Sarah A. Buhrow

Targeted Delivery of Gemcitabine to Pancreatic Adenocarcinoma Using Cetuximab as a Targeting Agent

One of the key challenges in anticancer therapy is the toxicity and poor bioavailability of the anticancer drugs. Nanotechnology can play a pivotal role by delivering drugs in a targeted fashion to the malignant cells that will reduce the systemic toxicity of the anticancer drug. In this report, we show a stepwise development of a nanoparticle-based targeted delivery system for in vitro and in vivo therapeutic application in pancreatic cancer. In the first part of the study, we have shown the fabrication and characterization of the delivery system containing gold nanoparticle as a delivery vehicle, cetuximab as a targeting agent, and gemcitabine as an anticancer drug for in vitro application. Nanoconjugate was first characterized physico-chemically. In vitro targeting efficacy, tested against three pancreatic cancer cell lines (PANC-1, AsPC-1, and MIA Paca2) with variable epidermal growth factor receptor (EGFR) expression, showed that gold uptake correlated with EGFR expression. In the second part, we showed the in vivo therapeutic efficacy of the targeted delivery system. Administration of this targeted delivery system resulted in significant inhibition of pancreatic tumor cell proliferation in vitro and orthotopic pancreatic tumor growth in vivo. Tumor progression was monitored noninvasively by measuring bioluminescence of the implanted tumor cells. Pharmacokinetic experiments along with the quantitation of gold both in vitro and in vivo further confirmed that the inhibition of tumor growth was due to targeted delivery. This strategy could be used as a generalized approach for the treatment of a variety of cancers characterized by overexpression of EGFR.

Phase II Study of Oral Capsular 4-Hydroxyphenylretinamide (4-HPR/Fenretinide) in Pediatric Patients with Refractory or Recurrent Neuroblastoma: A Report from the Children's Oncology Group

Purpose: To determine the response rate to oral capsular fenretinide in children with recurrent or biopsy proven refractory high-risk neuroblastoma. Experimental Design: Patients received 7 days of fenretinide: 2,475 mg/m2/d divided TID (<18 years) or 1,800 mg/m2/d divided BID (≥18 years) every 21 days for a maximum of 30 courses. Patients with stable or responding disease after course 30 could request additional compassionate courses. Best response by course 8 was evaluated in stratum 1 (measurable disease on CT/MRI ± bone marrow and/or MIBG avid sites) and stratum 2 (bone marrow and/or MIBG avid sites only). Results: Sixty-two eligible patients, median age 5 years (range 0.6–19.9), were treated in stratum 1 (n = 38) and stratum 2 (n = 24). One partial response (PR) was seen in stratum 2 (n = 24 evaluable). No responses were seen in stratum 1 (n = 35 evaluable). Prolonged stable disease (SD) was seen in 7 patients in stratum 1 and 6 patients in stratum 2 for 4 to 45+ (median 15) courses. Median time to progression was 40 days (range 17–506) for stratum 1 and 48 days (range 17–892) for stratum 2. Mean 4-HPR steady-state trough plasma concentrations were 7.25 μmol/L (coefficient of variation 40–56%) at day 7 course 1. Toxicities were mild and reversible. Conclusions: Although neither stratum met protocol criteria for efficacy, 1 PR + 13 prolonged SD occurred in 14/59 (24%) of evaluable patients. Low bioavailability may have limited fenretinide activity. Novel fenretinide formulations with improved bioavailability are currently in pediatric phase I studies. Clin Cancer Res; 17(21); 6858–66. ©2011 AACR.

