Effects of cytochrome P450 inhibitors and inducers on the metabolism and pharmacokinetics of ospemifene.

PURPOSE: The objectives were to determine the cytochrome P450 (CYP) enzymes involved in the metabolism of ospemifene and its main hydroxylated metabolites and to examine the effects of CYP inhibitors and inducers on ospemifene pharmacokinetics. METHODS: In vitro metabolism studies were conducted using human liver microsomes; CYP-selective inhibitors and CYP-specific substrates were used to determine the roles of nine CYP isoforms in ospemifene metabolism. Two Phase 1 clinical trials were conducted in healthy postmenopausal women; crossover designs examined the effects of pretreatment with the CYP modulators rifampicin, ketoconazole, fluconazole and omeprazole on ospemifene pharmacokinetics. RESULTS: Although several CYP inhibitors decreased the in vitro formation of ospemifene metabolites, none of them completely blocked metabolism. Roles for CYP3A4, CYP2C9, CYP2C19 and CYP2B6 in the metabolism of ospemifene and its two main metabolites, 4--hydroxyospemifene and 4'-hydroxyospemifene, were confirmed. The in vivo experiments demonstrated that ospemifene serum concentrations were decreased by rifampicin pretreatment, increased by ketoconazole or fluconazole pretreatment, and minimally affected by omeprazole pretreatment. CONCLUSIONS: The clinical pharmacokinetic findings and in vitro data suggest that CYP3A4 is important for ospemifene metabolism, but other CYP isoforms and metabolic pathways also contribute. Strong CYP3A or CYP2C9 inducers (e.g. rifampicin) would be expected to decrease the exposure to ospemifene. Ospemifene should be used with caution when coadministered with the modest CYP3A inhibitor ketoconazole and should not be coadministered with the potent CYP3A/CYP2C9/CYP2C19 inhibitor fluconazole. The potent CYP2C19 inhibitor omeprazole is unlikely to cause clinically significant changes in ospemifene pharmacokinetics.

Endoxifen, the active metabolite of tamoxifen, is a substrate of the efflux transporter P-glycoprotein (multidrug resistance 1).

Tamoxifen is widely prescribed to patients with estrogen receptor-positive breast cancer, and it is a prodrug that requires bioactivation by cytochrome P450 enzymes CYP2D6 and 3A4 to generate the active metabolite, endoxifen. Large interpatient variability in endoxifen plasma levels has been reported, and polymorphisms in CYP2D6 have been implicated as a major determinant of such variability. However, little is known regarding the role of drug transporters such as P-glycoprotein [multidrug resistance 1 (MDR1), ATP-binding cassette B1 (ABCB1)] to endoxifen disposition and response. Therefore, we determined the ability of P-glycoprotein to transport endoxifen in vitro, using a polarized human P-glycoprotein-overexpressing cell line. Markedly higher transport of endoxifen was observed in the basal-to-apical direction, which was abrogated in the presence of the potent and specific P-glycoprotein inhibitor (2R)-anti-5-{3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (LY335979). To validate the in vivo relevance of P-glycoprotein to endoxifen disposition, plasma and tissue concentrations were also determined in Mdr1a-deficient mice after oral administration of endoxifen. Plasma endoxifen levels did not significantly differ between wild-type and Mdr1a-deficient mice. However, brain concentrations of endoxifen were nearly 20-fold higher in Mdr1a-deficient mice compared to wild-type mice. Because P-glycoprotein is highly expressed at the blood-brain barrier and in some breast cancer tumors, variation in expression and function of this transporter may alter central nervous system entry and the attained intracellular concentration in such breast cancer cells and therefore may prove to be of relevance to therapeutic outcome.

Identifying subsets of complex mixtures most associated with complex diseases: polychlorinated biphenyls and endometriosis as a case study.

