Barry J. Maurer

Retinoid therapy of high-risk neuroblastoma

Retinoids are derivatives of vitamin A that include all trans-retinoic acid (ATRA), 13-cis-retinoic acid, (13-cis-RA), and fenretinide (4-HPR). High levels of either ATRA or 13-cis-RA can cause arrest of cell growth and morphological differentiation of human neuroblastoma cell lines, and phase I trials showed that higher and more sustained drug levels were obtained with 13-cis-RA relative to ATRA. A phase III randomized trial showed that high-dose, pulse therapy with 13-cis-RA given after completion of intensive chemoradiotherapy (with or without autologous bone marrow transplantation) significantly improved event-free survival in high-risk neuroblastoma. The cytotoxic retinoid 4-HPR achieved multi-log cell kills in neuroblastoma cell lines resistant to ATRA and 13-cis-RA, and a pediatric phase I trial has shown it to be well tolerated. Cytotoxicity of 4-HPR is mediated at least in part by increasing tumor cell ceramide levels and combining 4-HPR with ceramide modulators increased anti-tumor activity in pre-clinical models. Thus, further clinical trials of 4-HPR in neuroblastoma, and of 4-HPR in combination with ceramide modulators, are warranted.

Fenretinide Cytotoxicity for Ewing's Sarcoma and Primitive Neuroectodermal Tumor Cell Lines Is Decreased by Hypoxia and Synergistically Enhanced by Ceramide Modulators

Patients with disseminated Ewing’s family of tumors (ESFT) often experience drug-resistant relapse. We hypothesize that targeting minimal residual disease with the cytotoxic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR; fenretinide) may decrease relapse. We determined the following: (a) 4-HPR cytotoxicity against 12 ESFT cell lines in vitro; (b) whether 4-HPR increased ceramide species (saturated and desaturated ceramides); (c) whether physiological hypoxia (2% O2) affected cytotoxicity, mitochondrial membrane potential (ΔΨm) change, or ceramide species or reactive oxygen species levels; (d) whether cytotoxicity was enhanced by l-threo-dihydrosphingosine (safingol); (e) whether physiological hypoxia increased acid ceramidase (AC) expression; and (f) the effect of the AC inhibitor N-oleoyl-ethanolamine (NOE) on cytotoxicity and ceramide species. Ceramide species were quantified by thin-layer chromatography and scintillography. Cytotoxicity was measured by a fluorescence-based assay using digital imaging microscopy (DIMSCAN). Gene expression profiling was performed by oligonucleotide array analysis. We observed, in 12 cell lines tested in normoxia (20% O2), that the mean 4-HPR LC99 (the drug concentration lethal to 99% of cells) = 6.1 ± 5.4 μm (range, 1.7–21.8 μm); safingol (1–3 μm) synergistically increased 4-HPR cytotoxicity and reduced the mean 4-HPR LC99 to 3.2 ± 1.7 μm (range, 2.0–8.0 μm; combination index < 1). 4-HPR increased ceramide species in the three cell lines tested (up to 9-fold; P < 0.05). Hypoxia (2% O2) reduced ceramide species increase, ΔΨm loss, reactive oxygen species increase (P < 0.05), and 4-HPR cytotoxicity (P = 0.05; 4-HPR LC99, 19.7 ± 23.9 μm; range, 2.3–91.4). However, hypoxia affected 4-HPR + safingol cytotoxicity to a lesser extent (P = 0.04; 4-HPR LC99, 4.9 ± 2.3 μm; range, 2.0–8.2). Hypoxia increased AC RNA expression; the AC inhibitor NOE enhanced 4-HPR-induced ceramide species increase and cytotoxicity. The antioxidant N-acetyl-l-cysteine somewhat reduced 4-HPR cytotoxicity but did not affect ceramide species increase. We conclude the following: (a) 4-HPR was active against ESFT cell lines in vitro at concentrations achievable clinically, but activity was decreased in hypoxia; and (b) combining 4-HPR with ceramide modulators synergized 4-HPR cytotoxicity in normoxia and hypoxia.