Inhibition of Mitochondrial Respiration as a Source of Adaphostin-induced Reactive Oxygen Species and Cytotoxicity

Adaphostin is a dihydroquinone derivative that is undergoing extensive preclinical testing as a potential anticancer drug. Previous studies have suggested that the generation of reactive oxygen species (ROS) plays a critical role in the cytotoxicity of this agent. In this study, we investigated the source of these ROS. Consistent with the known chemical properties of dihydroquinones, adaphostin simultaneously underwent oxidation to the corresponding quinone and generated ROS under aqueous conditions. Interestingly, however, this quinone was not detected in intact cells. Instead, high performance liquid chromatography demonstrated that adaphostin was concentrated by up to 300-fold in cells relative to the extracellular medium and that the highest concentration of adaphostin (3000-fold over extracellular concentrations) was detected in mitochondria. Consistent with a mitochondrial site for adaphostin action, adaphostin-induced ROS production was diminished by >75% in MOLT-4 rho0 cells, which lack mitochondrial electron transport, relative to parental MOLT-4 cells. In addition, inhibition of oxygen consumption was observed when intact cells were treated with adaphostin. Loading of isolated mitochondria to equivalent adaphostin concentrations caused inhibition of uncoupled oxygen consumption in mitochondria incubated with the complex I substrates pyruvate and malate or the complex II substrate succinate. Further analysis demonstrated that adaphostin had no effect on pyruvate or succinate dehydrogenase activity. Instead, adaphostin inhibited reduced decylubiquinone-induced cytochrome c reduction, identifying complex III as the site of inhibition by this agent. Moreover, adaphostin enhanced the production of ROS by succinate-charged mitochondria. Collectively, these observations demonstrate that mitochondrial respiration rather than direct redox cycling of the hydroquinone moiety is a source of adaphostin-induced ROS and identify complex III as a potential target for antineoplastic agents.

First-in-Human Phase I Study of the Tamoxifen Metabolite Z-Endoxifen in Women With Endocrine-Refractory Metastatic Breast Cancer

Purpose Endoxifen is a tamoxifen metabolite with potent antiestrogenic activity. Patients and Methods We performed a phase I study of oral Z-endoxifen to determine its toxicities, maximum tolerated dose (MTD), pharmacokinetics, and clinical activity. Eligibility included endocrine-refractory, estrogen receptor-positive metastatic breast cancer. An accelerated titration schedule was applied until moderate or dose-limiting toxicity occurred, followed by a 3+3 design and expansion at 40, 80, and 100 mg per day. Tumor DNA from serum (circulating cell free [cf); all patients] and biopsies [160 mg/day and expansion]) was sequenced. Results Of 41 enrolled patients, 38 were evaluable for MTD determination. Prior endocrine regimens during which progression occurred included aromatase inhibitor (n = 36), fulvestrant (n = 21), and tamoxifen (n = 15). Patients received endoxifen once daily at seven dose levels (20 to 160 mg). Dose escalation ceased at 160 mg per day given lack of MTD and endoxifen concentrations > 1,900 ng/mL. Endoxifen clearance was unaffected by CYP2D6 genotype. One patient (60 mg) had cycle 1 dose-limiting toxicity (pulmonary embolus). Overall clinical benefit rate (stable > 6 months [n = 7] or partial response by RECIST criteria [n = 3]) was 26.3% (95% CI, 13.4% to 43.1%) including prior tamoxifen progression (n = 3). cfDNA mutations were observed in 13 patients ( PIK3CA [n = 8], ESR1 [n = 5], TP53 [n = 4], and AKT [n = 1]) with shorter progression-free survival ( v those without cfDNA mutations; median, 61 v 132 days; log-rank P = .046). Clinical benefit was observed in those with ESR1 amplification (tumor; 80 mg/day) and ESR1 mutation (cfDNA; 160 mg/day). Comparing tumor biopsies and cfDNA, some mutations ( PIK3CA, TP53, and AKT) were undetected by cfDNA, whereas cfDNA mutations ( ESR1, TP53, and AKT) were undetected by biopsy. Conclusion In endocrine-refractory metastatic breast cancer, Z-endoxifen provides substantial drug exposure unaffected by CYP2D6 metabolism, acceptable toxicity, and promising antitumor activity.