BACKGROUND: Exploratory statistical analyses have been conducted on an epidemiologic data set in which the relationship was examined between exposure to polychlorinated biphenyl (PCB) mixtures and risk of endometriosis in women. In that study, the association between endometriosis and the sum of 4 antiestrogenic PCBs (PCBs 105, 114, 126, and 169) was borderline significant (P = 0.079), whereas an association was not found (P = 0.681) with the sum of 12 estrogenic PCBs. This finding was inconsistent with the widely held notion that endometriosis is an estrogen-dependent disease, prompting further statistical analyses to explore these associations in more detail. METHODS: As an alternative method of data reduction, an optimization algorithm was developed to determine weights in a linear combination of scaled PCB levels that has the strongest possible association with the risk of endometriosis. RESULTS: Application of this method to the antiestrogenic PCB subgroup revealed that PCB 114 was responsible for nearly 100% of the association. The fact that PCB 114 is neither the most potent nor abundant antiestrogen in the mixture suggests that PCB 114 might be estrogenic or that the association may be driven by a different mechanism. Use of this statistical weighting method for further analyses of 12 estrogenic PCBs showed that any association with endometriosis was driven mainly by PCBs 99 and 188 and possibly a few others. CONCLUSION: Although the role of PCB mixtures in endometriosis remains unclear, these results demonstrate how the integration of refined statistical methods coupled with toxicologic and biologic interpretation can generate testable hypotheses that might not otherwise have been generated.

Evaluation of the safety and tolerability of oral TAS-108 in postmenopausal patients with metastatic breast cancer.

BACKGROUND: The potential of TAS-108 for the treatment of breast cancer has been shown by preclinical studies. We therefore investigated the safe dosage, tolerability, and effectiveness on hormone levels and bone metabolism markers and the pharmacokinetics of TAS-108 administered in postmenopausal Japanese women with metastatic breast cancer. PATIENTS AND METHODS: The subjects had previously undergone standard endocrine therapeutic modalities. TAS-108 was given repeatedly to five patients each, at three dose levels (40, 80, and 120 mg p.o.) once a day after the first daily meal for a scheduled 8 weeks. Plasma concentrations of TAS-108 and its metabolites were measured at the scheduled time points. RESULTS: Fifteen patients received TAS-108 treatment. Orally administered TAS-108 was well tolerated at doses up to 120 mg and did not cause notable changes either in hormone levels or bone metabolism markers. Pharmacokinetic results indicated dose-dependent increases in plasma levels of TAS-108 and its metabolites. A steady state was achieved by 2 weeks at all dose levels, suggesting no marked accumulation. Clinical benefits were confirmed in 5 of 15 patients. CONCLUSIONS: Repeated oral administration of TAS-108 at doses up to 120 mg was well tolerated, and the plasma level of this compound increased dose-dependently.

Therapeutic Drug Monitoring of Oral Anti-Hormonal Drugs in Oncology.

Oral anti-hormonal drugs are essential in the treatment of breast and prostate cancer. It is well known that the interpatient variability in pharmacokinetic exposure is high for these agents and exposure-response relationships exist for many oral anti-hormonal drugs. Yet, they are still administered at fixed doses. This could lead to underdosing and thus suboptimal efficacy in some patients, while other patients could be overdosed resulting in unnecessary side effects. Therapeutic drug monitoring (TDM), individualized dosing based on measured blood concentrations of the drug, could therefore be a valid option to further optimize treatment. In this review, we provide an overview of relevant clinical pharmacokinetic and pharmacodynamic characteristics of oral anti-hormonal drugs in oncology and translate these into practical guidelines for TDM. For some agents, TDM targets are not well established yet and as a reference the median pharmacokinetic exposure could be targeted (exemestane: minimum plasma concentration (Cmin) 4.1 ng/mL and enzalutamide: Cmin 11.4 mg/L). However, for most drugs, exposure-efficacy analyses could be translated into specific targets (abiraterone: Cmin 8.4 ng/mL, anastrozole: Cmin 34.2 ng/mL, and letrozole: Cmin 85.6 ng/mL). Moreover, prospective clinical trials have shown TDM to be feasible for tamoxifen, for which the exposure-efficacy threshold of its active metabolite endoxifen is 5.97 ng/mL. Based on the available data, we therefore conclude that individualized dosing based on drug concentrations is feasible and promising for oral anti-hormonal drugs and should be developed further and implemented into clinical practice.

