Safgren, Stephanie L.

Abstract Background: Breast cancer recurrence has been shown to differ based upon CYP2D6 genotype and endoxifen (Endx) exposure following treatment with tamoxifen (TAM) in the adjuvant setting. The average Endx Css is 29 nM (range, 2-80 nM) in TAM treated patients at a dose of 20 mg/day. Administration of Endx itself to patients is predicted to provide adequate, consistent, active drug levels in all treated patients, resulting in clinical benefit. As part of two phase 1 trials of oral Endx to determine the safety, tolerability, and MTD, we characterized the pharmacokinetics and in vivo metabolism of Endx. Methods: Patients were given escalating daily oral doses of Endx (28 day cycle) over the dose range of 20 - 160 mg/day in Phase I trials at the Mayo Clinic and NCI. Pharmacokinetics sampling was performed on days 1, 7, 14 and 28, and prior to subsequent cycles. Endx plasma concentrations and metabolite profiles were determined by HPLC and LC/MS/MS. Results: 44 patients with ECOG PS 0-1 have been enrolled to date encompassing 8 dose levels in the Mayo (25 women with AI refractory MBC; median age 58 yrs, range 41-83 yrs) and NCI (19 women with ovary (9), breast (4), desmoid (2), fallopian tube (2), endometrial cancer (2); median age 64 yrs, range 43-75 yrs) studies. Day 1 Endx peak concentrations (Cmax) were reached 4h after the Endx oral dose and mean values of Cmax, C24h, and AUC0-24h increased in proportion to dose. Css was achieved on day 7 and 3.5-fold accumulation (t1/2= 50.5 h) was observed on day 28. At the starting dose ( 20 mg/day) and highest dose level (160 mg/day), Endx Css values of 146 ng/ml (390 nM) and 1950 ng/ml (5200 nM), respectively, were achieved and maintained throughout the 28-day treatment. Endx Css values remained unchanged following continuous dosing for 8-10 months. The mean apparent steady-state clearance was 6.2 L/h. We also characterized the Endx metabolite profile in patient plasma and urine. Metabolites detected in plasma include nor-Endx, N-hydroxy-Endx, Endx-catechol, methoxy-Endx catechol, and Endx-glucuronide, and appear in amounts much lower compared to Endx. Like the parent drug, metabolites accumulated in plasma over the 28-day cycle. Characterization of the pharmacokinetics of Nor-Endx, a putative aromatase inhibitor, is ongoing and will be presented. Conclusion: In these ongoing Phase I studies, daily administration of Endx is well tolerated with Css consistently above those obtained following administration of standard dose tamoxifen. In the Mayo study, expansion at 20 and 100 mg/day has commenced to perform translational studies and further characterize Endx PK. In the NCI study, dose escalation continues to establish the maximum tolerated dose. Supported in part by R01CA 133049, U01 CA69912, P30CA 15083, P50CA 116201 and NCI Contracts CM52206 and HHSN261201100014C. Citation Format: Joel M. Reid, Matthew P. Goetz, Shivaani Kumar, Renee M. McGovern, Sarah A. Buhrow, Stephanie L. Safgren, Vera J. Suman, Travis J. Docktor, Charles Erlichman, Howard Streicher, James H. Doroshow, Jerry Collins, Matthew M. Ames. Pharmacokinetics and in vivo metabolism of Z-endoxifen: Results from two phase I studies in women with ER+ breast cancer, gynecologic malignancies and desmoids. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4631. doi:10.1158/1538-7445.AM2014-4631

