Liposome co-encapsulation of anti-cancer agents for pharmacological optimization of nanomedicine-based combination chemotherapy.

Aim: Co-encapsulation of anti-cancer agents in pegylated liposomes may provide an effective tool to maximize efficacy of combined drug therapy by taking advantage of the long circulation time, passive targeting, and reduced toxicity of liposome formulations. Methods: We have developed several liposome formulations of co-encapsulated drugs using various permutations of three active agents: doxorubicin (Dox), mitomycin-C lipidic prodrug (MLP), and alendronate (Ald). Dox and MLP are available in single drug liposomal formulations: pegylated liposomal Dox (PLD, Doxil®), clinically approved, and pegylated liposomal MLP (PL-MLP, Promitil®), in phase 1-2 clinical testing. We have previously shown that co-encapsulation of Dox and Ald in pegylated liposomes (PLAD) results in a formulation with valuable immuno-pharmacologic properties and superior antitumor properties over PLD in immunocompetent animal models. Building on the PLAD and PL-MLP platforms, we developed a new pegylated liposomal formulation of co-entrapped Dox and MLP (PLAD-MLP), with the former localized in the liposome water phase via remote loading with an ammonium alendronate and the latter passively loaded into the liposome lipid bilayer. An alternative formulation of co-entrapped MLP and Dox in which ammonium Ald was replaced with ammonium sulfate (PLD-MLP) was also tested for comparative purposes. Results: PLAD-MLP displays high loading efficiency of Dox and MLP nearing 100%, and a mean vesicle diameter of 110 nm. Cryo-transmission electron microscopy (cryo-TEM) of PLAD-MLP reveals round vesicles with an intra-vesicle Dox-alendronate precipitate. PLAD-MLP was tested in an in vitro MLP activation assay with the reducing agent dithiothreitol and found to be significantly less susceptible to thiolytic activation than PL-MLP. Alongside thiolytic activation of MLP, a significant fraction of encapsulated Dox was released from liposomes. PLAD-MLP is stable upon in vitro incubation in human plasma with nearly 100% drug retention. In mouse pharmacokinetic studies, PLAD-MLP extended MLP half-life in circulation when compared to that of MLP delivered as PL-MLP. In addition, the MLP levels in tissues were greater than those obtained with PL-MLP, indicating that PLAD-MLP slows down the cleavage of the prodrug MLP to MMC, thus resulting in a more sustained and prolonged exposure. The circulation half-life of Dox in PLAD-MLP was similar to the PLD Dox half-life. The pattern of tissue distribution was similar for the co-encapsulated drugs, although Dox levels were generally higher than those of MLP, as expected from cleavage of MLP to its active metabolite MMC. In mouse tumor models, the therapeutic activity of PLAD-MLP was superior to PL-MLP and PLD with a convenient safety dose window. The Ald-free formulation, PLD-MLP, displayed similar pharmacokinetic properties to PLAD-MLP, but its therapeutic activity was lower. Conclusion: PLAD-MLP is a novel multi-drug liposome formulation with attractive pharmacological properties and powerful antitumor activity and is a promising therapeutic tool for combination cancer chemotherapy.

Coencapsulation of alendronate and doxorubicin in pegylated liposomes: a novel formulation for chemoimmunotherapy of cancer.

We developed a pegylated liposome formulation of a dissociable salt of a nitrogen-containing bisphosphonate, alendronate (Ald), coencapsulated with the anthracycline, doxorubicin (Dox), a commonly used chemotherapeutic agent. Liposome-encapsulated ammonium Ald generates a gradient driving Dox into liposomes, forming a salt that holds both drugs in the liposome water phase. The resulting formulation (PLAD) allows for a high-loading efficiency of Dox, comparable to that of clinically approved pegylated liposomal doxorubicin sulfate (PLD) and is very stable in plasma stability assays. Cytotoxicity tests indicate greater potency for PLAD compared to PLD. This appears to be related to a synergistic effect of the coencapsulated Ald and Dox. PLAD and PLD differed in in vitro monocyte-induced IL-1ß release (greater for PLAD) and complement activation (greater for PLD). A molar ratio Ald/Dox of ∼1:1 seems to provide an optimal compromise between loading efficiency of Dox, circulation time and in vivo toxicity of PLAD. In mice, the circulation half-life and tumor uptake of PLAD were comparable to PLD. In the M109R and 4T1 tumor models in immunocompetent mice, PLAD was superior to PLD in the growth inhibition of subcutaneous tumor implants. This new formulation appears to be a promising tool to exploit the antitumor effects of aminobisphosphonates in synergy with chemotherapy.

