Downloaded from http://journals.lww.com/anti-cancerdrugs by BhDMf5ePHKbH4TTImqenVHwXmMsAVI5gwwTmSakDrhWvEgT1T2qZXHu9I6YkevtZ on 01/17/2019 Cytotoxicity and molecular activity of fenretinide and metabolites in T-cell lymphoid malignancy, neuroblastoma, and ovarian cancer cell lines in physiological hypoxia Michael M. Song a,b, *, Monish R. Makena a,b, *, Ashly Hindle a,b , Balakrishna Koneru a,b , Thinh H. Nguyen a,c , Dattesh U. Verlekar a,b , Hwangeui Cho a,b , Barry J. Maurer a,b,d,e , Min H. Kang a,b,c,d and C. Patrick Reynolds a,b,c,d,e Objective All-trans-N-(4-hydroxyphenyl)retinamide or fenretinide (4-HPR) acts by reactive oxygen species (ROS) and dihydroceramides (DHCers). In early-phase clinical trials 4-HPR has achieved complete responses in T-cell lymphomas (TCL) and neuroblastoma (NB) and signals of activity in ovarian cancer (OV). We defined the activity of 4-HPR metabolites in N-(4-methoxyphenyl)retinamide (MPR), 4-oxo-N-(4-hydroxyphenyl)retinamide (oxoHPR), and the 4-HPR isomer 13-cis-fenretinide (cis-HPR) in NB, OV, and TCL cell lines cultured in physiological hypoxia. Methods We compared the effect of 4-HPR, cis-HPR, oxoHPR, and MPR on cytotoxicity, ROS, and DHCers in a panel of TCL, NB, and OV cell lines cultured in bone marrow level physiological hypoxia (5% O 2 ), utilizing a fluorescence- based cytotoxicity assay (DIMSCAN), flow cytometry, and quantitative mass spectrometry. Results 4-HPR (10 μmol/l) achieved more than three logs of cell kill in nine of 15 cell lines. Cytotoxicity of 4-HPR and oxoHPR was comparable; in some cell lines, cis-HPR cytotoxicity was lower than 4-HPR, but additive when combined with 4-HPR. MPR was not cytotoxic. ROS and DHCers were equivalently increased by 4-HPR and oxoHPR in all cell lines (P < 0.01), to a lesser extent by cis-HPR (P < 0.01), and not increased in response to MPR (P > 0.05). Mitochondrial membrane depolarization, caspase-3 cleavage, and apoptosis (TUNEL) were all significantly increased by 4-HPR and oxoHPR (P < 0.01). Conclusion Cytotoxic and pharmacodynamic activity was comparable with 4-HPR and oxoHPR, lower with cis-HPR, and MPR was inactive. Neither MPR or cis-HPR antagonized 4-HPR activity. These data support focusing on achieving high 4-HPR exposures for maximizing antineoplastic activity. Anti-Cancer Drugs 30:117–127 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. Anti-Cancer Drugs 2019, 30:117–127 a Cancer Center, Departments of b Cell Biology and Biochemistry, c Pharmacology and Neuroscience, d Pediatrics and e Internal Medicine, Texas Tech University Health Sciences Center School of Medicine, Lubbock, Texas, USA Correspondence to C. Patrick Reynolds, MD, PhD, 3601 4th Street STOP 9445, Lubbock, TX 79430, USA Tel: +1 806 743 1558; fax: +1 806 743 2691; e-mail: patrick.reynolds@ttuhsc.edu *Michael M. Song and Monish R. Makena contributed equally to the writing of this article. Received 16 July 2018 Revised form accepted 30 August 2018 Introduction All-trans-N-(4-hydroxyphenyl)retinamide or fenretinide (4-HPR) is a synthetic retinoid with cytotoxicity to var- ious types of cancer cell lines in vitro, including neuro- blastoma (NB) [1–3], leukemia [4–8], and ovarian cancer (OV) [9–11]. 4-HPR formulated as micropulverized crystals in corn oil containing oral capsules showed lim- ited clinical activity in recurrent NB [12,13]. In an OV phase II trial of 4-HPR oral capsules, there were no objective responses but patients with 4-HPR plasma levels of at least 9 μmol/l had a significantly longer overall survival than patients with lower plasma levels [14]. These clinical trials showed the limited bioavailability of the corn oil capsule formulation with achieved mean peak plasma concentrations of ~ 1–10 μmol/l at doses in the range of 200 to 2475 mg/m 2 /day [15,16]. Recently, an intravenous emulsion formulation of 4-HPR [16], as well as an oral formulation with improved bio- availability [4-HPR/Lym-X-Sorb (LXS) oral powder] [17,18], entered clinical trials. In a pediatric phase I clinical trial, the 4-HPR/LXS formulation showed a 2–6-fold increased plasma concentration of 4-HPR at equivalent doses of the corn oil capsules [19]. In pre- clinical murine studies pharmacologic modulation of 4-HPR metabolism by ketoconazole (a cytochrome P450 3A4 inhibitor) showed a greater than two-fold increase in 4-HPR plasma concentrations [20] and anti-NB activity [21]. In a phase I clinical trial of refractory and relapsed high-risk NB, 4-HPR with concomitant ketoconazole was well-tolerated and increased 4-HPR plasma levels were obtained using 4-HPR/LXS oral powder [22]. In recur- rent hematopoietic malignancies, a phase I clinical trial of Preclinical report 117 0959-4973 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/CAD.0000000000000696 Copyright r 2018 Wolters Kluwer Health, Inc. All rights reserved.