Evaluation of CYP2D6 enzyme activity using a 13C-dextromethorphan breath test in women receiving adjuvant tamoxifen

Background In tamoxifen-treated patients, breast cancer recurrence differs according to CYP2D6 genotype and endoxifen steady-state concentrations (Endx Css). The 13C-dextromethorphan breath test (DM-BT), labeled with 13C at the O-CH3 moiety, measures CYP2D6 enzyme activity. We sought to examine the ability of the DM-BT to identify known CYP2D6 genotypic poor metabolizers and examine the correlation between DM-BT and Endx Css. Methods DM-BT and tamoxifen pharmacokinetics were obtained at baseline, 3, and 6 months following tamoxifen initiation. Potent CYP2D6 inhibitors were prohibited. The correlation between baseline DM-BT with CYP2D6 genotype and Endx Css was determined. The association between baseline DM-BT (where values ⩽0.9 is an indicator of poor in vivo CYP2D6 metabolism) and Endx Css (using values⩽11.2 known to be associated with poorer recurrence free survival) was explored. Results A total of 91 patients were enrolled and 77 were eligible. CYP2D6 genotype was positively correlated with baseline, 3, and 6 months DM-BT (r ranging from 0.457–0. 60; P<0.001). Both CYP2D6 genotype (r=0.47, 0.56, P<0.0001), and baseline DM-BT (r=0.60, 0.54, P<0.001) were associated with 3 and 6 months Endx Css, respectively. Seven (78%) of nine patients with low (⩽11.2 nmol/l) 3 month Endx Css also had low DM-BT (⩽0.9) including 2/2 CYP2D6 PM/PM and 5/5 IM/PM. In contrast, one (2%) of 48 patients with a low DM-BT had Endx Css more than 11.2 nmol/l. Conclusion In patients not taking potent CYP2D6 inhibitors, DM-BT was associated with CYP2D6 genotype and 3 and 6 months Endx Css but did not provide better discrimination of Endx Css compared with CYP2D6 genotype alone. Further studies are needed to identify additional factors which alter Endx Css.

Antibody-Targeted Chemotherapy for the Treatment of Melanoma.

Antibody-directed chemotherapy (ADC) offers an advantage over conventional chemotherapy because it provides antibody-directed targeting, with resultant improvement in therapeutic efficacy and reduced toxicity. Despite extensive research, with notable exceptions, broad clinical application of ADC remains elusive; major hurdles include the instability of antibody-chemotherapy linkers and reduced tumor toxicity of the chemotherapy when bound to the antibody. To address these challenges, we have developed a platform technology that utilizes the nab-paclitaxel formulation of paclitaxel, Abraxane, in which hydrophobic paclitaxel is suspended in 130-nm albumin nanoparticles and thus made water-soluble. We have developed a method to noncovalently coat the Abraxane nanoparticle with recombinant mAbs (anti-VEGF, bevacizumab) and guide Abraxane delivery into tumors in a preclinical model of human A375 melanoma. Here, we define the binding characteristics of bevacizumab and Abraxane, demonstrate that the chemotherapy agent retains its cytotoxic effect, while the antibody maintains the ability to bind its ligand when the two are present in a single nanoparticle (AB160), and show that the nanoparticle yields improved antitumor efficacy in a preclinical human melanoma xenograft model. Further data suggest that numerous therapeutic monoclonal IgG1 antibodies may be utilized in this platform, which has implications for many solid and hematologic malignancies. Cancer Res; 76(13); 3954-64. ©2016 AACR.