Increased antitumor potential of the raloxifene prodrug, raloxifene diphosphate.

Raloxifene (RAL) significantly reduced the incidence of breast cancer in women at high risk of developing the disease. Unlike tamoxifen (TAM), an increased incidence of endometrial cancer was not observed in women treated with RAL. However, RAL, having two hydroxyl moieties, can be conjugated rapidly through phase II metabolism and excreted, making it difficult to achieve adequate bioavailability by oral administration in humans. As a result, higher doses must be administered to obtain an efficacy equivalent to that achieved with TAM. To improve oral bioavailability and antitumor potential, RAL diphosphate was prepared as a prodrug. RAL diphosphate showed several orders of magnitude lower binding potential to both ER alpha and ER beta and weak antiproliferative potency on cultured human MCF-7 and ZR-75-1 breast cancer cells, as compared to RAL. However, RAL diphosphate has a much higher bioavailability than RAL, endowing it with higher antitumor potential than RAL against both 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats and human MCF-7 breast cancer implanted in athymic nude mice. The RAL prodrug may provide greater clinical benefit for breast cancer therapy and prevention.

Metabolism, distribution, and excretion of a next generation selective estrogen receptor modulator, lasofoxifene, in rats and monkeys.

Disposition of lasofoxifene (LAS; 6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalen-2-ol. tartrate) was investigated in rats and monkeys after oral administration of a single oral dose of [(14)C]LAS. Total mean recoveries of the radiocarbon were 96.7 and 94.3% from rats and monkeys, respectively. The major route of excretion in both species was the feces, and based on a separate study in the bile duct-cannulated rat, this likely reflects excretion in bile rather than incomplete absorption. Whole-body autoradioluminography suggested that [(14)C]LAS radioequivalents distributed rapidly in the rat with most tissues achieving maximal concentrations at 1 h. Half-life of radioactivity was longest in the uvea (124 h) and shortest in the spleen ( approximately 3 h). LAS was extensively metabolized in both rats and monkeys because no unchanged drug was detected in urine and/or bile. Based on area under the curve((0-24)) values, >78% of the circulating radioactivity was due to the metabolites. A total of 22 metabolites were tentatively identified by liquid chromatography-tandem mass spectrometry. Based on the structures of the metabolites, six metabolic pathways of LAS were identified: hydroxylation at the tetraline ring, hydroxylation at the aromatic ring attached to tetraline, methylation of the catechol intermediates by catechol-O-methyl transferase, oxidation at the pyrrolidine ring, and direct conjugation with glucuronic acid and sulfuric acid. LAS and its glucuronide conjugate (M7) were the major circulating drug-related moieties in both rats and monkeys. However, there were notable species-related qualitative and quantitative differences in the metabolic profiles. The catechol (M21) and its sulfate conjugate (M10) were observed only in monkeys, whereas the glucuronide conjugate of the methylated catechol (M8) and hydroxy-LAS (M9) were detected only in rats.

The growth-promoting action of individual women's sera on mammary carcinoma cells.

