Abstract 4392: Protein Interaction and Binding Studies of the Nitric Oxide-Generating Antineoplastic Agent JS-K

Abstract

Abstract The NO prodrug O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate or JS-K is active against acute myeloid leukemia, multiple myeloma, hepatocellular carcinoma, prostate cancer and non-small lung cancer. We have developed a nanoscale micelle formulation for JS-K using Pluronic® P123 polymers. We studied the interaction between P123-formulated JS-K and free JS-K formulated in 40% DMSO/PBS with 4% human serum albumin (HSA) and 0.09% alpha1 acid glycoprotein (AGP). Particle size was measured by dynamic light scattering. HSA had a single peak (8.2 ± 0.51 nm), but 2 peaks were observed when HSA was mixed with 2.25% P123 (peak 1: 10.65 ± 0.484 nm; peak 2: 133.15 ± 11.95 nm). AGP had 2 peaks (peak 1: 236.33 ± 10.71 nm; peak 2: 9.73 ± 0.12 nm) but a single peak (23.49 nm ± 0.09) was observed in the presence of 2.25% P123, suggesting interaction with P123. Binding studies of both P123 JS-K and free JS-K with HSA and AGP were carried out by dialysis at concentrations of 20 -700 µM for 2 hours. At that time point, saturation of drug-protein binding was observed with the P123 formulation but not with free JS-K. Both P123 JS-K and free JS-K were nearly 100% bound with HSA at concentrations ≤ 50 µM. Total recovery for P123 JS-K was higher than free JS-K, indicating stabilization of the drug. At concentrations of 100, 200, 300, 400, 500, 600, and 700 µM of P123 JS-K, 73.5% ± 15, 80.5% ± 5, 82.6% ± 5, 76.1% ± 11.5, 76.2% ± 9, 74.7% ± 0.4, and 71.9% ± 9.4 HSA binding was observed, respectively (average and SEM of 3 experiments). For free JS-K at similar concentrations, the percentage drug bound were 77.3% ± 11, 80.3% ± 5.5, 68.6% ± 3.1, 66.5% ± 1.6, 60.6% ± 1.9, 62.0% ± 1.5, and 54.7% ± 5.76, respectively (average and SEM of 3 experiments). Differences between the unbound fraction of P123-formulated JS-K and free JS-K were statistically significant (P < 0.001) for each JS-K concentration. The binding constants for P123 JS-K and free JS-K were 3.023 × 10-3/μM and 4.3 × 10-2/μM, respectively. We evaluated protein binding of JS-K by measuring protein fluorescence quenching of tryptophan. Fluorometric analysis with HSA was carried out for P123 JS-K or free JS-K concentrations ranging of 20 to 700 µM at 30 minutes. A Stern Volmer constant of 3.58 × 10-3/µM and 1.5 × 10-2/µM was obtained for P123 JS-K and free JS-K, respectively (3 different experiments, r2 =0.99 and 0.77, respectively). Fluorescence analysis of the interaction between P123 JS-K or free JS-K with AGP was carried out at similar concentrations after 30 minutes. The Stern Volmer constants for the interaction between P123 JS-K and AGP and free JS-K and AGP were 2 × 10-3/µM and 3 × 10-3/µM, respectively (average of three different experiments, r2 = 0.67 and 0.91 respectively). These experiments show that JS-K interacts with serum proteins. Pluronic® P123 micelles affect this interaction. Such interactions are likely to influence the in vivo pharmacokinetic properties of the drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4392. doi:10.1158/1538-7445.AM2011-4392