Pharmacologic Studies of a Prodrug of Mitomycin C in Pegylated Liposomes (Promitil(®)): High Stability in Plasma and Rapid Thiolytic Prodrug Activation in Tissues.

PURPOSE: Pegylated liposomal (PL) mitomycin C lipid-based prodrug (MLP) has recently entered clinical testing. We studied here the preclinical pharmacology of PL-MLP. METHODS: The stability, pharmacokinetics, biodistribution, and other pharmacologic parameters of PL-MLP were examined. Thiolytic cleavage of MLP and release of active mitomycin C (MMC) were studied using dithiothreitol (DTT), and by incubation with tissue homogenates. RESULTS: MLP was incorporated in the bilayer at 10% molar ratio with nearly 100% entrapment efficiency, resulting in a formulation with high plasma stability. In vitro, DTT induced cleavage of MLP with predictable kinetics, generating MMC and enhancing pharmacological activity. A long circulation half-life of MLP (10-15 h) was observed in rodents and minipigs. Free MMC is either extremely low or undetectable in plasma. However, urine from PL-MLP injected rats revealed delayed but significant excretion of MMC indicating in vivo activation of MLP. Studies in mice injected with H3-cholesterol radiolabeled PL-MLP demonstrated relatively greater tissue levels of H3-cholesterol than MLP. MLP levels were highest in tumor and spleen, and very low or undetectable in liver and lung. Rapid cleavage of MLP in various tissues, particularly in liver, was shown in ex-vivo experiments of PL-MLP with tissue homogenates. PL-MLP was less toxic in vivo than equivalent doses of MMC. Therapeutic studies in C26 mouse tumor models demonstrated dose-dependent improved efficacy of PL-MLP over MMC. CONCLUSIONS: Thiolytic activation of PL-MLP occurs in tissues but not in plasma. Liposomal delivery of MLP confers a favorable pharmacological profile and greater therapeutic index than MMC.

Liposome encapsulation of zoledronic acid results in major changes in tissue distribution and increase in toxicity.

BACKGROUND: Zoledronic acid (Zol) is a potent inhibitor of farnesyl-pyrophosphate synthase with broad clinical use in the treatment of osteoporosis, and bone metastases. We have previously shown that encapsulation of Zol in liposomes targeted to the folate receptor (FR) greatly enhances its in vitro cytotoxicity. To examine whether targeted liposomal delivery of Zol could be a useful therapeutic approach, we investigated here the in vivo pharmacology of i.v. administered liposomal Zol (L-Zol) in murine models. METHODS: Zol was passively entrapped in the water phase of liposomes containing a small fraction of either dipalmitoyl-phosphatidylglycerol (DPPG) or a polyethylene-glycol (PEG)-conjugated phospholipid with or without insertion of a folate lipophilic conjugate. Radiolabeled formulations were used for pharmacokinetic (PK) and biodistribution studies. Toxicity was evaluated by clinical, hematological, biochemical, and histopathological parameters. Therapeutic studies comparing free Zol, nontargeted and folate targeted L-Zol were performed in FR-expressing human tumor models. RESULTS: Encapsulation of Zol in liposomes resulted in major PK changes including sustained high plasma levels and very slow clearance. DPPG-L-Zol was cleared faster than PEG-L-Zol. Grafting of folate lipophilic conjugates on liposomes further accelerated the clearance of Zol. L-Zol caused a major shift in drug tissue distribution when compared to free Zol, with a major increase (20 to 100-fold) in liver and spleen, a substantial increase (7 to 10-fold) in tumor, and a modest increase (2-fold) in bone. Liposomal formulations proved to be highly toxic, up to 50-fold more than free Zol. PEG-L-Zol was more toxic than DPPG-L-Zol. Toxicity was non-cumulative and appears to involve macrophage/monocyte activation and release of cytokines. Co-injection of L-Zol with a large dose of blank liposomes, or injection of a very low Zol-to-phospholipid ratio liposome formulation reduced toxicity by 2-4-fold suggesting that diluting macrophage exposure below a threshold Zol concentration is important to overcome toxicity. L-Zol failed to significantly enhance the therapeutic activity of Zol vis-à-vis free ZOL and doxorubicin. Folate-targeted L-Zol was marginally better than other treatment modalities in the KB tumor model but toxic deaths greatly affected the outcome. CONCLUSIONS: Liposome delivery of Zol causes a major change in tissue drug distribution and an increase in tumor Zol levels. However, the severe in vivo toxicity of L-Zol seriously limits its dose and its utility for in vivo tumor cell targeting. This strategy is under evaluation using liposomes carrying less toxic bisphosphonates.