In vitro studies concerning the growth-stimulating effect of hormones, especially of estradiol and its metabolites, have mainly been performed using pure substances and breast cancer cell lines. In order to take into account the metabolism of inactive into active hormones or drugs and vice versa which occurs in several tissues, the influence of individual patients' sera on the growth of breast cancer cells in vitro was tested. Besides measuring the growth promoting action of several hormone replacement therapies, the antiestrogenic effect was determined by measuring the effect of 10(-10) M estradiol added to the culture medium (E2-sensitivity). Influence on proliferation and stimulatability was similar in MCF-7 and T47-D cells. Growth-promoting potential correlated significantly with patient age, being higher in young ladies than in older ones. The converse was true for E2 sensitivity. From the different steroid hormones tested, only higher estradiol levels were associated with increased growth stimulation and diminished E2 sensitivity. Hormone replacement therapy (HRT) of different types did not significantly increase growth potential of serum, however these results are preliminary. Treatment with tamoxifen of breast cancer patients led to a decrease of E2 sensitivity, whereas growth potential was not affected significantly. For the aromatase inhibitor Arimidex, a tendency towards growth inhibition and increased E2 sensitivity was observed. Our in vitro system allows identifying differences between individual persons and groups of women of different age or treatment with respect to stimulation of growth or influence on estrogen sensitivity of breast cancer cells by serum. It is speculated that results might reflect the personal risk or the risk under treatment to develop breast cancer.

Stearoyl-CoA desaturase-1, a novel target of omega-3 fatty acids for reducing breast cancer risk in obese postmenopausal women.

BACKGROUND/OBJECTIVES: Conversion of saturated fatty acids to monounsaturated fatty acids by the enzyme stearoyl-Co-A-desaturase (SCD-1) is emerging as a major factor in promoting carcinogenesis including breast cancer. The aim of our study was to explore the regulation of SCD-1 by Raloxifene and omega-3 fatty acids in women at increased risk of breast cancer based on high breast density. SUBJECTS/METHODS: As a reflection of SCD-1 activity, we measured the ratios of palmitoleic acid (C16:1n7) to palmitic acid (C16:0) (SCD-16) and oleic acid (C18:1n9) to steric acid (C18:0) (SCD-18) in plasma samples of postmenopausal women enrolled in our clinical trial (NCT00723398) designed to test the effects of the antiestrogen, Raloxifene and/or the omega-3 preparation Lovaza, on breast density, a validated biomarker of breast cancer risk. RESULTS: We report that Lovaza but not Raloxifene-reduced SCD-16 and SCD-18 for the 2-year duration of the trial. Importantly, decreasing levels of SCD-16 and SCD-18 were associated with a progressive reduction in breast density but only in obese women (body mass index ⩾30). CONCLUSIONS: Body mass index-related factors play an important role in the reduction of breast density and hence breast cancer risk by omega-3 fatty acids. SCD-1 may be a useful biomarker in future clinical trials testing the benefit of nutritional interventions in reducing obesity-associated breast cancer risk.

Pharmacodynamics of tamoxifen and its 4-hydroxy and N-desmethyl metabolites: activation of caspases and induction of apoptosis in rat mammary tumors and in human breast cancer cell lines.

The antiestrogen tamoxifen (TAM) is extensively metabolized by cytochrome P-450 in humans and rodents. The active, estrogen receptor-binding metabolites, 4-hydroxy TAM (OHT) and N-desmethyl TAM (DMT) have been well characterized. We showed that the s.c. injection of 1 mg/kg TAM in adult female Sprague Dawley rats bearing carcinogen-induced mammary tumors resulted in rapid serum decline of parent TAM but higher exposure of the metabolites, OHT and DMT. We found for the first time that the administration of TAM for a short time resulted in a delayed induction of caspase activity and apoptosis within the mammary tumors. When TAM, OHT, or DMT was added to human breast cancer cell lines in culture, each elicited a time- and dose-dependent induction of caspase activity, preceding apoptosis. Importantly, pretreatment of the cells with a pharmacological inhibitor of caspases [benzyloxy Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk)] blocked apoptosis induced by all three of the compounds, implicating a critical role of caspases in TAM-, OHT-, or DMT-induced apoptosis. The results obtained from these studies suggest that one possible mechanism of inhibition of mammary carcinogenesis and tumor growth in vivo may be the induction of caspase-dependent apoptosis, and that the metabolites OHT and DMT may contribute to the antitumor effect of TAM.

