munich, Germany; N-desmethyldroloxifene,trans-4-hydroxytamoxifen, racemic 4-hydroxytamoxifen, 4′-hydroxytamoxifen, N-desmethyltamoxifen,N,N-didesmethyltamoxifen and tamoxifen N-oxide).N,N-Didesmethyltoremifene was the generous gift of Orion-Farmos Corporation (Turku, Finland). Tamoxifen citrate, racemic and trans-4-hyroxytamoxifen, and nafoxidine were purchased from the Sigma-Aldrich (St. Louis, MO). All other chemicals were obtained from local suppliers at the highest quality commercially available.
Human Liver Microsomes.
Samples of human liver were obtained from organ donors according to procedures approved by University of Queensland ethics committees and frozen in liquid nitrogen for storage at −70°C prior to use. Microsomes were prepared according to the method of Guengerich (1994), with the addition of a final wash in resuspension buffer (10 mM Tris-acetate, 1 mM EDTA containing 20% glycerol) to remove residual drugs. P450 concentrations were determined as previously described (Guengerich, 1994).
Recombinant P450 Preparations.
Human P450 enzymes were expressed in bacteria in bicistronic format with hNPR as described previously (Gillam et al., 1997; Parikh et al., 1997; Cuttle et al., 2000). DH5α strain Escherichia coliwere transformed with bicistronic expression constructs each of which contained cDNAs encoding hNPR and one of the following recombinant P450s: CYP1A1, 1A2, 1B1 [four variants, designated RAVN, RALN, GSVN, GSLN expressing the following amino acids at positions 48 (Arg or Gly), 119 (Ala or Ser), and 432 (Val or Leu); all four variants contained Asn at position 453 (Shimada et al., 2000)], CYP2A6, 2B6, 2C9*1 (wild type), CYP2C19, 2D6 [full-length variant designated DB4 (Gillam et al., 1995), but with wild type (Ala) at position 374], CYP2E1, 3A4, 3A5, and 3A7. Cells were also transformed with the monocistronic expression vector containing the cDNA for hNPR alone and with the empty vector, pCW. Bacteria were cultured and harvested as described previously (Gillam et al., 1993) except that the expression of P450 forms CYP1A1, 2A6, 2B6, 2D6, and 3A7 was augmented by coexpression of bacterial chaperones. Briefly, E. coli strain DH5α was cotransformed with the relevant bicistronic expression vector and pGro7 (Nishihara et al., 1998) using the method of Inoue et al. (1990). Colonies harboring both plasmids were selected by growth on LB agar containing 100 μg/ml ampicillin and 20 μg/ml chloramphenicol. Isolated colonies were precultured at 37°C overnight in LB media containing both antibiotics then used to inoculate terrific broth containing 1 mM thiamine, trace elements, 100 μg/ml ampicillin, and 20 μg/ml chloramphenicol. Cultures were incubated for 5 h at 25°C, 200 rpm shaking speed before initiation of induction by the addition of arabinose (4 mg/ml), isopropyl β-d-thiogalactoside (1 mM), and δ−aminolevulinic acid (0.5 mM). Flasks were then incubated for a further 43 h at 25°C, 160 to 180 rpm before harvest. Membranes were prepared and characterized for P450 hemoprotein expression and hNPR activity. Bacterial membranes were used directly in enzyme assays.
Enzyme Assays.
Incubations contained 0.05 to 0.2 μM P450 from microsomes or bacterial membranes, substrate, an NADPH-generating system (consisting of 1 mM NADPH, 2.5 mM glucose 6-phosphate, and 0.5 U/ml glucose-6-phosphate dehydrogenase) in either 80 mM potassium phosphate, pH 7.4, with 0.46% (w/v) KCl (experiments with 18 μM tamoxifen) or 100 mM potassium phosphate, pH 7.4, with 2 mM ascorbic acid (experiments using 250 μM tamoxifen). Incubations using CYP3A and CYP2C forms were supplemented with membranes obtained from cells expressing hNPR alone. Control incubations were included in each assay run and contained membranes from cells expressing hNPR alone (“hNPR”) or from cells transformed with the empty expression vector (“pCW”). All procedures were undertaken under reduced light and using amber glass and plasticware to minimize photodegradation of tamoxifen.
Assay of Primary Metabolites.