N-(4-Hydroxyphenyl)retinamide increases dihydroceramide and synergizes with dimethylsphingosine to enhance cancer cell killing

Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is cytotoxic in many cancer cell types. Studies have shown that elevation of ceramide species plays a role in 4-HPR cytotoxicity. To determine 4-HPR activity in a multidrug-resistant cancer cell line as well as to study ceramide metabolism, MCF-7/AdrR cells (redesignated NCI/ADR-RES) were treated with 4-HPR and sphingolipids were analyzed. TLC analysis of cells radiolabeled with [3H]palmitic acid showed that 4-HPR elicited a dose-responsive increase in radioactivity migrating in the ceramide region of the chromatogram and a decrease in cell viability. Results from liquid chromatography/electrospray tandem mass spectrometry revealed large elevations in dihydroceramides (N-acylsphinganines), but not desaturated ceramides, and large increases in complex dihydrosphingolipids (dihydrosphingomyelins, monohexosyldihydroceramides), sphinganine, and sphinganine 1-phosphate. To test the hypothesis that elevation of sphinganine participates in the cytotoxicity of 4-HPR, cells were treated with the sphingosine kinase inhibitor d-erythro-N,N-dimethylsphingosine (DMS), with and without 4-HPR. After 24 h, the 4-HPR/DMS combination caused a 9-fold increase in sphinganine that was sustained through +48 hours, decreased sphinganine 1-phosphate, and increased cytotoxicity. Increased dihydrosphingolipids and sphinganine were also found in HL-60 leukemia cells and HT-29 colon cancer cells treated with 4-HPR. The 4-HPR/DMS combination elicited increased apoptosis in all three cell lines. We propose that a mechanism of 4-HPR–induced cytotoxicity involves increases in dihydrosphingolipids, and that the synergy between 4-HPR and DMS is associated with large increases in cellular sphinganine. These studies suggest that enhanced clinical efficacy of 4-HPR may be realized through regimens containing agents that modulate sphingoid base metabolism. [Mol Cancer Ther 2008;7(9):2967–76]

Improved Oral Delivery of N-(4-Hydroxyphenyl)Retinamide with a Novel LYM-X-SORB Organized Lipid Complex

Purpose: Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is a cytotoxic retinoid that suffers from a wide interpatient variation in bioavailability when delivered orally in a corn oil capsule. The poor bioavailability of the capsule formulation may have limited responses in clinical trials, and the large capsules are not suitable for young children. To support the hypothesis that a novel organized lipid matrix, LYM-X-SORB, can increase the oral bioavailability of fenretinide, fenretinide in LYM-X-SORB matrix and in a powderized LYM-X-SORB formulation was delivered to mice. Experimental Design: Fenretinide was delivered orally to mice as the contents of the corn oil capsule, in LYM-X-SORB matrix (4-HPR/LYM-X-SORB matrix) or in a LYM-X-SORB matrix powderized with sugar and flour (4-HPR/LYM-X-SORB oral powder). Levels of 4-HPR, and its principal metabolite, N-(4-methoxyphenyl)retinamide, were assayed in plasma and tissues. Results: In a dose-responsive manner, from 120 to 360 mg/kg/d, delivery to mice of 4-HPR in LYM-X-SORB matrix, or as 4-HPR/LYM-X-SORB oral powder, increased 4-HPR plasma levels up to 4-fold (P < 0.01) and increased tissue levels up to 7-fold (P < 0.01) compared with similar doses of 4-HPR delivered using capsule contents. Metabolite [N-(4-methoxyphenyl)retinamide] levels mirrored 4-HPR levels. Two human neuroblastoma murine xenograft models showed increased survival (P < 0.03), when treated with 4-HPR/LYM-X-SORB oral powder, confirming the bioactivity of the formulation. Conclusions: 4-HPR/LYM-X-SORB oral powder is a novel, oral drug delivery formulation, suitable for pediatric use, which warrants further development for the delivery of fenretinide in the treatment of cancer. A phase I clinical trial in pediatric neuroblastoma is in progress.