Effect of a standardized meal on the bioavailability of a single oral dose of tibolone 2.5 mg in healthy postmenopausal women.

STUDY OBJECTIVE: To assess the effect of food on absorption and pharmacokinetic disposition of tibolone. DESIGN: Open-label, randomized, crossover study with a 1-week washout period. SETTING: Institut für Klinische Pharmakologie, Hohenkirchen-Siegertsbrunn, Germany SUBJECTS: Twenty-four healthy, early postmenopausal women. INTERVENTION: Single doses of tibolone 2.5 mg were administered after subjects consumed a high-fat meal or fasted. MEASUREMENTS AND MAIN RESULTS: Plasma concentrations of tibolone and its three primary metabolites, delta4-tibolone, 3alpha-hydroxytibolone, and 3beta-hydroxytibolone, were assayed by gas chromatography with mass spectrometry. Peak plasma concentration (Cmax), time to Cmax, area under the plasma concentration versus time curve from time zero to infinity (AUC(0-infinity)), and elimination half-life were calculated, and food effects were evaluated. Plasma concentrations of tibolone and delta4-tibolone were too low to estimate AUC(0-infinity) and half-life. Absorption or formation of 3alpha-hydroxytibolone and 3beta-hydroxytibolone was slower in fed participants than in fasting participants, but this was of no clinical relevance because of the long-term nature of tibolone treatment. Meal consumption did not affect AUC(0-infinity) or half-life for 3alpha-hydroxytibolone and 3beta-hydroxytibolone. CONCLUSION: Food consumption decreased and delayed Cmax but had no effect on the exposure of tibolone and its metabolites. Tibolone therefore can be administered irrespective of meal timing.

Dose proportionality of three different doses of tibolone.

STUDY OBJECTIVE: To assess dose proportionality of tibolone tablets after multiple oral administration. DESIGN: Open-label, randomized, three-period crossover study SETTING: Department of Drug Metabolism and Kinetics, N.V. Organon, Oss, The Netherlands. SUBJECTS: Twenty-seven postmenopausal women aged 65 years or younger. INTERVENTION: Subjects received tibolone 1.25, 2.5, or 5.0 mg once/day for 7 days. MEASUREMENTS AND MAIN RESULTS: Plasma concentrations of tibolone and its three primary metabolites were assayed. Steady state was attained by day 5. Elimination half-life for 3alpha-hydroxytibolone was 7.2-8.5 hours. At steady state, the dose-normalized peak concentration and area under the curve satisfied bioequivalence requirements for the 1.25- and 2.5-mg doses, but not fully for the 5.0-mg dose. Parameters were proportionally slightly lower for the 5.0-mg dose compared with the 1.25- and 2.5-mg doses. CONCLUSION: Proportional bioequivalence was established for the 1.25- and 2.5-mg doses, but not between the 5.0-mg dose and the two lower doses of tibolone.

Mass spectrometric determination of p-nonylphenol metabolism and disposition following oral administration to Sprague-Dawley rats.

Isomers of 4-nonylphenol (NP), which are important industrial compounds and environmental breakdown products from widely used surfactants, have estrogenic activity in vitro and in vivo that has prompted interest in its potential for modulation of endocrine function in humans and wildlife. Mass spectrometry was used to quantify NP and metabolites in serum and endocrine-responsive tissues from dietary exposure in Sprague-Dawley rats. Tissue accumulation of NP aglycone was observed despite the predominance of glucuronidation in blood. Serum toxicokinetics of total NP, measured following gavage administration, showed rapid absorption and elimination (average half-times 0.8 and 3.5 h, respectively). NP was similarly administered by gavage to pregnant dams and total and aglycone NP were measured in dam serum and fetuses to show placental transfer into serum and brain. These data provide a basis for future correlations of biologic effects observed following dietary exposure in rats with those predicted from environmental exposures to humans.

The in vivo metabolism of tibolone in animal species.

