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.

A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations.

PURPOSE: Cytotoxicity assays in 96-well tissue culture plates allow rapid sample handling for multicondition experiments but have a limited dynamic range. Using DIMSCAN, a fluorescence digital image system for quantifying relative cell numbers in tissue culture plates, we have developed a 96-well cytotoxicity assay with a >4-log dynamic range. METHODS: To overcome background fluorescence that limits detection of viable cells with fluorescein diacetate, we used 2'4'5'6'-tetrabromofluorescein (eosin Y) to quench background fluorescence in the medium and in nonviable cells to enhance the reduction of background fluorescence achieved with digital image thresholding. The sensitivity and linearity of the new assay were tested with serial dilutions of neuroblastoma and leukemia cell lines. DIMSCAN was compared with other in vitro cytotoxicity assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and trypan blue dye exclusion. RESULTS: Without background fluorescence reduction, scans produced a nearly flat curve across various cell concentrations from 100 to 10(6) cells per well. Either digital image thresholding or eosin Y dramatically reduced background fluorescence, and combining them achieved a linear correlation (r > 0.9) of relative fluorescence to viable cell number over >4 logs of dynamic range, even in the presence of 4 x 10(4) nonviable cells per well. Cytotoxicity of deferoxamine for neuroblastoma cell lines measured by the DIMSCAN assay achieved dose-response curves similar to data obtained by manual trypan blue counts or colony formation in soft agar but with a wider dynamic range. Long-term cultures documented the clonogenic ability of viable cells detected by DIMSCAN over the entire dynamic range. The cytotoxicity of two drug combinations (buthionine sulfoximine + melphalan or fenretinide + safingol) was tested using both DIMSCAN and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the wider dynamic range of DIMSCAN facilitated detection of synergistic interactions. CONCLUSION: DIMSCAN offers the ability to rapidly and efficiently conduct cytotoxicity assays in 96-well plates with a dynamic range of >4 logs. This assay enables rapid testing of anticancer drug combinations in microplates.

External beam radiotherapy and high-dose brachytherapy combined with cisplatin and paclitaxel in patients with advanced cervical carcinoma.

OBJECTIVE: The aim of the present study was to evaluate toxicity and efficacy of concurrent chemoradiation with cisplatin and paclitaxel and high-dose rate (HDR) brachytherapy in patients with locally advanced cervical cancer. PATIENTS AND METHODS: 19 patients with locally advanced cervical carcinoma were treated with external beam radiotherapy (EBRT) to the pelvis +/- paraaortic nodes, HDR brachytherapy, cisplatin at doses of 50 mg/m2 in weeks 1 and 4, and weekly paclitaxel at 50 mg/m2 in weeks 1-5 during years 2000-2002. Chemotherapy was administered until leukopenia < or = 2500/mm3, thrombocytopenia <100,000/mm3, and/or hemoglobin level <100 g/l occurred. The median follow up was 36 months (range 25-47). RESULTS: Only four patients were able to tolerate the complete intended course of radiochemotherapy. Chemotherapy was stopped in two patients because of allergic reaction and in one patient because of deep thrombosis. In 12 other cases, chemotherapy was discontinued for hematological toxicity. The 3-year disease free survival was 66%, the 3-year overall survival was 74%. CONCLUSION: The hematological toxicity was the main factor limiting administration of cisplatin at 50 mg/m2 in weeks 1 and 4 and weekly paclitaxel at 50 mg/m2 in weeks 1-5 concomitantly with extended field radiotherapy of locally advanced cervical carcinoma.

DIMSCAN: a microcomputer fluorescence-based cytotoxicity assay for preclinical testing of combination chemotherapy.

DIMSCAN is a semiautomatic fluorescence-based digital image microscopy system that quantifies relative total (using a DNA stain) or viable (using fluorescein diacetate [FDA]) cell numbers in tissue culture multiwell plates ranging from 6 to 384 wells per plate. DIMSCAN is a rapid and efficient tool for conducting in vitro cytotoxicity assays across a 4 log dynamic range. The specificity of detecting viable cells with FDA is achieved by using digital image processing and chemical quenching of fluorescence in nonviable cells with eosin Y. Average scan time for the most commonly used format, a 96-well plate, is 6 min. Cytotoxicity for neuroblastoma cell lines measured by DIMSCAN was found to be comparable to manual Trypan blue dye exclusion counts or colony formation in soft agar, but with a significantly wider dynamic range, which enables drug combination studies used to detect synergistic or antagonistic interactions. The linearity of DIMSCAN was validated (r2 = 0.99967 +/- 0.0003) for cells stained with FDA deposited using a fluorescence-activated cell sorter, documenting a dynamic range > 4 logs, and the ability to detect a single viable cell in a well 93% of the time. DIMSCAN has been used to demonstrate preclinical activity of cytotostatic and cytotoxic drugs and drug combinations that have subsequently shown activity in clinical trials.

Liquid chromatography method for quantifying N-(4-hydroxyphenyl)retinamide and N-(4-methoxyphenyl)retinamide in tissues.

A simple and accurate high-performance liquid chromatography (HPLC) method was developed to measure levels of N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and its main metabolite N-(4-methoxyphenyl)retinamide (4-MPR) in tissue. Following ultrasonic extraction of fresh tissue in acetonitrile (ACN), 4-HPR and 4-MPR were measured by HPLC with UV absorbance detection at 340 nm, using isocratic elution with ACN, H(2)O, and acetic acid. N-(4-ethoxyphenyl)retinamide (4-EPR) was employed as an internal standard. The 4-HPR and 4-MPR recovery in bovine liver or bovine brain tissue samples spiked with known amounts of 4-HPR and 4-MPR ranged from 93 to 110%. The detection limit of the method was 50 ng/ml. The method was tested on actual samples from an athymic (nu/nu) mouse carrying a subcutaneous tumor xenograft originating from SMS-KCNR neuroblastoma cells. The tissues were harvested and analyzed following a 3 day long treatment with intraperitoneal injections of 4-HPR/Diluent-12. 4-HPR and the metabolite 4-MPR were detected and quantitated in the tested tissues including tumor, liver, and brain. This method can be used to quantify 4-HPR and 4-MPR in different tissues to determine the bioavailability of 4-HPR.