Zeruesenay Desta

Publications (61)279.92 Total impact

• Article: Genetic associations with toxicity-related discontinuation of aromatase inhibitor therapy for breast cancer.

  N Lynn Henry, Todd C Skaar, Jessica Dantzer, Lang Li, Kelley Kidwell, Christina Gersch, Anne T Nguyen, James M Rae, Zeruesenay Desta, Steffi Oesterreich, Santosh Philips, Janet S Carpenter, Anna M Storniolo, Vered Stearns, Daniel F Hayes, David A Flockhart
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  ABSTRACT: Up to 25 % of patients discontinue adjuvant aromatase inhibitor (AI) therapy due to intolerable symptoms. Predictors of which patients will be unable to tolerate these medications have not been defined. We hypothesized that inherited variants in candidate genes are associated with treatment discontinuation because of AI-associated toxicity. We prospectively evaluated reasons for treatment discontinuation in women with hormone receptor-positive breast cancer initiating adjuvant AI through a multicenter, prospective, randomized clinical trial of exemestane versus letrozole. Using multiple genetic models, we evaluated potential associations between discontinuation of AI therapy because of toxicity and 138 variants in 24 candidate genes, selected a priori, primarily with roles in estrogen metabolism and signaling. To account for multiple comparisons, statistical significance was defined as p < 0.00036. Of the 467 enrolled patients with available germline DNA, 152 (33 %) discontinued AI therapy because of toxicity. Using a recessive statistical model, an intronic variant in ESR1 (rs9322336) was associated with increased risk of musculoskeletal toxicity-related exemestane discontinuation [HR 5.0 (95 % CI 2.1-11.8), p < 0.0002]. An inherited variant potentially affecting estrogen signaling may be associated with exemestane-associated toxicity, which could partially account for intra-patient differences in AI tolerability. Validation of this finding is required.
  Breast Cancer Research and Treatment 04/2013; · 4.43 Impact Factor
• Article: In vitro Analysis and Quantitative Prediction of Efavirenz Inhibition of Eight Cytochrome P450 (CYP) Enzymes: Major Effects on CYPs 2B6, 2C8, 2C9 and 2C19.

  Cong Xu, Zeruesenay Desta
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  ABSTRACT: In order to quantitatively predict drug interactions associated with efavirenz-based anti-HIV therapy, we evaluated reversible and time-dependent inhibitions of efavirenz on eight cytochrome P450 (CYP) enzymes in vitro. Present study showed that efavirenz was a potent competitive inhibitor of CYP2B6 (average K(i)= 1.68 µM in HLMs and K(i)= 1.38 µM in expressed CYP2B6) and CYP2C8 (K(i )= 4.78 µM in pooled HLMs and K(i )= 4.80 μM in HLMs with CYP2C8*3/*3 genotype). Efavirenz was a moderate inhibitor of CYP2C9 (K(i)= 19.46 µM) and CYP2C19 (K(i)= 21.31 µM); and a weak inhibitor of CYP3A (K(i)= 40.33 µM). No appreciable inhibition was observed on CYP1A2, CYP2A6 or CYP2D6. No time-dependent inhibition of the CYPs by efavirenz was observed in this study. Quantitative predictions showed that single dose of efavirenz may substantially slow the elimination of drugs predominately cleared by CYP2B6, CYP2C19 or by both enzymes and may also lower the area under the plasma concentration time curve (AUC) of active metabolites of some pro-drugs (e.g. clopidogrel and proguanil ) by up to 30%. Depending on substrates, chronic administration of efavirenz may increase the AUC of CYP2C8 and CYP2C9 substrates about 3.5 ~ 4.4-fold and 1.7 ~ 2.0-fold at steady state.
  Drug Metabolism and Pharmacokinetics 02/2013; · 2.32 Impact Factor
• Article: Is (+)-[(13)C]-pantoprazole better than (±)-[(13)C]-pantoprazole for the breath test to evaluate CYP2C19 enzyme activity?

  David L Thacker, Anil Modak, David A Flockhart, Zeruesenay Desta
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  ABSTRACT: Recently, we have shown that the (+)-[(13)C]-pantoprazole is more dependent on CYP2C19 metabolic status than (-)-[(13)C]-pantoprazole. In this study, we tested the hypothesis that (+)-[(13)C]-pantoprazole is a more sensitive and selective probe for evaluating CYP2C19 enzyme activity than the racemic mixture. (+)-[(13)C]-pantoprazole (95 mg) was administered orally in a sodium bicarbonate solution to healthy volunteers. Breath and plasma samples were collected before and up to 720 min after dosing. The (13)CO(2) in exhaled breath samples was measured by infrared spectrometry. Ratios of (13)CO(2)/(12)CO(2) after (+)-[(13)C]-pantoprazole relative to (13)CO(2)/(12)CO(2) at baseline were expressed as delta over baseline (DOB). (+)-[(13)C]-pantoprazole concentrations were measured by HPLC. Genomic DNA extracted from whole blood was genotyped for CYP2C19*2, *3 and *17 using Taqman assays. Statistically significant differences in the area under the plasma concentration time curve (AUC(plasma(0-∞)) (p < 0.001) and oral clearance (<0.01) of (+)-[(13)C]-pantoprazole as well as in the breath test indices (delta over baseline, DOB(30); and area under the DOB versus time curve, AUC(DOB(0-120))) (p < 0.01) were observed among poor, intermediate and extensive metabolizer of CYP2C19. DOB(30) and AUC(DOB(0-120)) adequately distinguished poor metabolizer from intermediate and extensive metabolizer of CYP2C19. Breath test indices significantly correlated with plasma elimination parameters of (+)-[(13)C]-pantoprazole (Pearson correlations: -0.68 to -0.73). Although relatively higher breath test indices were observed after administration of (+)-[(13)C]-pantoprazole (this study) than after (±)-[(13)C]-pantoprazole (previous study), the performance of the racemic and the enantiomer as marker of CYP2C19 activity remained similar. Our data confirm that the metabolism of (+)-[(13)C]-pantoprazole is highly dependent on CYP2C19 metabolic status, but the breath test derived from it is not superior to the racemic [(13)C]-pantoprazole in evaluating CYP2C19 activity in vivo. Thus, racemic [(13)C]-pantoprazole which is relatively easy to synthesize and more stable than (+)-[(13)C]-pantoprazole is adequate as a probe of this enzyme.
  Journal of Breath Research 12/2012; 7(1):016001. · 2.54 Impact Factor
• Article: Stereoselective and regiospecific hydroxylation of ketamine and norketamine.