The in vivo tissue distribution and metabolism of tibolone was studied in different animals to further investigate the compound's tissue-specificity. Tibolone's metabolism was studied in vivo in rats and rabbits by administration of [16-3H]-tibolone and the metabolic pattern was determined in urine and faeces after oral administration to female rats and dogs. The main excretory pathway was found to be excretion in the faeces. Important phase-I metabolic routes were the reduction of the 3-keto to the 3a- or 3beta-hydroxy functions with a preference for 3alpha-OH in rats and for 3beta-OH in dogs. To a lesser extent, hydroxylation reactions at C2 and C7, and a shift of the delta5(10)-double bond to a delta4(5)-position also occurred. The main phase-II metabolic route was sulphate conjugation of the hydroxyl groups at C3 and C17. Since the oxidation reactions form only a minor part of the metabolism of tibolone, it is concluded that the cytochrome P450 enzymes do not play an important role in tibolone's metabolism. For both phases, quantitative differences were found between the species. In human similar metabolites are found. Profiling of the target organs in female rats and rabbits showed a tissue-specific distribution of metabolites. The majority of the metabolites existed as sulphate conjugates and no glucuronidated conjugates were observed. The same metabolites were found in both the circulation and the tissues. However, different tissues had quantitatively different metabolic profiles.

Tamoxifen and estradiol improved locomotor function and increased spared tissue in rats after spinal cord injury: their antioxidant effect and role of estrogen receptor alpha.

17ß-Estradiol is a multi-active steroid that imparts neuroprotection via diverse mechanisms of action. However, its role as a neuroprotective agent after spinal cord injury (SCI), or the involvement of the estrogen receptor-alpha (ER-α) in locomotor recovery, is still a subject of much debate. In this study, we evaluated the effects of estradiol and of Tamoxifen (an estrogen receptor mixed agonist/antagonist) on locomotor recovery following SCI. To control estradiol cyclical variability, ovariectomized female rats received empty or estradiol filled implants, prior to a moderate contusion to the spinal cord. Estradiol improved locomotor function at 7, 14, 21, and 28 days post injury (DPI), when compared to control groups (measured with the BBB open field test). This effect was ER-α mediated, because functional recovery was blocked with an ER-α antagonist. We also observed that ER-α was up-regulated after SCI. Long-term treatment (28 DPI) with estradiol and Tamoxifen reduced the extent of the lesion cavity, an effect also mediated by ER-α. The antioxidant effects of estradiol were seen acutely at 2 DPI but not at 28 DPI, and this acute effect was not receptor mediated. Rats treated with Tamoxifen recovered some locomotor activity at 21 and 28 DPI, which could be related to the antioxidant protection seen at these time points. These results show that estradiol improves functional outcome, and these protective effects are mediated by the ER-α dependent and independent-mechanisms. Tamoxifen׳s effects during late stages of SCI support the use of this drug as a long-term alternative treatment for this condition.

Synthesis, radiolabeling and In Vivo tissue distribution of an anti-oestrogen glucuronide compound, (99m)Tc-TOR-G.

Toremifene (TOR) has been used as an anti-oestrogen drug for the treatment and prevention of human breast cancer. The aim of this study was the addition of the hydrophilic groups diethylenetriamine pentaacetic acid (DTPA) and glucuronic acid to the starting substance TOR and to label it with technetium-99m ((99m)Tc) radionuclide and to investigate radiopharmaceutical potential of the new compound. The synthesis reactions are completed in four steps, including enzymatic reaction, with the following substeps; preparation of microsomal fraction from Hutu 80 cell line and subsequent purification of UDP-glucuronyl transferase (UDPGT), estimation of protein quantity in microsomal samples and glucuronidation reaction. The results indicate that (99m)Tc-TOR-G may be proposed as a new anti-oestrogen glucuronide imaging agent for ovarian tumours.

Environmental PCB exposure and risk of endometriosis.