Therapeutic efficacy of a lipid-based prodrug of mitomycin C in pegylated liposomes: studies with human gastro-entero-pancreatic ectopic tumor models.

BACKGROUND: A mitomycin-C lipid-based prodrug (MLP) formulated in pegylated liposomes (PL-MLP) was previously reported to have significant antitumor activity and reduced toxicity in mouse tumor models (Clin Cancer Res 12:1913-20, 2006). MLP is activated by thiolysis releasing mitomycin-C (MMC) which rapidly dissociates from liposomes. The purpose of this study was to examine the plasma stability, pharmacokinetics, and antitumor activity of PL-MLP in mouse models of human gastroentero-pancreatic tumors. METHODS: MLP was incorporated with almost 100% efficiency in pegylated liposomes composed of hydrogenated phosphatidylcholine, with or without cholesterol (Chol). Mean vesicle size was 45-65 nm for liposome preparations downsized by homogenization, and 80-100 nm when downsized by extrusion, the latter displaying narrower polydispersity. MLP to phospholipid mole ratio was 5% (~20 µg MMC-equivalents/µmol). Therapeutic studies were carried out in the N87 gastric carcinoma (Ca), HCT15 colon Ca, and Panc-1 pancreatic Ca models implanted s.c. in CD1 nude mice. Treatment was administered i.v. in mice with established tumors. RESULTS: PL-MLP was very stable when incubated in plasma, and whole blood with a maximum of 5% release and activation to free MMC after 24 h. In the presence of a strong reducing agent (dithiotreitol), MLP was almost entirely activated to free MMC. Pharmacokinetic studies revealed major differences in plasma clearance between free MMC and PL-MLP. The longest half-lives were observed for extruded and Chol-containing preparations. Using a liposome radiolabel, it was found that the plasma levels of liposomes and prodrug were nearly superimposable confirming the absence of drug leakage in circulation. In vivo prodrug activation was significantly increased by co-injection of a large dose of a biocompatible reducing agent, N-acetylcysteine. PL-MLP was significantly more effective in delaying tumor growth and resulted in more tumor regressions than irinotecan in the N87 and HCT15 models, and than gemcitabine in the Panc-1 model. PL-MLP was ~3-fold less toxic than free MMC at MMC-equivalent doses, and displayed mild myelosuppression at therapeutic doses. CONCLUSIONS: Delivery of MLP in pegylated liposomes is more effective than conventional chemotherapy in the treatment of gastroentero-pancreatic ectopic tumor models, and may represent an effective tool for treatment of these malignancies in the clinical setting with improved safety over free MMC. Reducing agents offer a tool for controlling in vivo prodrug release.

Fabrication principles and their contribution to the superior in vivo therapeutic efficacy of nano-liposomes remote loaded with glucocorticoids.

We report here the design, development and performance of a novel formulation of liposome- encapsulated glucocorticoids (GCs). A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate. We demonstrate fabrication principles that derive from the physicochemical properties of the GC and the liposomal lipids, which play a crucial role in GC release rate and kinetics. These principles allow fabrication of formulations that exhibit either a fast, second-order (t(1/2) ~1 h), or a slow, zero-order release rate (t(1/2) ~ 50 h) kinetics. A high therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies.

Delivery of zoledronic acid encapsulated in folate-targeted liposome results in potent in vitro cytotoxic activity on tumor cells.