  Zeruesenay Desta, Ruin Moaddel, Evan T Ogburn, Cong Xu, Anuradha Ramamoorthy, Swarajya Lakshmi Vattem Venkata, Mitesh Sanghvi, Michael E Goldberg, Marc C Torjman, Irving W Wainer
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  ABSTRACT: The objective was to determine the cytochrome P450s (CYPs) responsible for the stereoselective and regiospecific hydroxylation of ketamine [(R,S)-Ket] to diastereomeric hydroxyketamines, (2S,6S;2R,6R)-HK (5a) and (2S,6R;2R,6S)-HK (5b) and norketamine [(R,S)-norKet] to hydroxynorketamines, (2S,6S;2R,6R)-HNK (4a), (2S,6R;2R,6S)-HNK (4b), (2S,5S;2R,5R)-HNK (4c), (2S,4S;2R,4R)-HNK (4d), (2S,4R;2R,4S)-HNK (4e), (2S,5R;2R,5S)-HNK (4f). The enantiomers of Ket and norKet were incubated with characterized human liver microsomes (HLMs) and expressed CYPs. Metabolites were identified and quantified using LC/MS/MS and apparent kinetic constants estimated using single-site Michaelis-Menten, Hill or substrate inhibition equation.  5a was predominantly formed from (S)-Ket by CYP2A6 and N-demethylated to 4a by CYP2B6. 5b was formed from (R)- and (S)-Ket by CYP3A4/3A5 and N-demethylated to 4b by multiple enzymes. norKet incubation produced 4a, 4c and 4f and minor amounts of 4d and 4e. CYP2A6 and CYP2B6 were the major enzymes responsible for the formation of 4a, 4d and 4f, and CYP3A4/3A5 for the formation of 4e. The 4b metabolite was not detected in the norKet incubates.  5a and 4b were detected in plasma samples from patients receiving (R,S)-Ket, indicating that 5a and 5b are significant Ket metabolites. Large variations in HNK concentrations were observed suggesting that pharmacogenetics and/or metabolic drug interactions may play a role in therapeutic response.
  Xenobiotica 05/2012; 42(11):1076-87. · 1.79 Impact Factor
• Article: Effects of clopidogrel and itraconazole on the disposition of efavirenz and its hydroxyl-metabolites: exploration of a novel CYP2B6 phenotyping index.

  Fen Jiang, Zeruesenay Desta, Ji-Hong Shon, Chang-Woo Yeo, Ho-Sook Kim, Kwang-Hyeon Liu, Soo-Kyung Bae, Sang-Seop Lee, David A Flockhart, Jae-Gook Shin
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  ABSTRACT: AIMS: To evaluate the effects of clopidogrel and itraconazole on the disposition of efavirenz and its hydroxyl-metabolites in relation to the CYP2B6*6 genotype and explore potential phenotyping indices for CYP2B6 activity in vivo using a low dose of oral efavirenz. METHODS: We conducted a randomized 3-phase crossover study in 17 healthy Korean subjects pre-genotyped for the CYP2B6*6 allele (CYP2B6*1/*1, n= 6; *1/*6, n= 6; *6/*6, n= 5). Subjects were pretreated with clopidogrel (75 mg day(-1) for 4 days), itraconazole (200 mg day(-1) for 6 days), or placebo and then given a single dose of efavirenz (200 mg). The plasma (0-120 hours) and urine (0-24 hours) concentrations of efavirenz and its metabolites (7- and 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz) were determined by LC/MS/MS. RESULTS: This study is the first to quantitatively delineate the full (phase I and II) metabolic profile of efavirenz and its three hydroxyl-metabolites in humans. Clopidogrel pretreatment markedly decreased AUC(0-48) , C(max) , and Ae(0-24) for 8,14-dihydroxyefavirenz, compared with placebo; 95% CI of the ratios were 0.55-0.73, 0.30-0.45, and 0.25-0.47, respectively. The 8,14-dihydroxyefavirenz/efavirenz AUC(0-120 ) ratio was significantly correlated with the weight-adjusted CL/F of efavirenz (r(2)  ≈ 0.4; P < 0.05), differed with CYP2B6*6 genotype, and was affected by clopidogrel pretreatment (P < 0.05), but not by itraconazole pretreatment. CONCLUSIONS: The disposition of 8,14-dihydroxy-EFV appears to be sensitive to CYP2B6 activity alterations in human subjects. The 8,14-dihydroxyefaviremz/efavirenz AUC(0-120) ratio is attractive as a candidate phenotyping index for CYP2B6 activity in vivo. © 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.
  British Journal of Clinical Pharmacology 05/2012; · 2.96 Impact Factor
• Article: Rapid and Simultaneous Determination of Efavirenz, 8-Hydroxyefavirenz, and 8,14-Dihydroxyefavirenz Using LC–MS–MS in Human Plasma and Application to Pharmacokinetics in Healthy Volunteers