BACKGROUND: Hormonally active environmental agents have recently been associated with the development of endometriosis. METHODS: We undertook a study to assess the relationship between endometriosis, an estrogen-dependent gynaecological disease, and 62 individual polychlorinated biphenyl (PCBs) congeners. We enrolled 84 eligible women aged 18-40 years undergoing laparoscopy for study, which included an interview and blood specimen (n=79; 94%). Thirty-two women had visually confirmed endometriosis at laparoscopy while 52 did not. Blood specimens were run in batches of 14 including four quality control samples for toxicological analysis. Each PCB congener was adjusted for recovery; batch-specific reagent blanks were subtracted. All PCB concentrations were log transformed and expressed in ng/g serum first as a sum and then as tertiles by purported estrogenic or anti-estrogenic activity of PCB congeners. RESULTS: Using unconditional logistic regression analysis, a significantly elevated odds ratio (OR) was observed for women in the third tertile of anti-estrogenic PCBs [OR 3.77; 95% confidence interval (CI) 1.12-12.68]. Risk remained elevated after controlling for gravidity, current cigarette smoking and serum lipids (OR 3.30; 95% CI 0.87-12.46). CONCLUSIONS: These data suggest that anti-estrogenic PCBs may be associated with the development of endometriosis.

CYP2D6 genotype, antidepressant use, and tamoxifen metabolism during adjuvant breast cancer treatment.

BACKGROUND: The efficacy of tamoxifen therapy for the treatment of breast cancer varies widely among individuals. Plasma concentrations of the active tamoxifen metabolite endoxifen are associated with the cytochrome P450 (CYP) 2D6 genotype. We examined the effects of concomitant use of selective serotonin reuptake inhibitor antidepressants, which are CYP2D6 enzyme inhibitors commonly prescribed to treat hot flashes in women who take tamoxifen, and genotypes for genes that encode tamoxifen-metabolizing enzymes on plasma concentrations of tamoxifen and its metabolites. METHODS: Eighty patients with newly diagnosed with breast cancer who were beginning tamoxifen therapy (20 mg/day orally), 24 of whom were taking CYP2D6 inhibitors, were genotyped for common alleles of the CYP2D6, CYP2C9, CYP3A5, and sulfotransferase (SULT) 1A1 genes. Plasma concentrations of tamoxifen and its metabolites were measured after 1 and 4 months of tamoxifen therapy. Differences in plasma concentrations of tamoxifen and its metabolites between genotype groups were analyzed by the Wilcoxon rank sum test. All statistical tests were two-sided. RESULTS: Among all women, plasma endoxifen concentrations after 4 months of tamoxifen therapy were statistically significantly lower in subjects with a CYP2D6 homozygous variant genotype (20.0 nM, 95% confidence interval [CI] = 11.1 to 28.9 nM) or a heterozygous genotype (43.1 nM, 95% CI = 33.3 to 52.9 nM) than in those with a homozygous wild-type genotype (78.0 nM, 95%CI = 65.9 to 90.1 nM) (both P = .003). Among subjects who carried a homozygous wild-type genotype, the mean plasma endoxifen concentration for those who were using CYP2D6 inhibitors was 58% lower than that for those who were not (38.6 nM versus 91.4 nM, difference = -52.8 nM, 95% CI = -86.1 to -19.5 nM, P = .0025). The plasma endoxifen concentration was slightly reduced in women taking venlafaxine, a weak inhibitor of CYP2D6, whereas the plasma endoxifen concentration was reduced substantially in subjects who took paroxetine (a potent inhibitor of CYP2D6). Genetic variations of CYP2C9, CYP3A5, or SULT1A1 had no statistically significant associations with plasma concentrations of tamoxifen or its metabolites. CONCLUSION: Interactions between CYP2D6 polymorphisms and coadministered antidepressants and other drugs that are CYP2D6 inhibitors may be associated with altered tamoxifen activity.

Bioanalytical high-throughput selected reaction monitoring-LC/MS determination of selected estrogen receptor modulators in human plasma: 2000 samples/day.

