Topotecan and doxorubicin combination to treat recurrent ovarian cancer: the influence of drug exposure time and delivery systems to achieve optimum therapeutic activity.

PURPOSE: To provide proof-of-concept data to support use of Doxil-liposomal topotecan (Topophore C) combinations to treat ovarian cancer. EXPERIMENTAL DESIGN: ES-2, OVCAR-3, and SKOV-3 ovarian cancer cell lines were treated with doxorubicin-topotecan combinations by exposing the cells to drugs from 1 to 72 hours. Pharmacokinetic analysis was conducted following administration of liposomal formulations of these drugs alone and in combination. Efficacy assessments were completed in ES-2 and SKOV-3 ovarian cancer models. RESULTS: On the basis of drug doses capable of achieving 50% reduction in cell viability over 72 hours, doxorubicin-topotecan combinations were additive in SKOV-3 but highly synergistic in ES-2 and OVCAR-3 cells. Favorable drug-drug interactions increased with increased drug exposure time. Topophore C pharmacokinetic remained unaffected when co-administered with Doxil. In the ES-2 model, Doxil at maximum tolerated dose (MTD 7.5 mg/kg) in combination with free topotecan (MTD 15 mg/kg) did not enhance median survival time (MST) over that achieved with topotecan alone. In contrast, MST was increased to 52 days with combination of Topophore C (MTD 2.5 mg/kg) and Doxil (7.5 mg/kg) compared with untreated animals (MST 18 days) or those treated with Topophore C alone (MTD 5 mg/kg, MST 40 days). In the SKOV-3 model, combination treatments showed better therapeutic efficacy than the individual drugs. CONCLUSIONS: Topotecan-doxorubicin combinations produced additive or synergistic effects which were best achieved when the tumor cells were exposed to drugs over extended time. Doxil-Topophore C combinations are therapeutically superior as judged in two ovarian cancer models. Clin Cancer Res; 19(4); 865-77. ©2012 AACR.

Topophore C: a liposomal nanoparticle formulation of topotecan for treatment of ovarian cancer.

We have recently developed a liposomal nanoparticle (LNP) formulation of irinotecan based on loading method that involves formation of a complex between copper and the water soluble camptothecin. The loading methodology developed for irinotecan was evaluated to develop a LNP topotecan formulation (referred to herein as Topophore C) and test its activity in pre-clinical model of ovarian carcinoma. Topotecan was encapsulated into preformed liposomes containing 300 mM copper sulfate and the divalent metal ionophore A23187. Formulation optimization studies included assessments of loading efficiency, influence of temperature on drug loading and in vitro stability of the resulting formulation. In vivo assessments included drug and liposome pharmacokinetics, drug levels within plasma and the peritoneal cavity following intravenous (i.v.) administration in mice and efficacy studies on ES2 ovarian cancer model. Topotecan loading into liposomes was optimized with encapsulation efficiency of >98 % at a final drug-to-lipid (D/L) mole ratio of 0.1. Higher D/L ratios could be achieved, but the resulting formulations were less stable as judged by in vitro drug release studies. Following Topophore C administration in mice the topotecan plasma half-life and AUC were increased compared to free topotecan by 10-and 22-fold, respectively. Topophore C was 2-to 3-fold more toxic than free topotecan, however showed significantly better anti-tumor activity than free topotecan administered at doses with no observable toxic effects. Topophore C is a therapeutically interesting drug candidate and we are particularly interested in developing its use in combination with liposomal doxorubicin for treatment of platinum refractory ovarian cancer.

The role of the transition metal copper and the ionophore A23187 in the development of Irinophore C™.

PURPOSE: A liposomal irinotecan formulation referred to as Irinophore C relies on the ability of copper to complex irinotecan within the liposome. It is currently being evaluated for critical drug-loading parameters. Studies presented here were designed to determine the optimum copper concentration required for the effective encapsulation and retention of irinotecan into liposomes. METHODS: Distearoylphosphatidylcholine/cholesterol liposomes were formulated using buffers containing various copper or manganese concentrations, and irinotecan loading was determined in the presence and absence of divalent metal ionophore A23187. The rate and extent of irinotecan encapsulation and the rate of irinotecan release from the liposomes were assessed. The amount of copper retained inside liposomes following irinotecan loading and the effect of copper on membrane permeability were determined. RESULTS: Efficient (>98%) irinotecan loading was achieved using encapsulated copper concentrations of 50 mM. However, irinotecan release was copper concentration dependent, with a minimum 300 mM concentration required for optimal drug retention. The presence of copper increased liposomal membrane permeability. CONCLUSION: Results explain why irinotecan loading rates are enhanced in the presence of formulations prepared with copper, and we speculate that the Irinophore C formulation exhibits improved drug retention, due to generation of a complex between copper and irinotecan.