Tamoxifen was added to the incubation mixture from a methanolic stock solution to give a substrate concentration of 250 μM or from a stock in acetone/ethanol (1:1 v/v) to give a concentration of 18 μM. Final concentrations of solvent were maintained below 1% v/v in all cases. Reactions were initiated by the addition of the NADPH-generating system and incubated at 37°C with gentle agitation for 40 min (18 μM experiments) or 120 min (250 μM experiments) unless otherwise indicated.
For incubations carried out with 18 μM tamoxifen, incubations (200 μl) were terminated by addition of 1.8 ml of methyl-t-butyl ether and 50 μl of internal standard (100 μg/ml nafoxidine). The aqueous and organic phases were mixed vigorously then the upper 1.5 ml were removed and evaporated to dryness. Residues were reconstituted in 250 μl of a mixture of mobile phase and water (2:1 v/v) prior to analysis by HPLC (isocratic conditions).
For incubations carried out with 250 μM tamoxifen, incubations (1 ml) were terminated by addition of 2 ml of helium-purged ethyl acetate and 100 μl of internal standard (15 μM N,N-didesmethyl toremifene HCl). The phases were mixed vigorously, and the upper 1.4 ml were removed. A second extraction was performed with a further 1.2 ml of ethyl acetate. The combined extracts were evaporated to dryness under argon, and the residue was reconstituted in 100 μl of acetonitrile prior to analysis by HPLC (gradient conditions).
Isomerization of trans-4-Hydroxytamoxifen.
Membranes containing recombinant enzymes were incubated using the method described above with minor modifications.trans-4-Hydroxytamoxifen was added as substrate (from a stock in 1.15% w/v KCl) to a final concentration of 0.52 μM. Incubations (500 μl total volume) were allowed to proceed at 37°C for 40 min before being terminated by addition of 1.8 ml of ethyl acetate and 50 μl of internal standard (0.75 μg/mlN-desmethyldroloxifene). The phases were mixed vigorously, then the upper 1.4 ml was removed and evaporated under air. Residues were reconstituted in a mixture of mobile phase and water (2:1 v/v, 300 μl) prior to analysis by HPLC (isomerization conditions). Isomerization was also examined at atrans-4-hydroxytamoxifen concentration of 100 μM, using the conditions described above for incubations with tamoxifen at 250 μM, and isomers were analyzed by HPLC (gradient conditions).
HPLC Analysis of Metabolites.
Three sets of HPLC conditions were used according to the different incubations described above.
Isocratic Conditions.
HPLC was performed according to a previously described method (Crewe et al., 1997) and using a 5-μm Spherisorb ODS-1 reverse phase column (250 × 4.6 mm; Waters Corp., Milford, MA) maintained at 40°C. The mobile phase consisted of 10 mM KH2PO4, pH 3.7, buffer/methanol/acetonitrile (2.5:3:4.5 v/v), and the flow rate was 1.3 ml/min. The column eluant was subjected to UV irradiation, and metabolites were subsequently detected by fluorescence at excitation and emission wavelengths of 260 and 375 nm, respectively. The retention times of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen, 4′-hydroxytamoxifen, tamoxifen N-oxide, and nafoxidine were 27.5, 21.8, 14.6, 16.2, 24.4, and 31.5 min, respectively.
Gradient Conditions.
HPLC was performed using a Shimadzu HPLC system (Shimadzu Scientific Instruments Inc., Columbia, MD) fitted with an auto-injector and a 3.9 × 150 mm Waters Symmetry C8 reverse phase column (Waters Corp.). The mobile phase was 20 mM ammonium acetate/acetonitrile run on a gradient derived from that described inPoon et al. (1995) and modified by extension of the second gradient step to optimize peak separation. Initial conditions were 95:5 20 mM ammonium acetate/acetonitrile. The following steps were programmed: 0 to 4 min linear gradient to 80:20 ammonium acetate/acetonitrile; 4 to 24 min linear gradient to 60:40 ammonium acetate/acetonitrile; 24 to 60 min linear gradient to 35:65 ammonium acetate/acetonitrile; 60 to 70 min constant at 35:65 ammonium acetate/acetonitrile; 70 to 80 min linear gradient to 95:5 ammonium acetate/acetonitrile; and a final re-equilibration at 95:5 ammonium acetate/acetonitrile for 10 min. The flow rate was 0.75 ml/min. Metabolites were detected by absorbance at 280 nm. Under these conditions, the retention times of tamoxifen and its main metabolites were as follows:trans-4-hydroxytamoxifen, 43.8 min;cis-4-hydroxytamoxifen, 44.9 min; 4′-hydroxytamoxifen, 46.7 min; N,N-didesmethyltamoxifen, 52.7 min;N-desmethyltamoxifen, 55.7 min; tamoxifenN-oxide, 59.5 min; tamoxifen, 59.6 min. Quantification of metabolites was performed with reference to standard curves prepared using authentic metabolites after correction for recovery of the internal standard (N,N-didesmethyl toremifene HCl).