  Kwon-Bok Kim, Hyunmi Kim, Fen Jiang, Chang-Woo Yeo, Soo Kyung Bae, Zeruesenay Desta, Jae-Gook Shin, Kwang-Hyeon Liu
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  ABSTRACT: We developed a rapid and sensitive method for determining efavirenz, 8-hydroxyefavirenz, and 8,14-dihydroxyefavirenz in human plasma simultaneously using liquid chromatography–tandem mass spectrometry (LC–MS–MS). Three compounds and ritonavir, an internal standard, were extracted from plasma using ethyl acetate in the presence of 0.1M sodium carbonate after incubation of β-glucuronidase (500U). After drying the organic layer, the residue was reconstituted in mobile phase (acetonitrile:20mM ammonium acetate, 90:10, v/v) and injected onto a reversed-phase C18 column. The isocratic mobile phase was eluted at 0.2mLmin−1. The ion transitions monitored in multiple reaction-monitoring mode were m/z 314→244, 330→258, 346→262, and 721→296 for efavirenz, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and ritonavir, respectively. The retention time is 1.93, 1.70, 1.52, and 1.82min for efavirenz, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and ritonavir, respectively. The coefficients of variation of the assay precision were less than 10.7%, and the accuracy was 90–111%. The lower limits of quantification (LLOQ) were 5ngmL−1 for efavirenz and 8-hydroxyefavirenz. This method was used to measure the plasma concentrations of efavirenz and its metabolites from healthy volunteers after a single 600mg oral dose of efavirenz. This analytical method is a very rapid, sensitive, and accurate to determine the pharmacokinetic profiles of efavirenz including its metabolites. KeywordsColumn liquid chromatography–tandem mass spectrometry–Efavirenz–8-Hydroxyefavirenz–8,14-Dihydroxyefavirenz–Human plasma
  Chromatographia 04/2012; 73(3):263-271. · 1.20 Impact Factor
• Article: Inhibition of platelet aggregation by prostaglandin E1 (PGE1) in diabetic patients during therapy with clopidogrel and aspirin.

  Rolf P Kreutz, Perry Nystrom, Yvonne Kreutz, Jia Miao, Richard Kovacs, Zeruesenay Desta, David A Flockhart, Yan Jin
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  ABSTRACT: Diabetes mellitus (DM) is associated with increased platelet activation and reduced platelet inhibition by clopidogrel. Prostaglandin E1 (PGE1) stimulates adenyl cyclase activity in platelets and increases cyclic AMP concentrations, which inhibit Ca(2+)release and platelet aggregation induced by P2Y1 receptor activation. PGE1 is included in the VerifyNow P2Y12 assay to suppress P2Y1 induced platelet aggregation. We hypothesized that diabetes mellitus may be associated with altered response to PGE1 in subjects treated with clopidogrel. Subjects with established coronary artery disease who were taking clopidogrel 75 mg daily and aspirin for >14 days were enrolled (n = 96). Diabetic (n = 34) were compared with non-diabetic subjects (n = 62). VerifyNow P2Y12 assay and light transmittance aggregometry (LTA) were performed using ADP as agonist with and without addition of PGE1. Genomic DNA was genotyped for common cytochrome P450 (CYP) 2C19 variants using Taqman assays. Residual on-treatment platelet aggregation induced by 20 µM ADP was not significantly different between subjects with and without DM. Addition of 22 nM and 88 nM PGE1 to 20 µM ADP resulted in a significant reduction of maximal platelet aggregation (MPA). Residual LTA platelet aggregation with PGE1 and VerifyNow P2Y12 platelet reactivity were significantly higher in subjects with DM than those without DM and in carriers of CYP 2C19*2 polymorphism. We conclude that an impaired inhibitory response to PGE1 may contribute to the high platelet reactivity phenotype in subjects with DM treated with clopidogrel. Addition of PGE1 to ADP agonist platelet assays may identify subjects with blunted inhibitory response to prostaglandins and result in a higher proportion of subjects with DM being classified as non-responders.
  Platelets 03/2012; · 1.85 Impact Factor
• Article: Effects of the CYP2B6*6 allele on catalytic properties and inhibition of CYP2B6 in vitro: implication for the mechanism of reduced efavirenz metabolism and other CYP2B6 substrates in vivo.

