Abstract 2651: Cellular uptake and stability studies of the nitric oxide-generating pro-drug JS-K

Abstract

Abstract The nitric oxide (NO) prodrug O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate, or JS-K, has potent antineoplastic activity in vitro and in vivo. Potential targets of JS-K include acute myeloid leukemia, multiple myeloma, hepatocellular carcinoma, prostate cancer and non-small cell lung cancer. JS-K is sparingly soluble in aqueous media. Preliminary pharmacokinetic studies have shown that JS-K has a short half-life in vivo. We have developed a nanoscale micelle formulation for JS-K using Pluronic® P123 polymers. Pluronics® are polyethylene oxide / polypropylene oxide copolymers that are non-toxic and non-irritant. This formulation prolongs the half-life of JS-K in biologic media in vitro. The purpose of this study was to determine the effect of the P123 Pluronic® formulation on the cellular distribution of JS-K. We compared micelle-solubilized JS-K to free JS-K using the HL-60 human myeloid leukemia cell line. JS-K was added at a concentration of 50 μM to HL-60 cells incubated at 37°C in phosphate buffered saline (PBS), RPMI-1640 with 10% fetal bovine serum (FBS), or PBS with 10% FBS. JS-K recovery from the nuclei and cytoplasm was measured using High Pressure Liquid Chromatography (HPLC). In all experiments, cellular uptake of P123-formulated JS-K was greater than that of free JS-K. The highest levels of recovery of P123-formulated JS-K were from nuclei. Indeed, recovery of JS-K from the cytoplasm was minimal. When cells were incubated in PBS, the percent recovery of free JS-K from nuclei at 15, 30, 60 and 120 minutes was 5.03+2.41; 5.00+2.77, 10.83+5.32 and 0.58+0.22, respectively. Under the same conditions and at the same time points, the percent recovery of P123-formulated JS-K was 9.05+3.5, 20.00+7.07, 21.15+3.14 and 27.73+4.30, respectively (average of two different experiments; P<0.05). When cells were incubated in PBS/10%FBS, the percent recovery of free JS-K from nuclei at 30, 60, 120 and 240 minutes was 3.55+1.92, 3.15+ 1.37, 2.13+1.08, and 1.83+1.55, respectively. Under the same conditions and at the same time points, the percent recovery of P123-formulated JS-K was 20.74+6.57, 12.70+3.41, 16.03+3.59, and 13.22+1.55, respectively (average of three different experiments; P<0.05). When cells were incubated in RPMI/10%FBS, the percent recovery of free JS-K from nuclei at 15, 30, 60, and 120 minutes was 0.38+0.15, 0.45+0.20, 0.43+0.22, and 0.2+0.09, respectively. Under the same conditions and at the same time points, the percent recovery of P123-formulated JS-K was 2.93+1.43, 4.68+1.40, 9.30+1.37 and 11.90+3.81, respectively (average of two different experiments; P<0.05). We conclude that a Pluronic® P123 formulation increases the stability of JS-K in biologic media and facilitates penetration of the drug into the nucleus. This clinically useful formulation could therefore enhance the cytotoxic effects of JS-K and increase its therapeutic efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2651.