Targeting combinations of liposomal drugs to both tumor vasculature cells and tumor cells for the treatment of HER2-positive breast cancer.

PURPOSE: We used two ligand-modified liposomal drugs to selectively deliver two different chemotherapeutics to tumor cells (TC) and tumor vasculature endothelial (TV) cells, and examined the therapeutic effect of altering the order of treatment administration, and the effect of the temporal spacing of the treatments on the accumulation of a second dose of liposomes and therapeutic activity. METHODS: Studies were completed in an orthotopic mouse model of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, utilizing liposomal doxorubicin, targeted to TC via αHER2 Fab' fragments, and liposomal vincristine, targeted to CD13 on TV cells via NGR peptides. RESULTS AND DISCUSSION: Combination treatment with TV-targeted plus TC-targeted therapies was therapeutically superior to either single agent; switching the order of administration of the combination did not alter treatment efficacy. The tumor accumulation of a second dose of liposomes was increased if administered at 4 days after pre-treatment with TV-targeted therapy. Using a treatment schedule exploiting this increase, the dose of simultaneously administered combination therapy was halved without compromising therapeutic effect. CONCLUSION: Proof-of-concept studies revealed the therapeutic potential of a dual-targeted two drug approach against HER2-positive breast cancer, and may be applicable to the treatment of other solid tumors.

Acid-labile mPEG-vinyl ether-1,2-dioleylglycerol lipids with tunable pH sensitivity: synthesis and structural effects on hydrolysis rates, DOPE liposome release performance, and pharmacokinetics.

A family of 3-methoxypoly(ethylene glycol)-vinyl ether-1,2-dioleylglycerol (mPEG-VE-DOG) lipopolymer conjugates, designed on the basis of DFT calculations to possess a wide range of proton affinities, was synthesized and tested for their hydrolysis kinetics in neutral and acidic buffers. Extruded ∼100 nm liposomes containing these constructs in ≥90 mol % 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) produced dispersions that retained their calcein cargo for more than 2 days at pH 7.5, but released the encapsulated contents over a wide range of time scales as a function of the electronic properties of the vinyl ether linkage, the solution pH, and the mPEG-VE-DOG composition in the membrane. The in vivo performance of two different 90:10 DOPE:mPEG-VE-DOG compositions was also evaluated for blood circulation time and biodistribution in mice, using (125)I-tyraminylinulin as a label. The pharmacokinetic profiles gave a t(1/2) of 7 and 3 h for 90:10 DOPE:ST302 and 90:10 DOPE:ST502, respectively, with the liposomes being cleared predominantly by liver and spleen uptake. The behavior of these DOPE:mPEG-VE-DOG formulations is consistent with their relative rates of vinyl ether hydrolysis, i.e., the more acid-sensitive mPEG-VE-DOG derivatives produced faster leakage rates from DOPE:mPEG-VE-DOG liposomes, but decreased the blood circulation times in mice. These findings suggest that the vinyl ether-based PEG-lipid derivatives are promising agents for stabilizing acid-sensitive DOPE liposomes to produce formulations with a priori control over their pH responsiveness in vitro. Our data also suggest, however, that the same factors that contribute to enhanced acid sensitivity of the DOPE:mPEG-VE-DOG dispersions are also likely responsible for their reduced pharmacokinetic profiles.

siRNA-mediated down-regulation of P-glycoprotein in a Xenograft tumor model in NOD-SCID mice.

PURPOSE: The efficacy of chemotherapy is decreased due to over-expression of the drug transporter P-glycoprotein (P-gp). This study was conducted to determine the feasibility of down-regulating tumor P-gp levels with non-viral siRNA delivery in order to sensitize the tumors to drug therapy. METHODS: P-gp over-expressing MDA435/LCC6 MDR1 cells were used to establish xenografts in NOD-SCID mouse. Cationic polymers polyethylenimine (PEI) and stearic acid-substituted poly-L-lysine (PLL-StA) were formulated with P-gp- specific siRNAs and delivered intratumorally to explore the feasibility of P-gp down-regulation in tumors. Intravenous Doxil™ was administered to investigate tumor growth. RESULTS: PEI and PLL-StA effectively delivered siRNA to MDA435/LCC6 MDR1 cells in vitro to reduce P-gp expression for 3 days. Intratumoral injection of siRNA with the carriers resulted in 60-80% and 20-32% of siRNA retention in tumors after 24 and 96 hr, respectively. This led to ~29.0% and ~61.5% P-gp down-regulation with PEI- and PLL-StA-mediated siRNA delivery, respectively. The P-gp down-regulation by intratumoral siRNA injection led to better response to systemic Doxil™ treatment, resulting in slowed tumor growth in originally doxorubicin-resistant tumors. CONCLUSION: Effective P-gp down-regulation was feasible with polymeric siRNA delivery in a xenograft model, resulting in an enhanced response to the drug therapy.