  Cong Xu, Evan T Ogburn, Yingying Guo, Zeruesenay Desta
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  ABSTRACT: The mechanism by which CYP2B6*6 allele alters drug metabolism in vitro and in vivo is not fully understood. To test the hypothesis that altered substrate binding and/or catalytic properties contribute to its functional consequences, efavirenz 8-hydroxylation and bupropion 4-hydroxylation were determined in CYP2B6.1 and CYP2B6.6 proteins expressed without and with cytochrome b5 (Cyt b5) and in human liver microsomes (HLMs) obtained from liver tissues genotyped for the CYP2B6*6 allele. The susceptibility of the variant protein to inhibition was also tested in HLMs. Significantly higher V(max) and K(m) values for 8-hydroxyefavirenz formation and ∼2-fold lower intrinsic clearance (Cl(int)) were noted in expressed CYP2B6.6 protein (-b5) compared with that of CYP2B6.1 protein (-b5); this effect was abolished by Cyt b5. The V(max) and Cl(int) values for 4-hydroxybupropion formation were significantly higher in CYP2B6.6 than in CYP2B6.1 protein, with no difference in K(m), whereas coexpression with Cyt b5 reversed the genetic effect on these kinetic parameters. In HLMs, CYP2B6*6/*6 genotype was associated with markedly lower V(max) (and moderate increase in K(m)) and thus lower Cl(int) values for efavirenz and bupropion metabolism, but no difference in catalytic properties was noted between CYP2B6*1/*1 and CYP2B6*1/*6 genotypes. Inhibition of efavirenz 8-hydroxylation by voriconazole was significantly greater in HLMs with the CYP2B6*6 allele (K(i) = 1.6 ± 0.8 μM) than HLMs with CYP2B6*1/*1 genotype (K(i) = 3.0 ± 1.1 μM). In conclusion, our data suggest the CYP2B6*6 allele influences metabolic activity by altering substrate binding and catalytic activity in a substrate- and Cyt b5-dependent manner. It may also confer susceptibility to inhibition.
  Drug metabolism and disposition: the biological fate of chemicals 01/2012; 40(4):717-25. · 3.74 Impact Factor
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  Available from: Jeffrey A Breall

  Article: Influence of paraoxonase-1 Q192R and cytochrome P450 2C19 polymorphisms on clopidogrel response.

  Rolf P Kreutz, Perry Nystrom, Yvonne Kreutz, Jia Miao, Zeruesenay Desta, Jeffrey A Breall, Lang Li, Chienwei Chiang, Richard Kovacs, David A Flockhart, Yan Jin
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  ABSTRACT: The metabolic activation of clopidogrel is a two-step process. It has been suggested that paraoxonase-1 (PON1) is a rate-limiting enzyme in the conversion of 2-oxo- clopidogrel to an active thiol metabolite. Conflicting results have been reported in regard to (1) the association of a common polymorphism of PON1 (Q192R) with reduced rates of coronary stent thrombosis in patients taking clopidogrel and (2) its effects on platelet inhibition in patient populations of European descent. Blood samples from 151 subjects of mixed racial background with established coronary artery disease and who received clopidogrel were analyzed. Platelet aggregation was determined with light transmittance aggregometry and VerifyNow(®) P2Y12 assay. Genotyping for cytochrome P450 2C19 (CYP2C19)*2 and *3 and PON1 (Q192R) polymorphisms was performed. Carriers of CYP2C19*2 alleles exhibited lower levels of platelet inhibition and higher on-treatment platelet aggregation than noncarriers. There was no significant difference in platelet aggregation among PON1 Q192R genotypes. Homozygous carriers of the wild-type variant of PON1 (QQ192) had similar on-treatment platelet reactivity to carriers of increased-function variant alleles during maintenance clopidogrel dosing, as well as after administration of a clopidogrel 600 mg loading dose. CYP2C19*2 allele is associated with impaired platelet inhibition by clopidogrel and high on-treatment platelet aggregation. PON1 (Q192R) polymorphism does not appear to be a significant determinant of clopidogrel response.
  Clinical Pharmacology: Advances and Applications 01/2012; 4:13-20.
• Article: Comparison of Changes in the Lipid Profile of Postmenopausal Women With Early Stage Breast Cancer Treated With Exemestane or Letrozole.

  Lauren Nicole Bell, Anne Thi Phuong Nguyen, Lang Li, Zeruesenay Desta, N Lynn Henry, Daniel F Hayes, Antonio C Wolff, Vered Stearns, Anna Maria Storniolo, David A Flockhart
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  ABSTRACT: Effects of aromatase inhibitor (AI) therapy on the plasma lipid profile are not clear. Here the authors describe changes in fasting lipids (total cholesterol, high-density lipoprotein [HDL], low-density lipoprotein [LDL], and triglycerides) before and after 3 months of exemestane or letrozole treatment. HDL was reduced in the entire cohort (P < .001) and in the exemestane group (P < .001) but unchanged in the letrozole group (P = .169). LDL was increased in the entire cohort (P = .005) and in the letrozole group (P = .002) but unchanged in the exemestane group (P = .361). This effect was at least partially attributable to washout of tamoxifen as only patients with prior use of tamoxifen experienced a significant increase in LDL. Baseline HDL was an independent predictor of the change in HDL (r(2) = -0.128, P < .001), and prior tamoxifen use was associated with greater increases in LDL (r(2) = 0.057, P < .001). Use of lipid-altering medications did not protect against the exemestane-induced drop in HDL or the increase in LDL observed in women with prior use of tamoxifen taking letrozole. In conclusion, AI treatment and/or washout of tamoxifen induced detrimental changes in the lipid profile of postmenopausal women with breast cancer.
  The Journal of Clinical Pharmacology 12/2011; · 2.91 Impact Factor
• Article: Stereoselective pharmacokinetics of stable isotope (+/-)-[(13) C]-pantoprazole: Implications for a rapid screening phenotype test of CYP2C19 activity.