Phase I trial of fenretinide delivered orally in a novel organized lipid complex in patients with relapsed/refractory neuroblastoma: a report from the New Approaches to Neuroblastoma Therapy (NANT) consortium.

A phase I study was conducted to determine the maximum‐tolerated dose, dose‐limiting toxicities (DLTs), and pharmacokinetics of fenretinide (4‐HPR) delivered in an oral powderized lipid complex (LXS) in patients with relapsed/refractory neuroblastoma.

C22:0- and C24:0-dihydroceramides confer mixed cytotoxicity in T-cell acute lymphoblastic leukemia cell lines.

We previously reported that fenretinide (4-HPR) was cytotoxic to acute lymphoblastic leukemia (ALL) cell lines in vitro in association with increased levels of de novo synthesized dihydroceramides, the immediate precursors of ceramides. However, the cytotoxic potentials of native dihydroceramides have not been defined. Therefore, we determined the cytotoxic effects of increasing dihydroceramide levels via de novo synthesis in T-cell ALL cell lines and whether such cytotoxicity was dependent on an absolute increase in total dihydroceramide mass versus an increase of certain specific dihydroceramides. A novel method employing supplementation of individual fatty acids, sphinganine, and the dihydroceramide desaturase-1 (DES) inhibitor, GT-11, was used to increase de novo dihydroceramide synthesis and absolute levels of specific dihydroceramides and ceramides. Sphingolipidomic analyses of four T-cell ALL cell lines revealed strong positive correlations between cytotoxicity and levels of C22:0-dihydroceramide (ρ = 0.74–0.81, P ≤ 0.04) and C24:0-dihydroceramide (ρ = 0.84–0.90, P ≤ 0.004), but not between total or other individual dihydroceramides, ceramides, or sphingoid bases or phosphorylated derivatives. Selective increase of C22:0- and C24:0-dihydroceramide increased level and flux of autophagy marker, LC3B-II, and increased DNA fragmentation (TUNEL assay) in the absence of an increase of reactive oxygen species; pan-caspase inhibition blocked DNA fragmentation but not cell death. C22:0-fatty acid supplemented to 4-HPR treated cells further increased C22:0-dihydroceramide levels (P ≤ 0.001) and cytotoxicity (P ≤ 0.001). These data demonstrate that increases of specific dihydroceramides are cytotoxic to T-cell ALL cells by a caspase-independent, mixed cell death mechanism associated with increased autophagy and suggest that dihydroceramides may contribute to 4-HPR-induced cytotoxicity. The targeted increase of specific acyl chain dihydroceramides may constitute a novel anticancer approach.

Oral Fenretinide in Biochemically Recurrent Prostate Cancer: A California Cancer Consortium Phase II Trial

BACKGROUND Fenretinide is a synthetic retinoid that is cytotoxic to a variety of cancers. We conducted a phase II trial of oral fenretinide in patients with biochemically recurrent prostate cancer. PATIENTS AND METHODS Eligible patients had histologically confirmed prostate cancer and a confirmed rising prostate-specific antigen (PSA) >or= 2 ng/mL following either radical prostatectomy and/or pelvic radiation therapy, without clinical or radiographic evidence of metastasis. The primary endpoint was PSA response, which was defined as a confirmed decrease by >or=50%, and >or=5 ng/mL, from the pretreatment value. Treatment comprised oral fenretinide 900 mg/m2 twice daily for 1 week, every 3 weeks, for 1 year. RESULTS After a median follow-up of 17.7 months, out of 23 patients, 7 (30%) patients had PSA stable disease (SD), 11 (48%) patients had PSA progression within 3 months, 4 patients had minimal increases over 3 months that did not qualify as SD or progression (17%), and one patient (4%) was not evaluable. Median time to PSA progression was 4.6 months (95% CI, 3.2-8.2 months). Observed grade 3 toxicities included fatigue, pain, hypermagnesemia, a rise in lipase, and nyctalopia. CONCLUSION Although well-tolerated, oral fenretinide did not meet prespecified PSA criteria for response in biochemically recurrent prostate cancer; however, 30% of patients had SD, which suggests modest single-agent clinical activity. The role of different formulations of fenretinide, which might allow for higher serum concentrations of the drug, is currently under investigation.