INTRODUCTION: Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, is a potent inhibitor of farnesyl-pyrophosphate synthase with poor in vitro cytotoxic activity as a result of its limited diffusion into tumor cells. The purpose of this study was to investigate whether liposomes targeted to the folate receptor (FR) can effectively deliver ZOL to tumor cells and enhance its in vitro cytotoxicity. METHODS: ZOL was entrapped in the water phase of liposomes of various compositions with or without a lipophilic folate ligand. Stability and blood levels after i.v. injection were checked. The in vitro cytotoxic activity and cell uptake of liposomal ZOL (L-ZOL) were examined on various human and mouse cell lines. RESULTS: All formulations were highly stable and resulted in high blood levels in contrast to free ZOL which was rapidly cleared from plasma. Non-targeted L-ZOL was devoid of any in vitro activity at concentrations up to 200 microM. In contrast, potent cytotoxic activity of folate-targeted L-ZOL (FTL-ZOL) was observed, with optimal activity, reaching the sub-micromolar range, for dipalmitoyl-phosphatidylglycerol (DPPG)-containing liposomes and relatively lower activity for pegylated (PEG) formulations. IC50 values of FTL-ZOL on FR-expressing tumor cells were >100-fold lower than those of free ZOL. Compared to doxorubicin, the cytotoxicity of DPPG-FTL-ZOL was equivalent in drug-sensitive cell lines, and greatly superior in drug-resistant cell lines. When tested on the non-FR upregulated cell lines, the cytotoxicity of FTL-ZOL was lower but still superior to that of L-ZOL. The uptake of ZOL by FR-expressing tumor cells was enhanced approximately 25-fold with DPPG-FTL-ZOL, and only approximately 4-fold with PEG-FTL-ZOL. CONCLUSIONS: FR targeting of ZOL using liposomes is an effective means to exploit the tumor cell growth inhibitory properties of ZOL. DPPG-FTL-ZOL is significantly more efficient at intracellular delivery of ZOL than PEG-FTL-ZOL in FR-expressing tumor cells.

Improved therapeutic activity of folate-targeted liposomal doxorubicin in folate receptor-expressing tumor models.

PURPOSE: The folate receptor (FR) is overexpressed in a broad spectrum of malignant tumors and represents an attractive target for selective delivery of anti-cancer agents to FR-expressing tumors. Targeting liposomes to the FR has been proposed as a way to enhance the effects of liposome-based chemotherapy. METHODS: Folate-polyethylene glycol-distearoyl-phosphatidyl-ethanolamine conjugate was inserted into pegylated liposomal doxorubicin (PLD). The therapeutic activity of folate-targeted (FT-PLD) and non-targeted (PLD) pegylated liposomal doxorubicin was tested in two human tumor models (KB, KB-V) and in one mouse ascitic tumor model (FR-expressing J6456) by the i.v. systemic route in all models, and by the i.p. intracavitary route in the ascitic tumor model only. RESULTS: Consistent with previous studies, PLD was clearly superior to free doxorubicin in all tumor models. When targeted and non-targeted liposome formulations were compared, FT-PLD was more effective than PLD in the KB and KB-V xenograft models, and in the J6456 intra-cavitary therapy model. The therapeutic effect was dose-dependent in the KB model and schedule-dependent in the J6456 intra-cavitary therapy model. In some experiments, toxic deaths aggravated by folate-depleted diet were a major confounding factor. In a non-FR expressing J6456 model, FT-PLD was as active as PLD indicating that its activity is not limited to FR-expressing tumors. CONCLUSION: Folate-targeting confers a significant albeit modest therapeutic improvement to PLD in FR-expressing tumor models, which appears particularly valuable in intracavitary therapy. The potential clinical added value of this approach has yet to be determined.

Her2-targeted pegylated liposomal doxorubicin: retention of target-specific binding and cytotoxicity after in vivo passage.