  David L Thacker, Anil Modak, Phuong D Nguyen, David A Flockhart, Zeruesenay Desta
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  ABSTRACT: Aims: We have previously shown that the (±)-[(13) C]-pantoprazole breath test is a promising noninvasive probe of CYP2C19 activity. As part of that trial, plasma, breath test indices and CYP2C19 (*2, *3, and *17) genotype were collected. Here, we examined whether [(13) C]-pantoprazole exhibits enantioselective pharmacokinetics and whether this enantioselectivity is correlated with indices of breath test. Methods: Plasma (-)- and (+)-[(13) C]-pantoprazole that were measured using a chiral HPLC were compared between CYP2C19 genotypes and correlated with breath test indices. Results: The AUC(() (0-∞)) of (+)-[(13) C]-pantoprazole in PM (*2/*2, n = 4) was 10.1- and 5.6-fold higher that EM (*1/*1or *17, n = 10) and IM (*1/*2or *3, n = 10) of CYP2C19, respectively (P < 0.001). The AUC(() (0-∞)) of (-)-[(13) C]-pantoprazole only significantly differed between PMs and EMs (1.98-fold; P = 0.05). The AUC(() (0-∞)) ratio of (+)-/(-)-[(13) C]-pantoprazole was 3.45, 0.77, and 0.67 in PM, IM, and EM genotypes, respectively. Breath test index, delta over baseline show significant correlation with AUC(() (0-∞)) of (+)-[(13) C]-pantoprazole (Pearson's r = 0.62; P < 0.001). Conclusions: [(13) C]-pantoprazole exhibits enantioselective elimination. (+)-[(13) C]-pantoprazole is more dependent on CYP2C19 metabolic status and may serve as a more attractive probe of CYP2C19 activity than (-)-[(13) C]-pantoprazole or the racemic mixture. Chirality, 2011. © 2011 Wiley-Liss, Inc.
  Chirality 09/2011; · 2.35 Impact Factor
• Article: Tamoxifen and its metabolites cause acute vasorelaxation of aortic rings by inducing vasodilator prostanoid synthesis.

  Marcelo F Montenegro, Carla S Ceron, Maria C O Salgado, Zeruesenay Desta, David A Flockhart, Jose E Tanus-Santos
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  ABSTRACT: The vascular effects of tamoxifen (Tam) and its metabolites are poorly known. We compared the vasorelaxation induced by Tam and its metabolites (N-desmethyl-Tam, 4-hydroxy-Tam, and endoxifen) in aortic rings from rats using standardized organ bath procedures, and we investigated the mechanisms involved in this effect. Tam and its metabolite-induced vasorelaxation in a concentration-dependent manner. Although 4-hydroxy-Tam and Tam had similar potency (pD2 = 8.5 ± 0.1 vs. 8.8 ± 0.1, respectively) and maximum effect (Emax = 88.5% ± 1.3% vs. 92.6% ± 1.3%, respectively), N-desmethyl-Tam and endoxifen were more potent and showed higher Emax than Tam did (pD2 = 9.0 ± 0.1 and 8.9 ± 0.1; Emax = 101.1% ± 1.8% and 101.0% ± 1.8% for N-desmethyl-Tam and endoxifen, respectively). Although preincubation of aortic rings with the estrogen receptor antagonist ICI 182780 or with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride induced no changes in the vasorelaxation induced by Tam or 4-hydroxy-Tam, both drugs significantly reduced Emax in response to N-desmethyl-Tam or to endoxifen. Inhibition of cyclooxygenase with indomethacin or the incubation with the prostaglandin D2 and E2 receptor antagonist AH6809 reduced the vasorelaxation-induced Tam and its metabolites by approximately 50%. Preincubation with Nω-nitro-L-arginine methyl ester hydrochloride combined with indomethacin abolished the vasorelaxation-induced Tam and its metabolites. These results show that Tam and its metabolites cause acute vasorelaxation by inducing vasodilator prostanoids synthesis.
  Journal of cardiovascular pharmacology 08/2011; 58(6):647-53. · 2.83 Impact Factor
• Article: Genotype-guided tamoxifen dosing increases active metabolite exposure in women with reduced CYP2D6 metabolism: a multicenter study.

  William J Irvin, Christine M Walko, Karen E Weck, Joseph G Ibrahim, Wing K Chiu, E Claire Dees, Susan G Moore, Oludamilola A Olajide, Mark L Graham, Sean T Canale, [......], Steven W Corso, Jeffrey M Peppercorn, Steven M Anderson, Kenneth J Friedman, Evan T Ogburn, Zeruesenay Desta, David A Flockhart, Howard L McLeod, James P Evans, Lisa A Carey
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  ABSTRACT: We examined the feasibility of using CYP2D6 genotyping to determine optimal tamoxifen dose and investigated whether the key active tamoxifen metabolite, endoxifen, could be increased by genotype-guided tamoxifen dosing in patients with intermediate CYP2D6 metabolism. One hundred nineteen patients on tamoxifen 20 mg daily ≥ 4 months and not on any strong CYP2D6 inhibiting medications were assayed for CYP2D6 genotype and plasma tamoxifen metabolite concentrations. Patients found to be CYP2D6 extensive metabolizers (EM) remained on 20 mg and those found to be intermediate (IM) or poor (PM) metabolizers were increased to 40 mg daily. Eighty-nine evaluable patients had tamoxifen metabolite measurements repeated 4 months later. As expected, the median baseline endoxifen concentration was higher in EM (34.3 ng/mL) compared with either IM (18.5 ng/mL; P = .0045) or PM (4.2 ng/mL; P < .001). When the dose was increased from 20 mg to 40 mg in IM and PM patients, the endoxifen concentration rose significantly; in IM there was a median intrapatient change from baseline of +7.6 ng/mL (-0.6 to 23.9; P < .001), and in PM there was a change of +6.1 ng/mL (2.6 to 12.5; P = .020). After the dose increase, there was no longer a significant difference in endoxifen concentrations between EM and IM patients (P = .84); however, the PM endoxifen concentration was still significantly lower. This study demonstrates the feasibility of genotype-driven tamoxifen dosing and demonstrates that doubling the tamoxifen dose can increase endoxifen concentrations in IM and PM patients.
  Journal of Clinical Oncology 08/2011; 29(24):3232-9. · 18.37 Impact Factor
• Article: Tamoxifen metabolites as active inhibitors of aromatase in the treatment of breast cancer.