Abstract 4631: Pharmacokinetics and in vivo metabolism of Z-endoxifen: Results from two phase I studies in women with ER+ breast cancer, gynecologic malignancies and desmoids

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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

Phase II study of oral capsular 4-hydroxyphenylretinamide (4-HPR/fenretinide) in pediatric patients with refractory or recurrent neuroblastoma: A report from the Children’s Oncology Group NSC #374551; IND# 40294

Purpose: To determine the response rate to oral capsular fenretinide in children with recurrent or biopsy proven refractory high-risk neuroblastoma. Experimental Design: Patients received 7 days of fenretinide: 2,475 mg/m2/d divided TID (<18 years) or 1,800 mg/m2/d divided BID (≥18 years) every 21 days for a maximum of 30 courses. Patients with stable or responding disease after course 30 could request additional compassionate courses. Best response by course 8 was evaluated in stratum 1 (measurable disease on CT/MRI ± bone marrow and/or MIBG avid sites) and stratum 2 (bone marrow and/or MIBG avid sites only). Results: Sixty-two eligible patients, median age 5 years (range 0.6–19.9), were treated in stratum 1 (n = 38) and stratum 2 (n = 24). One partial response (PR) was seen in stratum 2 (n = 24 evaluable). No responses were seen in stratum 1 (n = 35 evaluable). Prolonged stable disease (SD) was seen in 7 patients in stratum 1 and 6 patients in stratum 2 for 4 to 45+ (median 15) courses. Median time to progression was 40 days (range 17–506) for stratum 1 and 48 days (range 17–892) for stratum 2. Mean 4-HPR steady-state trough plasma concentrations were 7.25 μmol/L (coefficient of variation 40–56%) at day 7 course 1. Toxicities were mild and reversible. Conclusions: Although neither stratum met protocol criteria for efficacy, 1 PR + 13 prolonged SD occurred in 14/59 (24%) of evaluable patients. Low bioavailability may have limited fenretinide activity. Novel fenretinide formulations with improved bioavailability are currently in pediatric phase I studies. Clin Cancer Res; 17(21); 6858–66. ©2011 AACR.

Tamoxifen, HER2, and Endoxifen: The Role of CYP2D6 as a Predictor of Tamoxifen Resistance in ER+/HER2+ Breast Cancer.

Background: Endoxifen, the active metabolite of tamoxifen (Tam), is currently being developed as a drug for the treatment of estrogen receptor (ER) + breast cancer (BC). HER2 expression in ER+ BC is associated with Tam resistance, and in vivo Tam administration to mice bearing ER+/HER2+ xenografts stimulates BC growth (Shou, JNCI 2004). In humans, endoxifen is the most important Tam metabolite responsible for inhibiting estrogen induced BC growth (Wu, Cancer Research 2009). CYP2D6 metabolism affects the concentrations (conc) of endoxifen (Stearns, JNCI, 2003) and associated with worse disease free survival (DFS) in Tam treated BC (Goetz, JCO, 2005). We sought to determine the activity of Tam and its metabolites in ER+/HER2+ BC cell lines and to evaluate the role of CYP2D6 metabolism in Tam-treated patients (pts) with ER+/HER2+ BC. Additionally, we sought to determine endoxifen conc in mice administered oral Tam.Methods: MCF7 (parental and HER2-expressing) and BT474 (ER+/HER2+) cells were used to compare the activity of Tam, 4HT, and endoxifen on estrogen- stimulated growth. Oral tam PK were characterized in mice treated with standard dose of Tam (4 mg/kg; 100 μg). Clinical data were obtained via a retrospective analysis of Tam-treated pts with ER+/ HER2+ BC randomized to receive 5 years of Tam (NCCTG 89-30-52). CYP2D6 metabolism (extensive or decreased) was based on CYP2D6 genotype (*3, 4, 6, 10, 17, 41) and co-administration of a CYP2D6 inhibitor (yes/no). HER2 was determined by immunohistochemistry (IHC) or FISH (tumors 0, 1, or 2+ by IHC). The association between CYP2D6 and DFS was assessed using the log-rank test and proportional hazards modeling.Results: Compared to Tam, endoxifen potently inhibited the growth of estrogen- stimulated BT474 cells. In MCF7 cells, expression of HER2 shifted the conc of endoxifen required for 50% inhibition of growth (IC50) from 54 nM (parental) to 131 nM (HER2 expressing). Using the range of conc of Tam and its metabolites observed in humans (Tam, 300-500 nM; 4HT, 5-10 nM; and endoxifen, 10-200 nM), only endoxifen potently inhibited estrogen- stimulated growth of MCF7HER2+ cells and only at conc achievable in CYP2D6 extensive metabolizers (>50nM). In mice, conc of 4HT and endoxifen were below 15 nM following an oral dose of 4 mg/kg. In NCCTG 89-30-52, both CYP2D6 phenotype and HER2 status was determined in 201/256 randomized pts. HER2 was expressed in 23/215 (11%) but not associated with DFS overall (p=0.62). In the HER2+ subset, pts with decreased CYP2D6 metabolism (n=10) had significantly shorter DFS compared to extensive metabolizers (n=9) (HR 9.5, p=0.03; 95% CI 1.16-76.9).Conclusions: Our in vitro and clinical data provide a simple pharmacological model for understanding HER2 resistance in Tam-treated breast cancer. Mice, which lack the CYP2D6 enzyme, may not be an appropriate model for understanding tam resistance given low conc of both 4HT and endoxifen. Given that the in vitro conc of endoxifen needed to inhibit the growth of ER+/HER-2+ BC are achievable in only a subset of humans (CYP2D6 extensive metabolizers), the primary administration of endoxifen could overcome de novo Tam resistance in ER+/HER2+ BC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2006.