Bioavailability and therapeutic efficacy of HER2 scFv-targeted liposomal doxorubicin in a murine model of HER2-overexpressing breast cancer.

Drugs that are entrapped in the interior of nanocarriers such as liposomes have no therapeutic activity, i.e. they are not bioavailable. In order to achieve therapeutic activity, drug release from the liposomes must occur at a rate sufficient to achieve therapeutic concentrations of drug at the cellular target. For ligand-targeted liposomes, directed against internalizing antigens, receptor-mediated internalization of the liposome package occurs and the entrapped drugs become active (bioavailable) upon their intracellular release from the lysosomal apparatus. We have examined, in a murine breast cancer model, the rate and the extent of bioavailability of doxorubicin (DXR) entrapped in liposomes targeted by a single-chain antibody fragment against the HER2/neu antigen, in comparison with free DXR and non-targeted liposomal DXR (DOXIL). Breast cancer tumors contained the highest total levels of DXR and the highest levels of bioavailable DXR when anti-HER2/neu-targeted liposomes were used, and the targeted liposomes also resulted in the greatest level of tumor control.

Improved therapeutic responses in a xenograft model of human B lymphoma (Namalwa) for liposomal vincristine versus liposomal doxorubicin targeted via anti-CD19 IgG2a or Fab' fragments.

PURPOSE: Monoclonal antibody-mediated targeting of liposomal anticancer drugs to surface antigens expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. In a murine model of human B-cell lymphoma, we have made in vitro and in vivo comparisons of long-circulating sterically stabilized (Stealth) immunoliposome (SIL) formulations of two anticancer drugs, vincristine (VCR) and doxorubicin (DXR), with different mechanisms of action and drug release rates. EXPERIMENTAL DESIGN: SIL formulations of VCR or DXR were conjugated to the monoclonal antibody anti-CD19 (SIL[alphaCD19]) or its Fab' fragments (SIL[Fab']). Specific binding of SILs to Namalwa cells was studied using radiolabeled liposomes, and cytotoxicities of DXR- or VCR-loaded SILs were quantitated by a tetrazolium assay. Pharmacokinetic and drug leakage experiments were performed in mice using dual-labeled liposomes, and the therapeutic responses of SILs were evaluated in a Namalwa (human B lymphoma) cell xenograft model. RESULTS: SIL[alphaCD19] or SIL[Fab'] had higher association with and cytotoxicity against Namalwa cells than nontargeted liposomes. SIL[Fab'] had longer circulation times than SIL[alphaCD19], and VCR had faster release rates from the liposomes than DXR. SIL formulations of either VCR or DXR had significantly better therapeutic outcomes than nontargeted liposomes or free drugs. SILs loaded with VCR were superior to those loaded with DXR. SIL[Fab'] had better therapeutic outcomes than SIL[alphaCD19] for the drug DXR but were equally efficacious for the drug VCR. CONCLUSIONS: Treatment of a B lymphoma model with single injections of anti-CD19-targeted liposomal formulations of VCR resulted in high levels of response and long-term survivors. Responses to anti-CD19-targeted liposomal DXR were more modest, although the longer circulation times of SIL[Fab'] versus SIL[alphaCD19] led to superior therapeutics for DXR-loaded immunoliposomes.