  Wenjie Jessie Lu, Zeruesenay Desta, David A Flockhart
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  ABSTRACT: The mechanism of tamoxifen action in the treatment of breast cancer is believed to be via active metabolites that act as potent estrogen receptor antagonists. Attempts to identify relationships between active metabolite concentrations and clinical outcomes have produced mixed results. Since anti-estrogenic effects may be brought about not only by estrogen antagonism, but also by reduced estrogen synthesis, we tested the ability of tamoxifen and its principal metabolites to inhibit aromatase in vitro. The activity of human aromatase in both recombinant and placental microsomal preparations was measured using the rate of generation of a fluorescent metabolite in the presence and absence of multiple concentrations of tamoxifen, endoxifen, N-desmethyl-tamoxifen, and Z-4-hydroxy-tamoxifen. Aromatase inhibition was further characterized by measuring the inhibition of testosterone metabolism to estradiol. The biochemical mechanisms of inhibition were documented and their inhibitory potency was compared. Using recombinant human aromatase, endoxifen, and N-desmethyl-tamoxifen were able to inhibit aromatase activity with K (i) values of 4.0 and 15.9 μM, respectively. Detailed characterization of inhibition by endoxifen and N-desmethyl-tamoxifen indicated non-competitive kinetics for both inhibitors. Similarly, endoxifen-inhibited testosterone metabolism via a non-competitive mechanism. No appreciable inhibition by tamoxifen or Z-4-hydroxy-tamoxifen was observed at similar concentrations. The relative inhibitory potency was: endoxifen > N-desmethyl-tamoxifen > Z-4-hydroxy-tamoxifen > tamoxifen. Similar data were obtained in human placental microsomes. Endoxifen and N-desmethyl-tamoxifen were found to be potent inhibitors of aromatase. Inhibition by these tamoxifen metabolites may contribute to the variability in clinical effects of tamoxifen in patients with breast cancer. Relationships between tamoxifen metabolite concentrations and clinical outcomes may be complex, and the biologic mechanisms that underlie these relationships may include aromatase inhibition.
  Breast Cancer Research and Treatment 03/2011; 131(2):473-81. · 4.43 Impact Factor
• Article: Contribution of N-glucuronidation to efavirenz elimination in vivo in the basal and rifampin-induced metabolism of efavirenz.

  Doo-Yeoun Cho, Evan T Ogburn, David Jones, Zeruesenay Desta
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  ABSTRACT: In this study, the contribution of efavirenz N-glucuronidation to efavirenz elimination in vivo was assessed. In a two-period placebo-controlled crossover trial design, a single 600-mg oral dose of efavirenz was administered to healthy volunteers (n = 10) pretreated with placebo pills or 600 mg/day rifampin orally for 10 days. Urine and plasma concentrations of efavirenz and 8-hydroxyefavirenz were measured by the liquid chromatography-tandem mass spectrometry method after enzymatic hydrolysis with β-glucuronidase (conjugated and unconjugated) and without enzymatic hydrolysis (unconjugated). Pharmacokinetic parameters of efavirenz within the placebo- or rifampin-treated group obtained after enzymatic hydrolysis did not show any statistically significant difference compared with those obtained without enzymatic hydrolysis (P > 0.05; paired t test, two-tailed). The amount of efavirenz excreted over 12 h was significantly larger after enzymatic hydrolysis in both the placebo (P = 0.007) and rifampin (P = 0.0001) treatment groups, supporting the occurrence of direct N-glucuronidation of efavirenz, but the relevance of this finding is limited because the amount of efavirenz excreted as unchanged or conjugated in urine is less than 1% of the dose administered. In both the placebo- and rifampin-treated groups, plasma concentrations of 8-hydroxyefavirenz and the amount excreted over 12 h were significantly larger (P < 0.00001) after enzymatic hydrolysis than without enzymatic hydrolysis. These findings suggest that although the occurrence of direct efavirenz N-glucuronidation is supported by the urine data, the abundance of efavirenz N-glucuronide in plasma is negligible and that the contribution of the N-glucuronidation pathway to the overall clearance of efavirenz seems minimal.
  Antimicrobial Agents and Chemotherapy 01/2011; 55(4):1504-9. · 4.84 Impact Factor
• Article: In vitro and in vivo oxidative metabolism and glucuronidation of anastrozole