Isomerization Conditions.
HPLC was performed according to a previously described method (Williams et al., 1994), using a Novapak (Waters Corp.) 4 μM C8 column. The mobile phase was 10 mM KH2PO4, pH 3.7, buffer/methanol/acetonitrile (3:3:4 v/v) and containing 0.02% (v/v) triethylamine. The flow rate was set to 1.5 ml/min. The column eluant was subjected to UV irradiation, and metabolites were detected by fluorescence using an excitation wavelength of 260 nm and a 375 nm emission filter. The retention times oftrans-4-hydroxytamoxifen, cis-4-hydroxytamoxifen, and N-desmethyldroloxifene were 13, 16, and 11 min, respectively.
Results
Metabolism of Tamoxifen to Its Primary Metabolites.
The metabolism of tamoxifen to 4-hydroxy and N-desmethyl metabolites was examined using a range of recombinant P450 forms. At the lower substrate concentration tested (18 μM), 4-hydroxylation was catalyzed predominantly by CYP2D6 (170 ± 20 pmol/40 min/0.2 nmol P450), with minor contributions by CYP2B6, 2C9, 3A4, 1A1, 1A2, and 3A5 (Fig. 2A). The relative contribution of CYP2B6 was increased at the high substrate concentration (250 μM; 28.1 ± 0.8 and 3.1 ± 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively; Fig. 2B). N-Demethylation of tamoxifen (Table 1) was catalyzed by CYP2D6, 1A1, 1A2, and 3A4 at the lower concentration, whereas CYP2D6 was the major catalysts at the high concentration, with smaller contributions by CYP1A1, 1A2, 1B1 (all variants), 2C9, 2C19, 3A4, and 3A5.
Incubations with recombinant CYP2B6 and 2D6 produced an additional HPLC peak not observed with any other forms, which had the same retention time as 4′-hydroxytamoxifen. This peak was quantified by comparison to a standard curve prepared with authentic metabolite. CYP2B6 generated 0.51 ± 0.08 pmol/40 min/0.2 nmol P450 (mean ± S.D.;n = 3) 4′-hydroxytamoxifen at a substrate concentration of 18 μM. At 250 μM tamoxifen, CYP2B6 and 2D6 generated 13.0 ± 1.9 and 1.4 ± 0.1 nmol/120 min/0.2 nmol P450, respectively.
A small amount of N,N-didemethytamoxifen was also observed in incubations of 250 μM tamoxifen with CYP1A2 and 2D6, two forms that were seen to produce significant N-desmethyltamoxifen.
Isomerization of trans-4-Hydroxytamoxifen.
The trans to cis isomerization of 4-hydroxytamoxifen was catalyzed predominantly by CYP1B1 and to a lesser extent CYP2B6 and 2C19 (Fig. 3). Isomerization was time- and NADPH-dependent as shown in previous work (Williams et al., 1994). For certain forms (3A4, 1A1, 2D6), interpretation of the isomerization data were complicated by the finding that substrate at the low concentrations employed appeared to be metabolized to secondary metabolites. Accordingly, isomerization oftrans-4-hydroxytamoxifen was also examined at high substrate concentrations (100 μM trans-4-hydroxytamoxifen). Under these conditions, CYP1A1, 1B1 (all variants), 2B6, 2D6, 3A4, and 3A5 all catalyzed isomerization of trans-4-hydroxytamoxifen (results not shown).