Fenretinide metabolism in humans and mice: utilizing pharmacological modulation of its metabolic pathway to increase systemic exposure

BACKGROUND AND PURPOSE High plasma levels of fenretinide [N‐(4‐hydroxyphenyl)retinamide (4‐HPR)] were associated with improved outcome in a phase II clinical trial. Low bioavailability of 4‐HPR has been limiting its therapeutic applications. This study characterized metabolism of 4‐HPR in humans and mice, and to explore the effects of ketoconazole, an inhibitor of CYP3A4, as a modulator to increase 4‐HPR plasma levels in mice and to increase the low bioavailability of 4‐HPR.

Toxicity Assessment of Molecularly Targeted Drugs Incorporated into Multiagent Chemotherapy Regimens for Pediatric Acute Lymphocytic Leukemia (ALL): Review from an International Consensus Conference

One of the challenges of incorporating molecularly targeted drugs into multi‐agent chemotherapy (backbone) regimens is defining dose‐limiting toxicities (DLTs) of the targeted agent against the background of toxicities of the backbone regimen. An international panel of 22 pediatric acute lymphocytic leukemia (ALL) experts addressed this issue (www.ALLNA.org). Two major questions surrounding DLT assessment were explored: (1) how toxicities can be best defined, assessed, and attributed; and (2) how effective dosing of new agents incorporated into multi‐agent ALL clinical trials can be safely established in the face of disease‐ and therapy‐related systemic toxicities. The consensus DLT definition incorporates tolerance of resolving Grade 3 and some resolving Grade 4 toxicities with stringent safety monitoring. This functional DLT definition is being tested in two Childrens Oncology Group (COG) ALL clinical trials. Pediatr Blood Cancer 2010;54:872–878

Fenretinide sensitizes multidrug-resistant human neuroblastoma cells to antibody-independent and ch14.18-mediated NK cell cytotoxicity

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Combining passive immunotherapy with an antibody to the disialoganglioside GD2 (ch14.18/SP2/0) and cytokines with 13-cis-retinoic acid for post-myeloablative maintenance therapy increased survival in high-risk NB, but the overall prognosis for these children is still in need of improvement. Fenretinide (4-HPR) is a synthetic retinoid that has shown clinical activity in recurrent NB and is cytotoxic to a variety of cancer cells, in part via the accumulation of dihydroceramides, which are precursors of GD2. We investigated the effect of 4-HPR on CHO-derived, ch14.18-mediated anti-NB effector functions, complement-dependent cytotoxicity (CDC), and antibody-dependent and antibody-independent cellular cytotoxicity (ADCC and AICC, respectively). Here, we demonstrate for the first time that pretreatment of fenretinide-resistant NB cells with 4-HPR significantly enhanced ch14.18/CHO-mediated CDC and ADCC and AICC by both human natural killer cells and peripheral blood mononuclear cells. Treatment with 4-HPR increased GD2 and death receptor (DR) expression in resistant NB cells and induced an enhanced granzyme B and perforin production by effector cells. Blocking of ganglioside synthesis with a glucosylceramide synthase inhibitor abrogated the increased ADCC response but had no effect on the AICC, indicating that GD2 induced by 4-HPR mediates the sensitization of NB cells for ADCC. We also showed that 4-HPR induced increased GD2 and DR expression in a resistant NB xenograft model that was associated with an increased ADCC and AICC response using explanted tumor target cells from 4-HPR-treated mice. In summary, these findings provide an important baseline for the combination of 4-HPR and passive immunotherapy with ch14.18/CHO in future clinical trials for high-risk NB patients.