  Landry K. Kamdem, Yong Liu, Vered Stearns, Susan A. Kadlubar, Jacqueline Ramirez, Stacie Jeter, Karineh Shahverdi, Bryan A. Ward, Evan Ogburn, Mark J. Ratain, David A. Flockhart, Zeruesenay Desta
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  ABSTRACT: WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Anastrozole is primarily cleared by hepatic metabolism via oxidative and conjugating enzymes.WHAT THIS STUDY ADDS• Anastrozole is oxidized to hydroxyanastrozole mainly by CYP3A4/5 and glucuronidated to anastrozole glucuronide predominantly by UGT1A4 in vitro.• Hydroxyanastrozole glucuronide and hydroxyanastrozole were quantified as the major metabolites of anastrozole in plasma of breast cancer patients.• This study describes for the first time anastrozole metabolic pathways and the enzymes involved, which may serve as the scientific basis for pharmacogenetic and drug-interaction assessments.AIMSLittle information is available regarding the metabolic routes of anastrozole and the specific enzymes involved. We characterized anastrozole oxidative and conjugation metabolism in vitro and in vivo.METHODSA sensitive LC-MS/MS method was developed to measure anastrozole and its metabolites in vitro and in vivo. Anastrozole metabolism was characterized using human liver microsomes (HLMs), expressed cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs).RESULTSHydroxyanastrozole and anastrozole glucuronide were identified as the main oxidative and conjugated metabolites of anastrozole in vitro, respectively. Formation of hydroxyanastrozole from anastrozole was markedly inhibited by CYP3A selective chemical inhibitors (by >90%) and significantly correlated with CYP3A activity in a panel of HLMs (r= 0.96, P= 0.0005) and mainly catalyzed by expressed CYP3A4 and CYP3A5. The Km values obtained from HLMs were also close to those from CYP3A4 and CYP3A5. Formation of anastrozole glucuronide in a bank of HLMs was correlated strongly with imipramine N-glucuronide, a marker of UGT1A4 (r= 0.72, P < 0.0001), while expressed UGT1A4 catalyzed its formation at the highest rate. Hydroxyanastrozole (mainly as a glucuronide) and anastrozole were quantified in plasma of breast cancer patients taking anastrozole (1 mg day−1); anastrozole glucuronide was less apparent.CONCLUSION Anastrozole is oxidized to hydroxyanastrozole mainly by CYP3A4 (and to some extent by CYP3A5 and CYP2C8). Once formed, this metabolite undergoes glucuronidation. Variable activity of CYP3A4 (and probably UGT1A4), possibly due to genetic polymorphisms and drug interactions, may alter anastrozole disposition and its effects in vivo.
  British Journal of Clinical Pharmacology 11/2010; 70(6):854 - 869. · 2.96 Impact Factor
• Article: In vitro cytochrome P450-mediated metabolism of exemestane.

  Landry K Kamdem, David A Flockhart, Zeruesenay Desta
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  ABSTRACT: Exemestane is a potent and irreversible steroidal aromatase inhibitor drug used for the treatment of estrogen receptor-positive breast cancer. Our aim was to identify and assess the contribution of the specific cytochromes P450 (P450s) responsible for exemestane primary in vitro metabolism. With the use of high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques, 17-hydroexemestane (MI) formation and 6-hydroxymethylexemestane (MII) formation were found to be the predominant exemestane metabolic pathways. In a bank of 15 well characterized human liver microsomes with known P450 isoform-specific activities, the MI formation rate correlated significantly with CYP1A2 (Spearman r = 0.60, p = 0.02) and CYP4A11 (Spearman r = 0.67, p = 0.01) isoform-specific activities, whereas the MII production rate significantly correlated with CYP2B6 (Spearman r = 0.57, p = 0.03) and CYP3A (Spearman r = 0.76, p = 0.005) isoform-specific activities. Recombinant CYP1A1 metabolized exemestane to MI with a catalytic efficiency (Cl(int)) of 150 nl/pmol P450 × min that was at least 3.5-fold higher than those of other P450s investigated. Recombinant CYP3A4 catalyzed MII formation from exemestane with a catalytic efficiency of 840 nl/pmol P450 × min that was at least 4-fold higher than those of other P450s investigated. Among a panel of 10 chemical inhibitors tested, only ketoconazole and troleandomycin (CYP3A-specific chemical inhibitors) significantly inhibited the formation of MII by 45 and 95%, respectively. None of them markedly inhibited the formation of MI. In summary, exemestane seems to be metabolized to MI by multiple P450s that include CYP4A11 and CYP1A1/2, whereas its oxidation to MII is primarily mediated by CYP3A.
  Drug metabolism and disposition: the biological fate of chemicals 09/2010; 39(1):98-105. · 3.74 Impact Factor
• Article: Impact of proton pump inhibitors on the effectiveness of clopidogrel after coronary stent placement: the clopidogrel Medco outcomes study.