Discussion
Previous studies have identified some of the P450 forms involved in the metabolism of tamoxifen. Studies in one of our laboratories (Crewe et al., 1997) using human liver microsomes and recombinant P450s expressed in lymphoblastoid cell membranes suggested that CYP2D6 plays a predominant role in the 4-hydroxylation of tamoxifen, with contributions from CYP2C9 and 3A4. In a later study using 100 μM tamoxifen, Dehal and Kupfer (1997) demonstrated 4-hydroxylation activity only with CYP2D6, using a combination of recombinant P450s expressed in E. coli and/or lymphoblastoid cell membranes. The current study extends these findings by using a wide range of forms and confirms the participation of CYP2C9 and 3A4 as well as CYP2D6 in tamoxifen 4-hydroxylation at low substrate concentrations. Additionally however, we have demonstrated the role of CYP1A1, CYP2B6, and CYP2C19 as minor contributors to 4-hydroxylation and of CYP2B6 as a significant catalyst at high substrate concentration. The present findings indicate that CYP2C9 and CYP3A4 are minor catalysts of tamoxifen 4-hydroxylation in vitro at low concentrations. However, their relative contributions in vivo may be more significant, due to the higher expression of these forms compared with CYP2D6. The lack of activity of other forms, such as CYP2B6 and 2C19, as observed by Dehal and Kupfer (1997) may be the result of inefficient coupling of the enzymes with NPR in the recombinant systems or due to limited assay sensitivity.
Previous studies have pointed to CYP3A4 being the major catalyst ofN-demethylation (Jacolot et al., 1991; Crewe et al., 1997) with potential contributions by CYP1A1 and 1A2 (Simon et al., 1993). The present work suggests CYP2D6, 1A1, and 1A2 may be more effective catalysts than CYP3A4 on an equivalent P450 basis and that several other P450s (CYP1B1, 2C9, 2C19, and 3A5) may also show significant activity at high substrate concentrations. Dehal and Kupfer (1997) have also provided evidence to support a role for a number of P450s inN-demethylation. However, it is unclear how the levels of activity found in the earlier study compared with controls carried out in the absence of P450 or cofactors, since all forms examined showed some apparent activity, including forms not seen to have activity here, when compared with P450-deficient controls. Most commercial tamoxifen stocks show low level contamination withN-desmethyltamoxifen, so some N-demethylated metabolite is evident in all incubations. Alternatively, the differences in the enzyme system (membrane environment, ratios of redox partners) may explain these conflicting results.
Our findings suggest CYP1B1 may be principally responsible for the isomerization of the antiestrogen trans-4-hydroxytamoxifen to the weakly estrogenic cis isomer, a reaction that may be associated with the drug-resistant phenotype in human breast tumors (Osborne et al., 1992). This NADPH-dependent isomerization is an atypical reaction for P450 enzymes, but a mechanism has been proposed that is consistent with known catalytic properties of these enzymes (Guengerich, 2001). Previous studies have suggested that CYP1B1 is up-regulated in breast tumors compared with normal breast tissue (Murray et al., 1997; Hellmold et al., 1998; McFayden et al., 1999), and such an effect may diminish the effectiveness oftrans-4-hydroxytamoxifen generated in situ or derived from hepatic metabolism.
Several different allelic variant forms of CYP1B1 have been described. Studies in one of our laboratories have shown a difference in the activity of CYP1B1 variants containing Leu versus Val at position 432 (Shimada et al., 1999, 2000). Certain substrates appear to be more effectively metabolized by Val variants whereas for other substrates the enzymes containing Leu at position 432 appear to be more efficient. In the current study, small differences in the ability of the four variants to catalyze trans-4-hydroxytamoxifen isomerization and tamoxifen N-demethylation were observed but did not reach statistical significance. Thus, it is unlikely that theCYP1B1 genotype would have any significant effect on the efficacy of tamoxifen treatment.
CYP2B6 was found to catalyze both 4-hydroxylation andtrans-4-hydroxytamoxifen isomerization, indicating that tamoxifen is a substrate for this as yet poorly characterized form. Tamoxifen has been shown to act as a phenobarbital-like inducer in rats, leading to up-regulation of members of the CYP2B subfamily (Nuwaysir et al., 1995). The possibility exists that tamoxifen treatment may, through induction of CYP2B6 over time, lead to changes in the relative proportion of metabolites formed. Accordingly, generation of trans-4-hydroxytamoxifen may be accelerated but with concomitant enhancement of isomerization to the cisform, limiting any enhancement of therapeutic effect. Induction of CYP2B6 could also contribute to the changes seen in the resistant phenotype. It would be of value to determine whether any changes in P450 expression in tumor cells occur upon development of tamoxifen resistance.
