Cytotoxicity and molecular activity of fenretinide and metabolites in T-cell lymphoid malignancy, neuroblastoma, and ovarian cancer cell lines in physiological hypoxia.

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% O2), 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.

A Phase I New Approaches to Neuroblastoma Therapy Study of Buthionine Sulfoximine and Melphalan With Autologous Stem Cells for Recurrent/Refractory High-Risk Neuroblastoma.

BACKGROUND: Myeloablative therapy for high-risk neuroblastoma commonly includes melphalan. Increased cellular glutathione (GSH) can mediate melphalan resistance. Buthionine sulfoximine (BSO), a GSH synthesis inhibitor, enhances melphalan activity against neuroblastoma cell lines, providing the rationale for a Phase 1 trial of BSO-melphalan. PROCEDURES: Patients with recurrent/resistant high-risk neuroblastoma received BSO (3 gram/m(2) bolus, then 24 grams/m(2) /day infusion days -4 to -2), with escalating doses of intravenous melphalan (20-125 mg/m(2) ) days -3 and -2, and autologous stem cells day 0 using 3 + 3 dose escalation. RESULTS: Among 28 patients evaluable for dose escalation, one dose-limiting toxicity occurred at 20 mg/m(2) melphalan (grade 3 aspartate aminotransferase/alanine aminotransferase) and one at 80 mg/m(2) (streptococcal bacteremia, grade 4 hypotension/pulmonary/hypocalcemia) without sequelae. Among 25 patients evaluable for response, there was one partial response (PR) and two mixed responses (MRs) among eight patients with prior melphalan exposure; one PR and three MRs among 16 patients without prior melphalan; one stable disease with unknown melphalan history. Melphalan pharmacokinetics with BSO were similar to reports for melphalan alone. Melphalan Cmax for most patients was below the 10 µM concentration that showed neuroblastoma preclinical activity with BSO. CONCLUSIONS: BSO (75 gram/m(2) ) with melphalan (125 mg/m(2) ) is tolerable with stem cell support and active in recurrent/refractory neuroblastoma. Further dose escalation is feasible and may increase responses.

Phase I trial of intravenous fenretinide (4-HPR) plus safingol in advanced malignancies.

PURPOSE: Fenretinide (4-HPR) is a synthetic retinoid that induces cytotoxicity through dihydroceramide production. Safingol, a stereochemical-variant dihydroceramide precursor, exhibits synergistic effects when administered with fenretinide in preclinical studies. We conducted a phase 1 dose-escalation clinical trial of this combination. METHODS: Fenretinide was administered as a 600 mg/m2 24-h infusion on Day 1 of a 21-day cycle followed by 900 mg/m2/day on Days 2 and 3. Safingol was concurrently administered as a 48-h infusion on Day 1 and 2 using 3 + 3 dose escalation. Primary endpoints were safety and maximum tolerated dose (MTD). Secondary endpoints included pharmacokinetics and efficacy. RESULTS: A total of 16 patients were enrolled (mean age 63 years, 50% female, median three prior lines of therapy), including 15 patients with refractory solid tumors and one with non-Hodgkin lymphoma. The median number of treatment cycles received was 2 (range 2-6). The most common adverse event (AE) was hypertriglyceridemia (88%; 38% ≥ Grade 3), attributed to the fenretinide intralipid infusion vehicle. Other treatment-related AEs occurring in ≥ 20% of patients included anemia, hypocalcemia, hypoalbuminemia, and hyponatremia. At safingol dose 420 mg/m2, one patient had a dose-limiting toxicity of grade 3 troponinemia and grade 4 myocarditis. Due to limited safingol supply, enrollment was halted at this dose level. Fenretinide and safingol pharmacokinetic profiles resembled those observed in monotherapy trials. Best radiographic response was stable disease (n = 2). CONCLUSION: Combination fenretinide plus safingol commonly causes hypertriglyceridemia and may be associated with cardiac events at higher safingol levels. Minimal activity in refractory solid tumors was observed. TRIAL REGISTRATION NUMBER: NCT01553071 (3.13.2012).

Suaeda glauca Attenuates Liver Fibrosis in Mice by Inhibiting TGFß1-Smad2/3 Signaling in Hepatic Stellate Cells.

