Effect of a Pluronic(®) P123 formulation on the nitric oxide-generating drug JS-K

Abstract

Purpose: O(2)-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] or JS-K is a nitric oxide-producing prodrug of the arylated diazeniumdiolate class with promising anti-tumor activity. JS-K has challenging solubility and stability properties. We aimed to characterize and compare Pluronic(®) P123-formulated JS-K (P123/JS-K) with free JS-K.

Methods: We determined micelle size, shape, and critical micelle concentration of Pluronic(®) P123. Efficacy was evaluated in vitro using HL-60 and U937 cells and in vivo in a xenograft in NOD/SCID IL2Rγ (null) mice using HL-60 cells. We compared JS-K and P123/JS-K stability in different media. We also compared plasma protein binding of JS-K and P123/JS-K. We determined the binding and Stern Volmer constants, and thermodynamic parameters.

Results: Spherical P123/JS-K micelles were smaller than blank P123. P123/JS-K formulation was more stable in buffered saline, whole blood, plasma and RPMI media as compared to free JS-K. P123 affected the protein binding properties of JS-K. In vitro it was as efficacious as JS-K alone when tested in HL-60 and U937 cells and in vivo greater tumor regression was observed for P123/JS-K treated NOD/SCID IL2Rγ (null) mice when compared to free JS-K-treated NOD/SCID IL2Rγ (null) mice.

Conclusions: Pluronic(®) P123 solubilizes, stabilizes and affects the protein binding characteristics of JS-K. P123/JS-K showed more in vivo anti-tumor activity than free JS-K.

Figure 1

Structure of JS-K.

Figure 2

TEM image (667x) of 1 mM JS-K loaded in 2.25% P123 micelles.

Figure 3

Critical micelle concentration (CMC) value of Pluronic® P123 as determined by plotting Absorbance (AU) with log Pluronic® % weight/volume. The CMC was obtained from the break point calculated on R statistical software. CMC was calculated from the average of two independent experiments with each concentration prepared in triplicates.

Figure 4

Stern Volmer Plot for JS-K and HSA or P123/JS-K and HSA at room temperature. y-axis : Fluorescence of HSA/Fluorescence of drug; x-axis: JS-K or P123/JS-K concentration. Plots obtained from averages of 3 independent experiments.

Figure 5

Stern Volmer plot for JS-K and AGP or P123/JS-K and AGP at room temperature. y-axis : Fluorescence of HSA/Fluorescence of drug; x-axis: JS-K or P123/JS-K concentration. Plots obtained from averages of 3 independent experiments.

Figure 6

Stern Volmer plot for JS-K and HSA at 298, 303 and 310 K. Each point and standard error of the mean (SEM) is a representative of two independent experiments with nine replicates.

Figure 7

Stern Volmer plot for P123/JS-K at temperatures 298, 303 and 310 K. Each point and standard error of the mean (SEM) is a representative of two independent experiments with nine replicates.

Figure 8

In vivo tumor regression analysis: NOD/SCID IL2Rγ^null mice were injected with HL-60 cells subcutaneously. Treatments started after tumors became palpable. A total of 8 injections were given. Treatment groups consisted of no treatment control, Pluronic® P123 treatment control, free JS-K injected at 4 μmol/kg, P123/JS-K injected at 4 μmol/kg, or P123/JS-K injected at 5 μmol/kg.