Abstract Background: CYP2D6 is the rate limiting enzyme responsible for the metabolic activation of tamoxifen (tam) to endoxifen. Compared to CYP2D6 poor metabolizers (PM), tam-treated CYP2D6 extensive metabolizers (EM) have higher endoxifen concentrations, more vasomotor symptoms (Goetz, MP J Clin Oncol 2005), and are more likely to discontinue tam (Rae, JM 2009. Pharmacogenomics J). Additionally, higher endoxifen concentrations are associated with a stepwise increase in tam side-effects (Lorizio, W Breast Cancer Res Treat 2012). The data regarding CYP2D6 genotype and recurrence is mixed. Venlafaxine is a weak CYP2D6 inhibitor not known to alter tam pharmacokinetics (PK) and commonly recommended for tam induced hot flashes. We conducted a multicenter pharmacological study to determine whether venlafaxine altered the PK of tam and to determine the distribution of CYP2D6 genotypes in this population Methods: Women taking tam for at least 4 weeks and for whom venlafaxine was recommended for the treatment of hot flashes were eligible. Blood samples were collected prior to and 8–16 weeks following initiation of venlafaxine for steady state tam and metabolites. Genotyping was performed for alleles associated with no (PM; *3, *4, *5,*6); reduced (intermediate, IM; *10, 17 and *41); and ultra-rapid (UM; *1×2) metabolism. Power calculations demonstrated that 17 patients with paired samples were required (two-sided alpha=0.05 t-test, 90% power) to detect a 25% change in endoxifen levels after at least 8 weeks of concurrent treatment. Results: 30 women (median age 48.5) initiated venlafaxine. CYP2D6 genotypes were within Hardy Weinberg Equilibrium (HWE). CYP2D6 UM allele frequency (6.7%) was higher while CYP2D6 *4 (13.3%) was lower than expected compared to an unselected population (0.5 and 21% respectively; Sachse Am. J. Hum. Genet. 1997), resulting in the absence of CYP2D6 PM/PM. Mean (min/max) baseline endoxifen concentrations (8.73; 1.5–20.5 ng/ml) were correlated with CYP2D6 phenotype as follows: intermediate (EM/PM, PM/IM): 6.8 (1.5–11.2); extensive (EM/EM, EM/IM): 9.4 (1.5–20.5) and ultra-rapid (UM/EM: 11.0; 7.8–14) (r2 = 0.35 p = 0.05). In patients with paired samples (n = 20), venlafaxine resulted in a 23% decrease in endoxifen (−2.06 ng/ml; 95% CI −0.69 to −3.04; p = 0.004), but not tam, NDMT, or 4HT concentrations. Following initiation of venlafaxine, CYP2D6 genotype was no longer associated with endoxifen concentrations (r2 = 0.28 p = 0.23). For women with reduced CYP2D6 metabolism [EM/PM (n = 9) or PM/IM (n = 1)], venlafaxine lowered endoxifen concentrations (−2.98 ng/ml) to a level (5.41 ng/ml) reported to be associated with a higher risk of recurrence in adjuvant tam treated patients (Madlensky, L Clin Pharmacol Ther 2011). Conclusions: In this study, women with tam-induced vasomotor symptoms requiring venlafaxine were comprised predominantly of CYP2D6 EM and UM metabolizers. Venlafaxine significantly decreased endoxifen concentrations. Although the optimal concentration of endoxifen is unknown, given prior data linking low endoxifen concentrations with recurrence, venlafaxine should be used with caution in tam treated patients. (Supported by R01CA133049) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD10-08.