Only CYP2B6 appeared to 4′-hydroxylate tamoxifen at low substrate concentrations. The biological effects of this metabolite have not been well characterized. One study has suggested it has a higher affinity for the estrogen receptor than tamoxifen (Ruenitz et al., 1982). To date, 4′-hydroxytamoxifen has been detected only in the rat (Ruenitz et al., 1984).
The data presented here raise the possibility that extrahepatic forms of P450 such as CYP1A1 and CYP1B1 may catalyze the clearance or bioactivation of tamoxifen in tissues such as the breast and endometrium. Accordingly, the P450 profile of various target tissues such as the breast may influence the therapeutic and/or toxic effects of tamoxifen. It follows that alterations in P450 expression in breast tissue (or particularly in breast tumor tissue) may underlie temporal alterations in response to tamoxifen, such as seen in the development of resistance. As noted above, clinical resistance to tamoxifen therapy has been associated with decreased tumor concentrations of tamoxifen and an increase in the cis/trans ratio of 4-hydroxytamoxifen (Osborne et al., 1992).
Various studies have demonstrated that CYP1B1 is expressed constitutively within the breast at both the protein and mRNA level, whereas CYP1A1 is present at only low levels or is not detectable (Huang et al., 1996; Eltom et al., 1998; Hellmold et al., 1998; Larsen et al., 1998; Williams et al., 1998). Both forms appear to be subject to induction in this tissue. Expression of CYP1B1 protein has also been detected in breast tumors (McKay et al., 1995; Murray et al., 1997;McFayden et al., 1999) and may be enhanced relative to adjacent normal tissue in the same patient (Murray et al., 1997; Hellmold et al., 1998;McFayden et al., 1999). Detection of various other P450s (CYP2A6, 2B6, 2E1, 2C, 2D6, and 3A) has been reported at the mRNA and/or protein level in normal breast tissue and tumors (Huang et al., 1996; Hellmold et al., 1998). In particular, CYP2D6, a polymorphic form shown to have a predominant role in both 4-hydroxylation andN-demethylation of tamoxifen, was found to be apparently expressed constitutively in extensive metabolizers but may be subject to tissue-specific regulation at the level of mRNA splicing (Huang et al., 1996). It would be of key interest to determine whether expression of any of these P450s was altered in resistance compared with sensitive tumors, especially that of CYP1B1 and 2B6 as key catalysts oftrans-4-hydroxytamoxifen isomerization.
Endometrial expression of P450s may influence some of the adverse effects of the drug in this tissue. The expression of CYP1B1 mRNA has been consistently detected in the human uterus (Liehr et al., 1995;Shimada et al., 1996) and specifically in the endometrium (Hakkola et al., 1997; Vadlamuri et al., 1998). However, Murray et al. (1997)observed CYP1B1 protein expression only in uterine tumors and not in normal tissue. In addition, there is controversy over the detection of mRNAs for other P450 forms in the uterus. Schuetz et al. (1993)reported the detection of CYP3A7 but not CYP3A4 or CYP3A5, andVadlamuri et al. (1998) and Shimada et al. (1996) reported CYP1A1 expression. In contrast, Hukkanen et al. (1998) failed to find either CYP1A1 or CYP3A7 but did detect CYP2B6, 2C, 2E1, 3A4, and 3A5. Further studies are required to confirm the expression of these forms in breast and uterus, at both the mRNA and protein levels, and to determine whether and to what extent individual P450 forms may mediate tamoxifen metabolism in these tissues. Investigations are currently underway to determine the extent to which extrahepatic tissues can metabolize tamoxifen. Such information should help in minimizing individual patient risk of developing resistance to tamoxifen and of developing endometrial cancer.
Acknowledgments
Grateful thanks are extended to Drs. I. N. H. White and P. Jank and to the Orion Farmos Corporation for the gift of chemicals used in this study.
Footnotes
-
Funding for this study was provided by the Kathleen Cuningham Foundation for Breast Cancer Research and the Australian Cancer Fund.
- Abbreviations used are::
- P450
- cytochrome P450 (heme-thiolate protein P450)
- HPLC
- high-performance liquid chromatography
- hNPR
- human NADPH-cytochrome P450 reductase
- Received December 17, 2001.
- Accepted April 18, 2002.
- The American Society for Pharmacology and Experimental Therapeutics