BACKGROUND: Receptor-directed targeting of ligand-bearing liposomes to tumor cells may enhance therapeutic efficacy by intracellular delivery of a concentrated payload of liposomal drug. The goal of this study was to assess whether Her2-targeted pegylated liposomal doxorubicin (PLD) retains its binding ability to Her2-expressing target cells through circulation in the blood and extravasation to tumor interstitial fluid. METHODS: PLD was grafted with a lipophilic conjugate of an anti-Her2 scFv antibody fragment at an approximate ratio of 7.5, 15, or 30 ligands per liposome. BALB/c mice were injected with J6456 lymphoma cells into the peritoneal cavity to generate malignant ascites used as a model for tumor interstitial fluid. When abdominal swelling developed, Her2-targeted (HT-) PLD and non-targeted PLD were injected into the mice i.v. at a dose of 15 mg/kg. The ascitic fluid was collected 48 h later, ascitic tumor cells were removed, and the doxorubicin levels in the cell-free ascitic fluid and plasma were determined. Binding of the cell-free ascitic fluid was tested in vitro against two Her2-expressing human tumor cell lines (N87, SKBR-3) and compared to the binding of shelf formulations (not passaged in vivo) of HT-PLD and PLD, by measuring the amount of cell-associated doxorubicin. RESULTS: Plasma and ascitic fluid levels of HT-PLD were only slightly below those of PLD indicating that, the Her2 ligand did not cause any significant change in the clearance rate of PLD. The in vitro binding of HT-PLD containing ascitic fluid to Her2-expressing cells was increased 10 to 20-fold above that of PLD-containing ascitic fluid, similarly to the 20-fold difference in binding between shelf Her2-PLD and PLD. The in vitro cytotoxicity of ascitic fluid containing HT-PLD tested against Her2-expressing tumor cells was far greater than that of PLD, and similar to that of the shelf formulations, indicating that the selective pharmacological activity of HT-PLD is preserved after in vivo passage. Optimal results were obtained with HT-PLD formulated with 15 ligands per liposome. CONCLUSIONS: HT-PLD retains most of its original binding capacity to Her2-expressing cells after in vivo passage indicating that the ligand is stably maintained in vivo in association with the doxorubicin liposomal carrier, and confirming the validity of the post-formulation ligand grafting approach for liposome targeting. Targeting of PLD using a Her2 antibody fragment provides an important means of in vivo selective drug delivery to tumors expressing the Her2 receptor.

An open-label study to evaluate dose and cycle dependence of the pharmacokinetics of pegylated liposomal doxorubicin.

PURPOSE: There are no definitive data in humans on the dose dependence and/or cycle dependence of the pharmacokinetics (PK) of pegylated liposomal doxorubicin (PLD). This study examined the PK of PLD across a twofold dose variation and along 3 cycles. METHODS: Fifteen patients received PLD in successive doses of 60, 30, and 45 mg/m(2) (Arm A) and 30, 60, and 45 mg/m(2) (Arm B), every 4 weeks. Twelve patients, six on each arm, completed all three cycles and were fully evaluable. Plasma levels of doxorubicin were analyzed by HPLC and fluorimetry. PK analysis was done by non-compartmental method. Repeated measures ANOVA and paired tests were used for statistical analysis. RESULTS: There was no significant difference in the PK parameters examined when the dose was increased from 30 to 60 mg/m(2). However, when we analyzed the effect of cycle number on the PK, we found a gradual and significant inhibition of clearance (P < 0.0001) from the 1st through the 3rd cycle of PLD, with a geometric mean increase of 43% in dose-normalized AUC (P = 0.0003). Dose-normalized C(max) and T(1/2) mean values increased by 17 and 18%, respectively between the 1st and 3rd cycles, but only the increase in T(1/2) was statistically significant (P = 0.0017). CONCLUSIONS: While the PK of PLD is not dose-dependent within the dose range of 30-60 mg/m(2), there is evidence of a cycle-dependent effect that results in inhibition of clearance when patients receive successive cycles of PLD. These results suggest the need for dose adjustments of PLD upon retreatment to minimize the risk of toxicity.

Intracellular uptake and intracavitary targeting of folate-conjugated liposomes in a mouse lymphoma model with up-regulated folate receptors.