Chronic liver injury due to various hepatotoxic stimuli commonly leads to fibrosis, which is a crucial factor contributing to liver disease-related mortality. Despite the potential benefits of Suaeda glauca (S. glauca) as a natural product, its biological and therapeutic effects are barely known. This study investigated the effects of S. glauca extract (SGE), obtained from a smart farming system utilizing LED lamps, on the activation of hepatic stellate cells (HSCs) and the development of liver fibrosis. C57BL/6 mice received oral administration of either vehicle or SGE (30 or 100 mg/kg) during CCl4 treatment for 6 weeks. The supplementation of SGE significantly reduced liver fibrosis induced by CCl4 in mice as evidenced by histological changes and a decrease in collagen accumulation. SGE treatment also led to a reduction in markers of HSC activation and inflammation as well as an improvement in blood biochemical parameters. Furthermore, SGE administration diminished fibrotic responses following acute liver injury. Mechanistically, SGE treatment prevented HSC activation and inhibited the phosphorylation and nuclear translocation of Smad2/3, which are induced by transforming growth factor (TGF)-ß1 in HSCs. Our findings indicate that SGE exhibits anti-fibrotic effects by inhibiting TGFß1-Smad2/3 signaling in HSCs.

Limonium tetragonum Promotes Running Endurance in Mice through Mitochondrial Biogenesis and Oxidative Fiber Formation.

The purpose of this study was to examine whether Limonium tetragonum, cultivated in a smart-farming system with LED lamps, could increase exercise capacity in mice. C57BL/6 male mice were orally administered vehicle or Limonium tetragonum water extract (LTE), either 30 or 100 mg/kg, and were subjected to moderate intensity treadmill exercise for 4 weeks. Running distance markedly increased in the LTE group (100 mg/kg) by 80 ± 4% compared to the vehicle group, which was accompanied by a higher proportion of oxidative fibers (6 ± 6% vs. 10 ± 4%). Mitochondrial DNA content and gene expressions related to mitochondrial biogenesis were significantly increased in LTE-supplemented gastrocnemius muscles. At the molecular level, the expression of PGC-1α, a master regulator of fast-to-slow fiber-type transition, was increased downstream of the PKA/CREB signaling pathway. LTE induction of the PKA/CREB signaling pathway was also observed in C2C12 cells, which was effectively suppressed by PKA inhibitors H89 and Rp-cAMP. Altogether, these findings indicate that LTE treatment enhanced endurance exercise capacity via an improvement in mitochondrial biosynthesis and the increases in the formation of oxidative slow-twitch fibers. Future study is warranted to validate the exercise-enhancing effect of LTE in the human.

Sirtuin 6 promotes eosinophil differentiation by activating GATA-1 transcription factor.

There is evidence emerging that exposure to cold temperatures enhances alternative activation of macrophages in white adipose tissue (WAT), which promotes adipocyte beiging and adaptive thermogenesis. Although we recently reported that NAD+ -dependent deacetylase sirtuin 6 (Sirt6) drives alternatively activated (M2) macrophage polarization, the role of myeloid Sirt6 in adaptive thermogenesis had remained elusive. In this study, we demonstrate that myeloid Sirt6 deficiency impaired both thermogenic responses and M2 macrophage infiltration in subcutaneous WAT (scWAT) during cold exposure. Moreover, the infiltration of Siglec-F-positive eosinophils in scWAT and Th2 cytokines levels was reduced in myeloid Sirt6 knockout mice. An ex vivo bone marrow-derived cell culture experiment indicated that Sirt6 was required for eosinophil differentiation independent of its deacetylase activity. Data from our in vitro experiments show that Sirt6 acted as a transcriptional cofactor of GATA-1, independent of its catalytic function as a deacetylase or ADP-ribosyltransferase. Specifically, Sirt6 physically interacted with GATA-1, and enhanced GATA-1's acetylation and transcriptional activity by facilitating its cooperation with p300. Overall, our results suggest that myeloid Sirt6 plays an important role in eosinophil differentiation and fat beiging/adaptive thermogenesis, which is at least in part due to its ability to bind GATA-1 and stimulate its transcriptional activity.

Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia.