Abstract Background: In AI refractory MBC, TAM anti-tumor activity is limited (median PFS 4.5 months). In patients (pts) receiving TAM in the adjuvant and metastatic setting, breast cancer recurrence has been shown to differ based upon CYP2D6 genotype and Endx exposure. In collaboration with NCI, Z-Endx (NSC #750393) was synthesized and preclinical pharmacology studies demonstrated that oral Z- Endx resulted in > 20 fold Endx exposure compared to an equivalent dose of TAM and significantly greater anti-tumor activity than TAM in AI sensitive and resistant MCF7 xenografts. We conducted a phase I study of Z-Endx in women with ER+, AI refractory MBC to identify dose-limiting toxicities (DLT), a maximum tolerated dose (MTD) and a dose associated with steady state concentrations (Css) of ≥ 2 uM, based on the IC50 of recently identified Endx substrates: PKC and PI3K. Methods: Pts were enrolled to an accelerated titration schedule (2 pts/dose level) until moderate toxicity or DLT, and then to a 3+3 design. Z-Endx was administered orally once daily (28 day cycle). Eye exams were performed at baseline, end of cycle 2, and after 6 cycles. PK was performed on days 1-2, 3, 7, 14, and 28, and prior to subsequent cycles. Results: 23 women with AI refractory MBC (median 56 yrs, range 41-83) received Z-Endx once daily encompassing 7 dose levels (Table 1). The median number of prior chemotherapies and hormonal therapies in the metastatic setting were 2 and 2, respectively. Dose escalation continues at 160 mg/day and the MTD has yet to be determined. Cmax and AUC increased in a dose-dependent manner. A 20 and 100 mg/day dose yields Css of 0.39 and 2.48 uM, respectively. Significant anti-tumor activity was observed including 1 pt (100 mg/day) with a PR lasting 225 days; 1 pt (80 mg/day) with a 30% reduction in tumor size (PFS 169 days) and 2 pts (60 and 80 mg/day) with stable disease for >270 days. No eye toxicity was observed. Conclusions: In women with AI refractory MBC, the MTD of Z-Endx HCl has not been determined but Endx Css concentrations of > 2 uM and substantial anti-tumor activity has been observed. Following completion of the 160 mg/day dose, expansion at 20 and 100 mg/day will commence to perform translational studies and further characterize Endx pk. A randomized phase II examining two different doses of Z-Endx is planned in AI refractory MBC. Supported in part by CA 133049, CA69912, CA15083, CA116201, and CA15083. Table 1Dose Level (mg/day)Number of Treatment CyclesModerate or Severe Toxicities ReportedTumor ResponseProgression-free Survival (days)28 day Z-Endx Css (uM) 20 (n = 2)2; 3Gr 2 hot flashes (1 pt)----60; 850.39 40 (n = 2)2; 6noneStable (1 pt)61; 1670.66 60 (n = 6)1; 2; 2; 5; 8; 14Gr 4 Triglycerides (1 pt);Gr 3 thromboembolic event (1 pt); Gr 2 hot flashes, anemia, and hypoalbuminemia (1 pt)Stable (3 pts)125+; 56; 57; 132; 232; 4331.01 80 (n = 3)4; 6; 10Gr 2 hot flashesStable (3 pts)113; 169; 2961.61 100 (n = 3)1; 2; 8Gr 2 nausea (1 pt); Gr 2 irritability (1 pt)PR (1 pt)30; 56; 2252.48 120 (n = 3)1; 2; 4Gr 2 hypersomnia, paresthesia, and peripheral sensory neuropathy (1 pt)—39; 54; 1082.18 * Bold face toxicities occurred during cycle 1 Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD3-4.

Endoxifen (4-hydroxy-N-desmethyl-tamoxifen), one of the major active metabolites of tamoxifen, has substantially greater estrogen antagonist properties and antiproliferative effects in breast tumor cells than tamoxifen, a mixed estrogen agonist/antagonist. An associated risk of endometrial cancer and hyperplasia has been linked to the estrogen agonist properties of tamoxifen. We evaluated endoxifen using a classic uterotrophic effects method. Rats were given endoxifen or tamoxifen orally for 3 days. Estradiol was the positive control. Endoxifen and tamoxifen plasma levels exceeded those previously observed clinically. Uterine weight was 3-fold higher in the estradiol group than in the tamoxifen or endoxifen groups, which did not differ from vehicle controls. Tamoxifen and endoxifen caused a greater increase in luminal epithelial cell height than estradiol. Both tamoxifen and endoxifen produced an increase in the stromal BrdU labeling index (LI) that was ≤ estradiol and inversely related to dose, but did not affect luminal epithelial cell BrdU LI. As expected, estradiol increased luminal epithelial cell proliferation. These results indicate that endoxifen induces uterotrophic effects, but is less potent than estradiol in eliciting these effects. Given prior preclinical observations that endoxifen has superior antitumor activity than tamoxifen, the observations of similar uterine effects suggest that the endoxifen risk/benefit ratio may be superior to tamoxifen.

