Iron complexation to histone deacetylase inhibitors SAHA and LAQ824 in PEGylated liposomes can considerably improve pharmacokinetics in rats

Abstract

The formulation of histone deacetylase inhibitors (HDACi) is challenging due to poor water solubility and rapid elimination of drugs in vivo. This study investigated the effects of complexing iron (Fe3+) to the HDACi suberoylanilide hydroxamic acid (SAHA) and LAQ824 (LAQ) prior to their encapsulation into PEGylated liposomes, and investigated whether this technique could improve drug solubility, in vitro release and in vivo pharmacokinetic (PK) properties. METHODS. The reaction stoichiometry, binding constants and solubility were measured for Fe complexes of SAHA and LAQ. The complexes were passively encapsulated into PEGylated liposomes and characterized by size distribution, zeta-potential, encapsulation efficiency (EE), and in vitro drug release studies. PC-3 cells were used to verify the in vitro anticancer activity of the formulations. In vivo pharmacokinetic properties of liposomal LAQ-Fe (L-LAQ-Fe) was evaluated in rats. RESULTS. SAHA and LAQ form complexes with Fe at 1:1 stoichiometric ratio, with a binding constant on the order of 104 M-1. Fe complexation improved the aqueous solubility and the liposomal encapsulation efficiency of SAHA and LAQ (29-35% EE, final drug concentration > 1 mM). Liposomal encapsulated complexes (L-HDACi-Fe) exhibited sustained in vitro release properties compared to L-HDACi but cytotoxicity on PC-3 cells was comparable to free drugs. The PK of L-LAQ-Fe revealed 15-fold improvement in the plasma t1/2 (12.11 h)and 211-fold improvement in the AUC∞ (105.7 µg·h/ml) compared to free LAQ (0.79 h, 0.5 µg·h/ml). Similarly, the plasma t1/2 of Fe was determined to be 11.83 h in a separate experiment using radioactive Fe-59. The majority of Fe-59 activity was found in liver and spleen of rats and correlates with liposomal uptake by the mononuclear phagocyte system. CONCLUSIONS. We have demonstrated that encapsulation of Fe complexes of HDACi into PEGylated liposomes can improve overall drug aqueous solubility, in vitro release and in vivo pharmacokinetic properties.

Figure 1

Chemical structure of SAHA and LAQ, and their hypothesized structures upon complexation with Fe at a stoichiometric ratio of 1:1.

Figure 2

Characterization of HDACi-Fe complexes. (a) UV-Vis spectrum showing the characteristic absorption peak at ca. 500 nm for SAHA-Fe complex. (b) UV-Vis spectrum showing the characteristic absorption peak at ca. 500 nm for LAQ-Fe complex. (c) SAHA complexes with Fe at a 1:1 molar ratio. (d) LAQ complexes with Fe at a 1:1 molar ratio. (e) The binding constant of SAHA-Fe complex was found to be 2.0×10⁴ M⁻¹. (f) The binding constant of LAQ-Fe complex was found to be 2.2×10⁴ M⁻¹.

Figure 3

In vitro release study for SAHA (a) and LAQ (b) in pH 7.4 buffer at 37°C (n=3, mean ± SD).

Figure 4

Dose response curves for SAHA, L-SAHA, SAHA-Fe, L-SAHA-Fe (a), LAQ, L-LAQ, LAQ-Fe, L-LAQ-Fe (b), FeCl₃ and L-Fe (c) in PC-3 human prostate cancer cells (n=4, mean ± SD). IC₅₀ values were calculated by fitting the data to a nonlinear logarithm curve using GraphPad Prism 5.0 software and are summarized in Table 3.

Figure 5

Oxidative stress and cell viability studies in PC-3 cells (n=8, mean ± SD). Cells were stained with H₂DCFDA, and then treated with L-Fe, L-SAHA, and L-SAHA-Fe at 1 μM and 10 μM concentrations. At 24 h, the fluorescence intensity was plotted as the fold-increase with respect to controls (a); corresponding cell viability at these concentrations was measured with the resazurin-metabolic assay (c). Similar experiments were conducted for L-LAQ and L-LAQ-Fe (b), (d); “n.s.” stands for not significant; ** p < 0.01, *** p < 0.001.

Figure 6

Flow cytometry study. PC-3 cells were untreated (a), or treated with 100 μM L-Fe (b) 10 μM L-SAHA (c), 10 μM L-SAHA-Fe (d), 10 μM L-LAQ (e), or 10 μM L-LAQ-Fe (f). After incubation for 72 h, both live and dead cells were collected. Untreated cells were primarily Annexin V-FITC and PI negative, indicating that they were viable and not undergoing apoptosis. Cells treated with L-SAHA, L-SAHA-Fe, L-LAQ, L-LAQ-Fe and L-Fe were all found to be undergoing early apoptosis (Annexin V-FITC positive and PI negative) or late apoptosis (Annexin V-FITC and PI positive). No necrosis was observed for any of the treatments (Annexin V-FITC negative and PI positive).

Figure 7

Plasma LAQ concentration (ng/ml) for free LAQ, L-LAQ and L-LAQ-Fe following I.V. bolus administration (5 mg/kg) to rats (n=5, mean ± SD). PK parameters were calculated with PKSolver based on non-compartmental analysis and summarized in Table 4.

Figure 8

PK and biodistribution of radioactive Fe-59 in L-Fe and L-LAQ-Fe after I.V. bolus administration to rats (n=5, mean ± SD). Each injection into animals contained 6–7 μCi of radioactivity. Plotted is the time course of %ID/g of plasma and blood cells as a function of time (a), the biodistribution of Fe-59 as %ID/g (b), and biodistribution of Fe-59 as %ID per whole organ (c); “n.s.” stands for not significant; ** p < 0.01, *** p < 0.001. Plasma t_1/2 of Fe-59 in both L-Fe and L-LAQ-Fe were calculated with PKSolver based on non-compartmental analysis.