Role of phospholipid transfer protein on the plasma distribution of amphotericin B following the incubation of different amphotericin B formulations.

PURPOSE: The purpose of this study was to investigate the role of phospholipid transfer protein (PLTP) on the plasma distribution of amphotericin B (AmpB) following incubation with different AmpB formulations in human plasmas with varying lipid profiles. METHODS: In a first set of experiments, plasma distribution profiles of AmpB were determined following the incubation of Fungizone and lipid-based formulations (Abelcet and AmBisome) at a concentration of 20 microg AmpB/mL for 5-120 min at 37 degrees C in the plasma obtained from six different individuals (total cholesterol concentrations range between 62 and 332 mg/dL). In a second set of experiments, Abelcet, and AmBisome at a concentration of 20 microg AmpB/mL were incubated for 5 min at 37 degrees C in human plasma (total cholesterol = 163 mg/dL) that had been pretreated with an antibody raised up against PLTP (1:400 v/v dilution from stock solution) for 20 min at 37 degrees C. Following incubation, the human plasma was separated into its lipoprotein and lipoprotein-deficient fractions by density gradient ultracentrifugation and analyzed for AmpB content by high-performance liquid chromatography. RESULTS: The majority of AmpB was covered in the lipoprotein-deficient plasma and high-density lipoprotein (HDL) fractions following incubation of Fungizone in human plasma. The majority of AmpB (48.7-87.2%) was recovered in the HDL fraction following incubation of Abelcet and AmBisome in human plasma. The presence of the PLTP antibody resulted in a 20% decrease in the percentage AmpB recovered in the HDL fraction following the incubation of Abelcet. However, the plasma distribution of AmpB remained unchanged following the incubation of AmBisome in plasma containing the PLTP antibody. CONCLUSIONS: Taken together, these findings suggest indirect evidence that PLTP may play an important role in the plasma distribution profile of AmpB following the incubation of Abelcet and may be one of the factors responsible for the preferential association of AmpB with HDL when administered as Abelcet.

Unidirectional inhibition of lipid transfer protein I-mediated transfer of cholesteryl esters between high-density and low-density lipoproteins by amphotericin B lipid complex.

PURPOSE: The purpose of this study was to determine whether Fungizone or amphotericin B lipid complex (ABLC; ABELCET) affects the transfer of cholesteryl ester (CE) by lipid transfer protein I (LTP I; also known as cholesteryl ester transfer protein) between HDL and LDL (bidirectional transfer HDL to LDL and LDL to HDL). METHODS: Increasing concentrations of either Fungizone or ABELCET (1.25-12.5 microg AmpB/ml) were incubated with HDL and [3H]CE-LDL or [3H]CE-HDL and LDL (the amount of each fraction added was equivalent to 10 microg of cholesterol) and LTP I in delipidated human plasma at 37 degrees C for 90 min. As a positive control, TP2, a monoclonal antibody directed against LTP-1, was added instead of drug. After incubation, manganese and phosphate reagents were then added to precipitate out all of the LDL. The supernatant, consisted of only HDL, was counted for radioactivity to determine the amount of CE transferred from LDL. Similarly, the precipitate consisted of only LDL, was counted for radioactivity to determine the amount of CE transferred from HDL. RESULTS: For Fungizone, the transfer of cholesteryl ester (CE) between HDL and LDL were not significantly different compared to nontreated controls. For ABELCET, CE transfer from HDL to LDL was significantly decreased at 12.5 microg AmpB/ml compared to control. However, transfer from LDL to HDL was not significantly different compared to non-treated controls. Similar results were observed with the major lipid component of ABELCET, dimyristoylphosphatidylcholine. CE transfer from HDL to LDL and LDL to HDL was significantly decreased when using the positive control (TP2). CONCLUSIONS: Fungizone does not affect LTP I-mediated transfer of CE between HDL and LDL. ABELCET inhibits transfer from HDL to LDL, but has no effect on CE transfer from LDL to HDL. This uni-directional inhibition may contribute to the high recovery of AmpB in HDL but the very low presence of drug in the LDL fraction following ABELCET incubation.