Abstract Purpose: Controversy exists about CYP2D6 genotype and tamoxifen efficacy. Experimental Design: A matched case–control study was conducted using the Austrian Breast and Colorectal Cancer Study Group Trial 8 (ABCSG8) that randomized postmenopausal women with estrogen receptor (ER)-positive breast cancer to tamoxifen for 5 years (arm A) or tamoxifen for 2 years followed by anastrozole for 3 years (arm B). Cases had disease recurrence, contralateral breast cancer, second non–breast cancer, or died. For each case, controls were identified from the same treatment arm of similar age, surgery/radiation, and tumor–node—metastasis (TNM) stage. Genotyping was conducted for alleles associated with no (PM; *3, *4, *6), reduced (IM; *10, and *41), and extensive (EM: absence of these alleles) CYP2D6 metabolism. Results: The common CYP2D6*4 allele was in Hardy–Weinberg equilibrium. In arm A during the first 5 years of therapy, women with two poor alleles [PM/PM: OR, 2.45; 95% confidence interval (CI), 1.05–5.73, P = 0.04] and women with one poor allele (PM/IM or PM/EM: OR, 1.67; 95% CI, 0.95–2.93; P = 0.07) had a higher likelihood of an event than women with two extensive alleles (EM/EM). In years 3 to 5 when patients remained on tamoxifen (arm A) or switched to anastrozole (arm B), PM/PM tended toward a higher likelihood of a disease event relative to EM/EM (OR, 2.40; 95% CI, 0.86–6.66; P = 0.09) among women on arm A but not among women on arm B (OR, 0.28; 95% CI, 0.03–2.30). Conclusion: In ABCSG8, the negative effects of reduced CYP2D6 metabolism were observed only during the period of tamoxifen administration and not after switching to anastrozole. Clin Cancer Res; 19(2); 500–7. ©2012 AACR.

Abstract Background: In tamoxifen (TAM) treated patients, both CYP2D6 metabolism and low steady state concentrations of endoxifen (END), the active metabolite of TAM, have been associated with a higher risk of recurrence (Madlensky L, Clin Pharmacol Ther. 2011; 89(5):718-25). END drug development is ongoing at Mayo and NCI, and our previous data demonstrated that 1) END had high oral bioavailability in mice and yielded plasma concentrations and total exposure 20-fold greater as compared to an equivalent oral dose of TAM (MM Ames et al, Cancer Res 2011;70(8 Suppl 1):Abstract nr 3603) and 2) END is superior to TAM in both HER2 (Reinicke AACR 2011) and aromatase expressing (Goetz, SABC 2011) MCF-7 xenograft models. While subcutaneous (SQ) TAM is used by most researchers to study and develop models of TAM resistance, little is known about 1) whether murine and human TAM pharmacokinetics are comparable and 2) whether the route of administration affects concentrations of the active metabolites. Therefore, we conducted murine pharmacokinetic studies evaluating both the oral and SQ administration of TAM and END. Results: Following administration of TAM, concentrations of the active metabolites (END and 4-hydroxy TAM [4HT]) were therapeutic (Cmax- END 35.9 nM, 4HT 62.8 nM) only after a 0.5 mg oral dose of TAM. In contrast, following all doses of SQ TAM, concentrations of the active metabolites (END and 