  Rolf P Kreutz, Eric J Stanek, Ronald Aubert, Jianying Yao, Jeffrey A Breall, Zeruesenay Desta, Todd C Skaar, J Russell Teagarden, Felix W Frueh, Robert S Epstein, David A Flockhart
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  ABSTRACT: To investigate the potential impact of proton pump inhibitors (PPIs) on the effectiveness of clopidogrel in preventing recurrent ischemic events after percutaneous coronary intervention (PCI) with stent placement. Population-based, retrospective cohort study. National medical and pharmacy benefit claims database comprising approximately 19 million members. A total of 16,690 patients who had undergone PCI with stent placement and who were highly adherent to clopidogrel therapy alone (9862 patients) or to clopidogrel with a PPI (6828 patients) between October 1, 2005, and September 30, 2006. The primary end point was the occurrence of a major adverse cardiovascular event during the 12 months after stent placement. These events were defined as hospitalization for a cerebrovascular event (stroke or transient ischemic attack), an acute coronary syndrome (myocardial infarction or unstable angina), coronary revascularization (PCI or coronary artery bypass graft), or cardiovascular death. A composite event rate was compared between patients who received clopidogrel alone and those who received concomitant clopidogrel-PPI therapy. Baseline differences in covariates were adjusted by using Cox proportional hazards models. In the 9862 patients receiving clopidogrel alone, 1766 (17.9%) experienced a major adverse cardiovascular event compared with 1710 patients (25.0%) who received concomitant clopidogrel-PPI therapy (adjusted hazard ratio 1.51, 95% confidence interval 1.39-1.64, p<0.0001). Similar associations of increased risk were observed for each PPI studied (omeprazole, esomeprazole, pantoprazole, and lansoprazole). Concomitant use of a PPI and clopidogrel compared with clopidogrel alone was associated with a higher rate of major adverse cardiovascular events within 1 year after coronary stent placement.
  Pharmacotherapy 08/2010; 30(8):787-96. · 2.90 Impact Factor
• Article: Methadone: a substrate and mechanism-based inhibitor of CYP19 (aromatase).

  Wenjie Jessie Lu, Robert Bies, Landry K Kamden, Zeruesenay Desta, David A Flockhart
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  ABSTRACT: The peripheral conversion of testosterone to estradiol by aromatase is the primary source of endogenous estrogen in postmenopausal women. Studies indicating that placental aromatase is able to metabolize methadone to its primary metabolite, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidin (EDDP), led us to test the hypothesis that methadone is able to act as an inhibitor of aromatase. Using recombinant human CYP19, we examined the ability of methadone to bring about either reversible or mechanism-based inhibition of the conversion of testosterone to estradiol. To test for reversible inhibition, racemic methadone or its metabolite EDDP or 2-ethyl-5-methyl-3, 3-diphenylpyrroline (EMDP) was incubated for 30 min with testosterone at the K(m) (4 microM). To test for mechanism-based inhibition, microsomal preincubations were performed for up to 30 min using racemic methadone (1-1000 microM), R- or S-methadone (0.5-500 microM), or EDDP or EMDP (10 and 100 microM) followed by incubation with testosterone at a V(max) concentration (50 microM). Racemic methadone, EDDP, and EMDP did not act as competitive inhibitors of CYP19. Preincubation of methadone, EDDP, or EMDP with CYP19 resulted in time- and concentration-dependent inhibition, indicating a mechanism-based reaction that destroys CYP19 activity. The K(I) and k(inact) values for racemic methadone were calculated to be 40.6 +/- 2.8 microM and 0.061 +/- 0.001 min(-1), respectively. No stereoselectivity was observed. Methadone is metabolized by CYP19 and may act as a potent inhibitor of CYP19 in vivo. These findings may contribute to variability in methadone clearance, to drug-drug interactions, and to side effects observed in individual patients.
  Drug metabolism and disposition: the biological fate of chemicals 08/2010; 38(8):1308-13. · 3.74 Impact Factor
• Article: Composite functional genetic and comedication CYP2D6 activity score in predicting tamoxifen drug exposure among breast cancer patients.

  Silvana Borges, Zeruesenay Desta, Yan Jin, Azzouz Faouzi, Jason D Robarge, Sanosh Philips, Santosh Philip, Anne Nguyen, Vered Stearns, Daniel Hayes, James M Rae, Todd C Skaar, David A Flockhart, Lang Li
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  ABSTRACT: Accurate assessment of CYP2D6 phenotypes from genotype is inadequate in patients taking CYP2D6 substrate together with CYP2D6 inhibitors. A novel CYP2D6 scoring system is proposed that incorporates the impact of concomitant medications with the genotype in calculating the CYP2D6 activity score. Training (n = 159) and validation (n = 81) data sets were obtained from a prospective cohort tamoxifen pharmacogenetics registry. Two inhibitor factors were defined: 1 genotype independent and 1 genotype based. Three CYP2D6 gene scoring systems, and their combination with the inhibitor factors, were compared. These 3 scores were based on Zineh, Zanger, and Gaedigk's approaches. Endoxifen/NDM-Tam plasma ratio was used as the phenotype. The overall performance of the 3 gene scoring systems without consideration of CYP2D6-inhibiting medications in predicting CYP2D6 phenotype was poor in both the training set (R(2) = 0.24, 0.22, and 0.18) and the validation set (R(2) = 0.30, 0.24, and 0.15). Once the CYP2D6 genotype-independent inhibitor factor was integrated into the score calculation, the R(2) values in the training and validation data sets were nearly twice as high as the genotype-only scoring model: (0.44, 0.43, 0.38) and (0.53, 0.50, 0.41), respectively. The integration of the inhibitory effect of concomitant medications with the CYP2D6 genotype into the composite CYP2D6 activity score doubled the ability to predict the CYP2D6 phenotype. However, endoxifen phenotypes still varied substantially, even with incorporation of CYD2D6 genotype and inhibiting factors, suggesting that other, as yet unidentified factors must be involved in tamoxifen activation.
  The Journal of Clinical Pharmacology 04/2010; 50(4):450-8. · 2.91 Impact Factor