The high-throughput determination of small molecules in biological matrixes has become an important part of drug discovery. This work shows that increased throughput LC/MS/MS techniques can be used for the analysis of selected estrogen receptor modulators in human plasma where more than 2000 samples may be analyzed in a 24-h period. The compounds used to demonstrate the high-throughput methodology include tamoxifen, raloxifene, 4-hydroxytamoxifen, nafoxidine, and idoxifene. Tamoxifen and raloxifene are used in both breast cancer therapy and osteoporosis and have shown prophylactic potential for the reduction of the risk of breast cancer. The described strategy provides LC/MS/MS separation and quantitation for each of the five test articles in control human plasma. The method includes sample preparation employing liquid-liquid extraction in the 96-well format, an LC separation of the five compounds in less than 30 s, and selected reaction monitoring detection from low nano- to microgram per milliter levels. Precision and accuracy are determined where each 96-well plate is considered a typical "tray" having calibration standards and quality control (QC) samples dispersed through each plate. A concept is introduced where 24 96-well plates analyzed in 1 day is considered a "grand tray", and the method is cross-validated with standards placed only at the beginning of the first plate and the end of the last plate. Using idoxifene-d5 as an internal standard, the results obtained for idoxifene and tamoxifen satisfy current bioanalytical method validation criteria on two separate days where 2112 and 2304 samples were run, respectively. Method validation included 24-h autosampler stability and one freeze-thaw cycle stability for the extracts. Idoxifene showed acceptable results with accuracy ranging from 0.3% for the high quality control (QC) to 15.4% for the low QC and precision of 3.6%-13.9% relative standard deviation. Tamoxifen showed accuracy ranging from 1.6% to 13.8% and precision from 7.8% to 15.2%. The linear dynamic range for these compounds was 3 orders of magnitude. The limit of quantification was 5 and 50 ng/ mL for tamoxifen and idoxifene, respectively. The other compounds in this study in general satisfy the more relaxed bioanalytical acceptance criteria for modern drug discovery. It is suggested that the quantification levels reported in this high-throughput analysis example are adequate for many drug discovery and related early pharmaceutical studies.

Glucuronidation of the nonsteroidal antiestrogen EM-652 (SCH 57068), by human and monkey steroid conjugating UDP-glucuronosyltransferase enzymes.

EM-652 (SCH 57068) is a new orally active antiestrogen that demonstrates pure antagonistic effects in the mammary gland and endometrium. In vivo studies have shown that EM-652 is primarily glucuronidated at the 7-hydroxy position in rats and that the metabolite is present in the plasma of female monkeys and human subjects after EM-800 (SCH 57050) or EM-652.HCl oral administration. Using hepatic microsomes from rat, monkey, and human, the formation of two EM-652 monoglucuronides at positions 4' and 7 was demonstrated by a liquid chromatographic tandem mass spectrometric method. Although no difference in EM-652 conjugation was observed between male and female monkey livers, an interindividual variation of hepatic EM-652 glucuronidation was shown with female human donors. Using microsome preparations from human embryonic kidney 293 cells stably expressing each of the 12 human and 11 monkey UGT enzymes cloned to date, the two EM-652-monoglucuronides were detected after incubation with microsomes containing human UGT1A1, UGT1A3, UGT1A8, UGT1A9, and monkey monUGT1A01, monUGT1A03, and monUGT1A09. Despite human UGT1A1 and monkey monUGT1A09 favored formation of EM-652-7-glucuronide, other active UGT1A enzymes formed both 4'- and 7-glucuronide derivatives in equal amounts. Kinetic analysis of EM-652 glucuronidation by these enzymes showed Michaelis constant (K(m)) values between 36 and 302 microM for EM-652-4'-glucuronide and 19 and 233 microM for EM-652-7-glucuronide. The present results demonstrate the importance of UGT1A isoforms, mainly UGT1A1, for EM-652 metabolism in humans.