4HT) were sub-therapeutic (Cmax- END 5.2 nM; 4HT 18.7 nM). In contrast to humans, the Cmax of N-desmethyl TAM, the most abundant human TAM metabolite, was < 16.5 nM in mice after oral and SQ TAM. SQ administration of END and TAM resulted in substantially greater bioavailability of the parent drug compared with oral dosing. Following administration of 0.25-5 mg oral END and 0.0625-0.625 mg subcutaneous END, the increase in AUC was greater than dose proportional and active concentrations (>80 nM) were detected in plasma at every dose and both dosing routes. Comparable END and TAM plasma concentrations were achieved with a 0.0625 mg SQ END and a 0.5 mg SQ TAM, respectively. Conclusions: Our data strongly suggest that SQ dosing of TAM results in non-therapeutic concentrations of the active hydroxylated metabolites, and may be a simple pharmacokinetic factor influencing response and resistance in a murine in vivo xenograft model. Given the nonlinear pharmacokinetics and differences in metabolism, the dose and route of END must be carefully selected for comparative studies with TAM. These data support the ongoing human studies designed to bypass the need for TAM metabolism by the direct administration of END. Supported by the Mayo Comprehensive Cancer Center Grant (CA15083) and the Mayo Clinic Breast Cancer SPORE (CA 116201; MMA, JMR, MPG). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3781. doi:1538-7445.AM2012-3781

Abstract Background: Controversy exists regarding the association between CYP2D6 enzyme activity and tamoxifen effectiveness in the adjuvant treatment of invasive breast cancer; however, this association in the primary prevention of breast cancer is unknown. Methods: We conducted a nested case–control study in the context of the NSABP P1 and P2 prevention trials to determine the impact of CYP2D6 genotype, CYP2D6 inhibitor use, and metabolizer status (CYP2D6 genotype combined with CYP2D6 inhibitor use), on breast cancer events. Women who developed breast cancer (both noninvasive and invasive) while on 5 years of selective estrogen receptor modulators therapy (cases) were matched to controls free of breast cancer. Comprehensive CYP2D6 genotyping was conducted for alleles associated with absent (*3, *4, *5, and *6), reduced (*10, *17, and *41), and increased (*1XN and *2XN) enzyme activity. Information regarding the use of CYP2D6 inhibitors was recorded. Results: A total of 591 cases were matched to 1,126 controls and DNA was genotyped in more than 97%. In patients treated with tamoxifen, there was no association of CYP2D6 genotype [OR (extensive/poor metabolizer): 0.90; 95% CI: 0.46–1.74, P = 0.74), use of a potent CYP2D6 inhibitor (OR 0.92; 95% CI: 0.575–1.486), or CYP2D6 metabolizer status (OR 1.03; 95% CI: 0.669–1.607) with breast cancer occurrence. Likewise, there was no association between any CYP2D6 metabolism parameter with breast cancer events in raloxifene-treated patients. Conclusion: In the NSABP P1 and P2 clinical trials, alterations in CYP2D6 metabolism are not associated with either tamoxifen or raloxifene efficacy. Clin Cancer Res; 17(21); 6944–51. ©2011 AACR.