The folate receptor is overexpressed in a broad spectrum of malignant tumors and represents an attractive target for selective delivery of anticancer agents to folate receptor-expressing tumors. This study examines folate-lipid conjugates as a means of enhancing the tumor selectivity of liposome-encapsulated drugs in a mouse lymphoma model. Folate-derivatized polyethylene glycol (PEG3350)-distearoyl-phosphatidylethanolamine was post-loaded at various concentrations into the following preparations: radiolabeled PEGylated liposomes, PEGylated liposomes labeled in the aqueous compartment with dextran fluorescein, and PEGylated liposomal doxorubicin (PLD, Doxil). We incubated folate-targeted radiolabeled or fluorescent liposomes with mouse J6456 lymphoma cells up-regulated for their folate receptors (J6456-FR) to determine the optimal ligand concentration required in the lipid bilayer for liposomal cell association, and to examine whether folate-targeted liposomes are internalized by J6456-FR cells in suspension. Liposomal association with cells was quantified based on radioactivity and fluorescence-activated cell sorting analysis, and internalization was assessed by confocal fluorescence microscopy. We found an optimal ligand molar concentration of approximately 0.5% using our ligand. A substantial lipid dose-dependent increase in cell-associated fluorescence was found in folate-targeted liposomes compared with nontargeted liposomes. Confocal depth scanning showed that a substantial amount of the folate-targeted liposomes are internalized by J6456-FR cells. Binding and uptake of folate-targeted PLD by J6456-FR cells were also observed in vivo after i.p. injection of folate-targeted PLD in mice bearing ascitic J6456-FR tumors. The drug levels in ascitic tumor cells were increased by 17-fold, whereas those in plasma were decreased by 14-fold when folate-targeted PLD were compared with nontargeted PLD in the i.p. model. Folate-targeted liposomes represent an attractive approach for the intracellular delivery of drugs to folate receptor-expressing lymphoma cells and seem to be a promising tool for in vivo intracavitary drug targeting.

In vivo fate of folate-targeted polyethylene-glycol liposomes in tumor-bearing mice.

PURPOSE: To compare the in vivo tissue distribution of folate-targeted liposomes (FTLs) injected i.v. in mice bearing folate receptor (FR)-overexpressing tumors (mouse M109 and human KB carcinomas, and mouse J6456 lymphoma) to that of nontargeted liposomes (NTLs) of similar composition. EXPERIMENTAL DESIGN: A small fraction of a folate-polyethylene-glycol (PEG)-distearoyl-phosphatidylethanolamine conjugate was incorporated in FTLs. Both FTLs and NTLs were PEGylated with a PEG-distearoyl-phosphatidylethanolamine conjugate to prolong circulation time. Liposomes were labeled with [(3)H]cholesterol hexadecyl ether with or without doxorubicin loading. Liposome levels in plasma, tissues, or ascites were assessed by the number of [(3)H] counts. For doxorubicin-loaded formulations, we also determined the tissue doxorubicin levels by fluorimetry. To estimate the amount of liposomes directly associated with tumor cells in vivo, we determined the [(3)H]radiolabel counts in washed pellets of ascitic tumor cells using the ascitic J6456 lymphoma RESULTS: FTLs retained the folate ligand in vivo, as demonstrated by their ability to bind ex vivo to FR-expressing cells after prolonged circulation and extravasation into malignant ascitic fluid. In comparison with NTLs, FTLs were cleared faster from circulation as a result of greater liver uptake. Despite the lower plasma levels, tumor levels of FTL-injected mice were not significantly different from those of NTL-injected mice. When NTLs and FTLs were loaded with doxorubicin, liver uptake decreased because of liver blockade, and uptake by spleen and tumor increased. When tumor-to-tissue liposome uptake ratios were analyzed, the targeting profile of FTLs was characterized by higher tumor:skin, and tumor:kidney ratios but lower tumor:liver ratio than NTLs. After a concomitant dose of free folic acid, FTLs (but not NTLs) plasma clearance and liver uptake were inhibited, indicating that accelerated clearance was mediated by the folate ligand. Surprisingly tumor uptake was not significantly affected by a codose of folic acid. In the J6456 ascitic tumor model, tumor cell-associated liposome levels were significantly greater for FTL-injected mice than for NTL-injected mice, despite slightly higher levels of the latter in whole ascites. CONCLUSIONS: Whereas folate targeting does not enhance overall liposome deposition in tumors, the targeting profile of tumor versus other tissues is substantially different and intratumor liposome distribution in ascitic tumors is affected favorably with a selective shift toward liposome association with FR-expressing cells.