Doxorubicin-loaded Fab' fragments of anti-disialoganglioside immunoliposomes selectively inhibit the growth and dissemination of human neuroblastoma in nude mice.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Intensive therapeutic intervention does not prolong the overall disease-free survival rate for this tumor. NB tumor, but not normal tissues, overexpress the disialoganglioside (GD(2)) at the cell surface. Anti-GD(2) whole antibodies (aGD(2)) or their corresponding Fab' fragments were covalently coupled to Stealth immunoliposomes (aGD(2)-SIL or Fab'-SIL), and their binding to GD(2)-positive NB cells was measured. Cytotoxic effects of immunoliposomes loaded with doxorubicin (DXR) were determined. Radiolabelled immunoliposomes were used to evaluate pharmacokinetics (PK). The effectiveness of different liposomal formulations of DXR was tested against a metastatic model of human NB in nude mice. aGD(2)-SIL and Fab'-SIL showed concentration-dependent specific binding and uptake by GD(2)-positive NB cells. DXR entrapped in aGD(2)-SIL or Fab'-SIL (aGD(2)-SIL[DXR], Fab'-SIL[DXR]) showed higher cytotoxicities than nontargeted liposomes (SL[DXR]). DXR-loaded Fab'-SIL (Fab'-SIL[DXR]) also showed specific binding, uptake, and cytotoxic effects on several GD(2)-positive NB cells in vitro. PK studies showed that Fab'-SIL had long-circulating profiles in blood compared with aGD(2)-SIL, with the PK profile for Fab'-SIL being almost identical to that obtained with nontargeted Stealth liposomes. In vivo, long-term survivors were obtained in mice treated with Fab'-SIL[DXR] but not in untreated animals, or those treated with free aGD(2) Fab', Fab'-SIL (no drug), free-DXR, or nontargeted Stealth liposomes[DXR] (no antibody; P < 0.0001). Immunoliposomes containing DXR prevented the establishment and growth of the tumor in all of the organs examined. In conclusion, Fab'-SIL[DXR] formulations led to the total inhibition of metastatic growth of human NB in a nude mouse metastatic model. This formulation should receive clinical evaluation as adjuvant therapy of NB.

Use of the post-insertion method for the formation of ligand-coupled liposomes.

A new technique is described for the formation of ligand-targeted liposomes that can be used with whole antibodies, antibody fragments, peptides or other ligands. The ligands are coupled to polyethylene glycol micelles and then transferred in a simple incubation step from the micelles into the outer monolayer of pre-formed, drug-loaded liposomes. This versatile method allows a combinatorial approach to the design of targeted liposomes that minimises manufacturing complexities, allowing a variety of ligands to be inserted into a variety of pre-formed liposomes containing a variety of drugs. This allows the ligand-targeted therapeutics to be tailored to the needs of individual patients.

Adventures in targeting.

An overview of our experiences in the field of immunoliposomal anticancer drugs is provided with respect to choice of ligand, and choice of model system, in order to provide some guidance as to the rational use of this new technology. Liposomes targeted by either peptide or monoclonal antibodies showed significantly higher binding to their respective target cells in vitro compared to non-targeted liposomes in all model systems examined. This higher binding led to higher cytotoxicities relative to non-targeted liposomes. For the immunoliposomes to deliver their entrapped drug to target cell in vivo, long circulations half-lives are required. We have evaluated the pharmacokinetics of liposomes prepared by several different coupling techniques, and have found significant differences in the clearance of these immunoliposomes from the circulation. Immunoliposomes prepared with whole anti-CD19 IgG coupled by the Mal-PEG-DSPE method demonstrated a short plasma half-life, which may reflect the random orientation of the MAb on the liposome surface. Coupling methods that mask or eliminate the Fc region result in immunoliposomes that have clearance rates more similar to untargeted liposomes. Insertion of peptides or antibodies into pre-formed liposomes through incubation with ligand-coupled PEG micelles resulted in immunoliposomes, termed post-insertion liposomes, that demonstrated comparable in vitro binding, pharmacokinetic profiles and in vivo therapeutic efficacy to liposomes made by conventional coupling methods. The therapeutic efficacy of liposomes, prepared by various coupling methods and targeted by different ligands, was compared in several different animal models of either haematological malignancies, pseudometastatic disease or solid tumours. In our hands, successful in vivo targeting has been obtained when the target is either small or readily accessible from the vasculature, where the liposomes have longer circulating half-lives and/or where a ligand against an internalizing epitope has been chosen. These results should aid in the rational design of applications for immunoliposomal drugs in the future.