Comparative Metabolism of Batracylin (NSC 320846) and N-acetylbatracylin (NSC 611001) Using Human, Dog, and Rat Preparations In Vitro

BACKGROUND Batracylin is a heterocyclic arylamine topoisomerase inhibitor with preclinical anticancer activity. Marked species differences in sensitivity to the toxicity of batracylin were observed and attributed to differential formation of N-acetylbatracylin by N-acetyltransferase. A Phase I trial of batracylin in cancer patients with slow acetylator genotypes identified a dose-limiting toxicity of hemorrhagic cystitis. To further explore the metabolism of batracylin and N-acetylbatracylin across species, detailed studies using human, rat, and dog liver microsomal and hepatocyte preparations were conducted. METHODS Batracylin or N-acetylbatracylin was incubated with microsomes and hepatocytes from human, rat, and dog liver and with CYP-expressing human and rat microsomes. Substrates and metabolites were analyzed by HPLC with diode array, fluorescence, radiochemical, or mass spectrometric detection. Covalent binding of radiolabeled batracylin and N-acetylbatracylin to protein and DNA was measured in 3-methylcholanthrene-induced rat, human, and dog liver microsomes, and with recombinant human cytochromes P450. RESULTS In microsomal preparations, loss of batracylin was accompanied by formation of one hydroxylated metabolite in human liver microsomes and five hydroxylated metabolites in rat liver microsomes. Six mono- or di-hydroxy-N-acetylbatracylin metabolites were found in incubations of this compound with 3MC rat liver microsomes. Hydroxylation sites were identified for some of the metabolites using deuterated substrates. Incubation with recombinant cytochromes P450 identified rCYP1A1, rCYP1A2, hCYP1A1 and hCYP1B1 as the major CYP isoforms that metabolize batracylin and N-acetylbatracylin. Glucuronide conjugates of batracylin were also identified in hepatocyte incubations. NADPH-dependent covalent binding to protein and DNA was detected in all batracylin and most N-acetylbatracylin preparations evaluated. CONCLUSIONS Microsomal metabolism of batracylin and N-acetylbatracylin results in multiple hydroxylated products (including possible hydroxylamines) and glutathione conjugates. Incubation of batracylin with hepatocytes resulted in production primarily of glucuronides and other conjugates. There was no clear distinction in the metabolism of batracylin and N-acetylbatracylin across species that would explain the differential toxicity.