A Novel Targeted Drug Delivery System for Reducing Irritation of a Poorly Soluble Anticancer Drug

Abstract

Background and Aim: Asulacrine (ASL) is an inhibitor of topoisomerase II with a broad anti-tumour spectrum. It has been advanced to Phase II clinical trials but further development was terminated due to the occurrence of phlebitis during intravenous administration. The aim of this study was to develop and evaluate a long-circulating nano-liposomal formulation as a drug delivery system to reduce the venous irritation of ASL. Methods: A stability-indicating HPLC assay with diode array detection was developed for the determination of ASL concentrations. Physicochemical properties were characterised for ASL. ASL loaded liposomes coated with Poloxamer 188 (P188-ASL-Lipo) were prepared by thin film hydration in conjugation with active drug loading using ammonium sulphate and post-insertion with P188. Effort was made to maximise the entrapment efficiency (EE) and drug loading (DL) into liposomes by optimising loading conditions such as extraliposomal pH to create a driving force for liposomal drug-uptake based on the Henderson-Hasselbalch equation. Physicochemical characteristics, physical and chemical stability and in vitro drug release profiles of P188-ASL-Lipo were determined. The irritancy such as pain on injection was evaluated using a rat paw-lick model (n = 8), and venous tissue tolerance and pharmacokinetics (plasma) were simultaneously evaluated following intravenous infusion to rabbits (n = 1). The ASL solution (1 mg/mL) was used as a control formulation. Results and discussion: The U-shaped pH-solubility profile in aqueous solutions with lowest solubility around pH 7.4 (0.84 μg/mL) indicated that ASL is an ampholyte. The basic pKa value was 6.72. The log D value at pH 3.8 was 1.15 and increased to 3 as pH increased to 7.4. ASL was found to be the most stable in acidic conditions. Setting extraliposomal pH at 5.6 during drug loading was found to be optimal to achieve the highest DL (4.76 %) and EE (99.9 %). At this pH, ASL was stable, and > 90% ASL was ionised conferring high drug solubility (1 mg/mL) and acting as a reservoir of unionised ASL for transmembrane transport. Cryo- TEM images showed that ASL precipitated inside the spherical liposomes. P188-ASL-Lipo with a size of 144 nm and a zeta potential of -20 mV, showed good physical and chemical stability at least for five months at 4 °C. The rabbit ear vein irritation study showed that ASL loaded liposomes coated with 7% P188 (7% P188-ASL-Lipo) reduced the venous irritation of the free drug solution. In addition the liposomes had a longer half-life (193 min) than the 5% P188-ASL-Lipo (95 min) and the ASL solution (84 min). In the rat paw-lick test only 25% of the rats from 7% P188-ASL-Lipo group had paw-lift upon injection, compared with 100% in the ASL solution group. Moreover, rats in the liposome group did not show any more severe responses such as paw-lick or swelling in footpad, in contrast to the ASL solution group. Conclusion: Preformulation work provided important information in optimising drug loading into liposomes. P188-ASL-Lipo formulation has been demonstrated to have the potential to reduce irritation at the injection site, and to minimise the incidence of phlebitis while increasing the blood circulation time in vivo.