Abstract Endoxifen (END), the active metabolite of tamoxifen (TAM), is currently being developed as a drug for the treatment of estrogen receptor positive breast cancer based on recent in vitro and clinical data demonstrating that TAM drug effect is substantially related to circulating plasma concentrations of END, a secondary metabolite produced by human CYP2D6 metabolism. Women with deficient CYP2D6 metabolism have lower END concentrations and a higher risk of breast cancer recurrence. We characterized the pharmacokinetics and oral bioavailability of END in female rats and dogs. We further compared END exposure following equivalent doses of END and TAM. Rats were administered 2 mg/kg i.v. END, 20 − 200 mg/kg oral END or 80 mg/kg oral TAM. Fasted dogs were administered 0.5 mg/kg i.v. END, 15 − 100 mg/kg oral END or 30 mg/kg oral TAM. Plasma END concentrations were also measured in rats or dogs after oral dosing once per day for 4 consecutive days. Plasma samples were analyzed using a validated reverse-phase HPLC method with fluorescence detection. In the single-dose administration study, terminal elimination half-life and plasma clearance values in rats given 2 mg/kg i.v. END were 6.3 h and 2.4 L/h/kg, respectively. Plasma concentrations above 0.1 µM and 1 µM END were achieved with 20 mg/kg and 200 mg/kg doses, respectively, and those concentrations were maintained for longer than 24 h. Oral absorption of END was linear over the range of 20 − 140 mg/kg with a bioavailability greater than 67%. END exposure in rats following 80 mg/kg oral END was 100-fold greater than END exposure following 80-mg/kg oral TAM. Following 0.5 mg/kg i.v. END to dogs, terminal elimination half-life and plasma clearance values were 9.2 h and 0.4 L/h/kg, respectively. Plasma concentrations above 1 µM and 10 µM were achieved with the 15 mg/kg dose and 100 mg/kg, respectively, and those concentrations were maintained for longer than 24 h. Absorption was linear over the range of 15 − 100 mg/kg with a bioavailability greater than 50%. END exposure in dogs following 30 mg/kg oral END was 10-fold greater than END exposure following 30-mg/kg oral TAM. In the repeated-dose administration study, the day 4 END peak plasma concentration in rats increased from 1 to 9 µM as the daily END dose was increased from 20 to 200 mg/kg. The day 4 END peak plasma concentration in dogs exceeded 20 µM and plasma concentrations remained above 2 µM for 24 hours following 4 daily doses of 30 mg/kg END. Based on these data, we conclude that END has high oral bioavailability; high concentrations may be maintained after repeated dosing in dogs and rats; and substantially higher END concentrations are achievable following an oral dose of END in comparison to a similar dose of TAM. Supported by NCI contract N01-CM-52206. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2607.

Abstract In collaboration with NCI, we are developing endoxifen (END), the active metabolite of tamoxifen (TAM), as a drug for the treatment of estrogen receptor positive breast cancer. Tamoxifen treated women with reduced or absent CYP2D6 enzyme activity have significantly lower END plasma concentrations. In a retrospective analysis of two different prospective tamoxifen trials (NCCTG and ABCSG), we have demonstrated that genetic or drug-induced reductions in CYP2D6 metabolism are associated with a higher risk of breast cancer recurrence in Tam treated ER positive breast cancer. To test the hypothesis that clinically relevant concentrations and exposures of END are achieved following oral administration of END, we characterized the pharmacokinetics of i.v. and oral END and oral TAM in female mice. Endoxifen was synthesized and provided by NCI/DTP. Plasma samples were analyzed using a validated reverse-phase HPLC method with fluorescence detection. Following i.v. administration of a 1 mg/kg dose, peak plasma concentration, terminal elimination half-life and plasma clearance values were 0.26 µM, 6.5 h and 11.8 L/h/kg, respectively. Oral pharmacokinetics and bioavailability were determined following doses of 50 and 200 mg/kg. Peak plasma concentrations of 0.76 µM were achieved with a 50 mg/kg dose and plasma concentrations above 0.1 µM were maintained for longer than 8 h. Peak plasma concentrations of 8 µM were achieved with a 200 mg/kg dose and plasma concentrations above 2 µM were maintained for 24 h. Bioavailability was 50% and greater than 100% following the 50 mg/kg and 200 mg/kg doses, respectively. Based on pharmacokinetic data for oral END and TAM normalized to a 50 mg/kg oral dose, END plasma concentrations and total exposure were 20-fold greater following oral END as compared to oral TAM. In conclusion, END has high oral bioavailability in mice and substantial plasma concentrations are achieved and maintained after a single oral END dose. Compared to an equivalent oral dose of Tam, substantially higher END concentrations are achieved following oral END. These data suggest that the primary administration of End may overcome the limitations related to human CYP2D6 metabolism, and support the ongoing development of endoxifen as a primary therapy for ER breast cancer. Supported by the Mayo Comprehensive Cancer Center Grant (CA15083) and the Mayo Clinic Breast Cancer SPORE (CA 116201; MMA, JMR, MPG). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3603.