Ceramide synthases (CERS) produce ceramides which are key intermediators in the biosynthesis of complex sphingolipids and play an important role in cell proliferation, differentiation, apoptosis and senescence. CERS6 is an isoform of ceramide synthases known to generate ceramides with C16 acyl chain (C16-Cer). CERS6 and C16-Cer levels were significantly higher in acute lymphoblastic leukemia (ALL) cells in comparison to peripheral blood mononuclear cells and T lymphocytes derived from healthy human volunteers. We investigated the role of CERS6 in chemo-resistance in T-ALL cell lines. Stable knockdown of CERS6 in CCRF-CEM and MOLT-4 cells resulted in increased sensitivity to ABT-737, a pan-BCL-2 inhibitor, while CCRF-CEM cells with exogenous CERS6 expression showed resistance to ABT-737 relative to the vector control. The cytotoxic activity of ABT-737 in CERS6 knockdown cells was significantly reduced by the addition of a caspase-8 inhibitor Z-IETD, suggesting that CERS6 alters the cytotoxicity via extrinsic pathway of apoptosis. By co-immunoprecipitation of CERS6 in CCRF-CEM cells, we identified CD95/Fas, a mediator of extrinsic apoptotic pathway, as a novel CERS6 binding partner. In Fas pull-down samples, FADD (Fas-associated protein with death domain) was detected at higher levels in cells with CERS6 knockdown compared with control cells when treated with ABT-737, and this was reversed by the overexpression of CERS6, demonstrating that CERS6 interferes with Fas-FADD DISC assembly. CERS6 may serve as a biomarker in determining the effectiveness of anticancer agents acting via the extrinsic pathway in T-ALL.

Determination of eperisone in human plasma by liquid chromatography-ESI-tandem mass spectrometry.

A sensitive and selective method for the determination of 4'-ethyl-3-methyl-3-piperidinopropiophenone hydrochloride (eperisone hydrochloride) in human plasma was developed and validated. The procedure employed an internal standard and a solvent extraction step followed by chromatography on a Xterra C18 minibore column. Detection was by electrospray ionization tandem mass spectrometry with multiple reaction monitoring. The mass transitions of eperisone and tolperisone (IS) were m/z 260 --> 98 and m/z 246 --> 98, respectively. The method has a limit of detection of 0.1 pg/mL for eperisone based on the three times signal-to-noise value with a linear range from 0.01 to 10.0 ng/mL for the analyte. Extraction recovery was on average 98.6 +/- 7.2% (SD) for eperisone. The Intra- and inter-day assay accuracy ranged from 93 to 114% and precision (RSD) was better than 8.5%. The method was successfully employed to analyze plasma samples and evaluate pharmacokinetics of eperisone in healthy male volunteers.

Clinical development of fenretinide as an antineoplastic drug: Pharmacology perspectives.

Fenretinide (4-HPR) is a synthetic retinoid that has cytotoxic activity against cancer cells. Despite substantial in vitro cytotoxicity, response rates in early clinical trials with 4-HPR have been less than anticipated, likely due to the low bioavailability of the initial oral capsule formulation. Several clinical studies have shown that the oral capsule formulation at maximum tolerated dose (MTD) achieved <10 µmol/L concentrations in patients. To improve bioavailability of 4-HPR, new oral powder (LYM-X-SORB®, LXS) and intravenous lipid emulsion (ILE) formulations are being tested in early-phase clinical trials. ILE 4-HPR administered as five-day continuous infusion achieved over 50 µmol/L at MTD with minimal systemic toxicities; multiple complete and partial responses were observed in peripheral T cell lymphomas. The LXS oral powder 4-HPR formulation increased plasma levels approximately two-fold at MTD in children without dose-limiting toxicities and demonstrated multiple complete responses in recurrent neuroblastoma. The clinical activity observed with new 4-HPR formulations is attributed to increased bioavailability. Phase I and II clinical trials of both LXS 4-HPR and ILE 4-HPR are in progress as a single agent or in combination with other drugs. Impact statement One of the critical components in drug development is understanding pharmacology (especially pharmacokinetics) of the drugs being developed. Often the pharmacokinetic properties, such as poor solubility leading to poor bioavailability, of the drug can limit further development of the drug. The development of numerous drugs has often halted at clinical testing stages, and several of them were due to the pharmacological properties of the agents, resulting in increased drug development cost. The current review provides an example of how improved clinical activity can be achieved by changing the formulations of a drug with poor bioavailability. Thus, it emphasizes the importance of understanding pharmacologic characteristics of the drug in drug development.