Elaine H. Moase

Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells

Dioleoylphosphatidylethanolamine (DOPE)-containing liposomes that demonstrated pH-dependent release of their contents were stabilized in the bilayer form through the addition of a cleavable lipid derivative of polyethylene glycol (PEG) in which the PEG was attached to a lipid anchor via a disulfide linkage (mPEG-S-S-DSPE). Liposomes stabilized with either a non-cleavable PEG (mPEG-DSPE) or mPEG-S-S-DSPE retained an encapsulated dye at pH 5.5, but treatment at pH 5.5 of liposomes stabilized with mPEG-S-S-DSPE with either dithiothreitol or cell-free extracts caused contents release due to cleavage of the PEG chains and concomitant destabilization of the DOPE liposomes. While formulations loaded with doxorubicin (DXR) were stable in culture media, DXR was rapidly released in human plasma. pH-Sensitive liposomes, targeted to the CD19 epitope on B-lymphoma cells, showed enhanced DXR delivery into the nuclei of the target cells and increased cytotoxicity compared to non-pH-sensitive liposomes. Pharmacokinetic studies suggested that mPEG-S-S-DSPE was rapidly cleaved in circulation. In a murine model of B-cell lymphoma, the therapeutic efficacy of an anti-CD19-targeted pH-sensitive formulation was superior to that of a stable long-circulating formulation of targeted liposomes despite the more rapid drug release and clearance of the pH-sensitive formulation. These results suggest that targeted pH-sensitive formulations of drugs may be able to increase the therapeutic efficacy of entrapped drugs.

Therapeutic opportunities for targeted liposomal drug delivery

Abstract One way to increase the therapeutic index of drugs such as anticancer drugs, which have low therapeutic indices would be by specifically targeting the drugs to the diseased cells. This can be accomplished by associating the drugs with liposomes and coupling a targeting antibody or ligand to the liposome surface. A variety of coupling methods can be used to attach antibodies or ligands to the liposomes, and, to date, the best targeting results have been obtained when the targeting moiety is attached at the terminus of a hydrophillic polymer such as polyethylene glycol (PEG). Targeted liposomes have been demonstrated to have specific binding and increased cytotoxicity to cells in vitro compared to non-targeted liposomes. The best therapeutic results in vivo have been obtained to date when the liposomes are targeted to cells easily accessible within the vasculature, or to micrometastatic cells. Treatment of more advanced solid tumours with targeted liposomes presents a challenge to overcome the ‘binding site barrier’ at the tumour surface. Evidence is accumulating that targeting to internalizing receptors is more successful than targeting to non-internalizing receptors.

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[αCD19]) 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[αCD19] or SIL[Fab′] had higher association with and cytotoxicity against Namalwa cells than nontargeted liposomes. SIL[Fab′] had longer circulation times than SIL[αCD19], 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[αCD19] 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[αCD19] led to superior therapeutics for DXR-loaded immunoliposomes.

Anti-MUC-1 immunoliposomal doxorubicin in the treatment of murine models of metastatic breast cancer.

The fate of breast cancer patients is dependent upon elimination or control of metastases. We studied the effect of antibody-targeted liposomes containing entrapped doxorubicin (DXR) on development of tumours in two models of breast cancer, pseudometastatic and metastatic, in mice. The former used the mouse mammary carcinoma cell line GZHI, which expresses the human MUC-1 gene (L. Ding, E.N. Lalani, M. Reddish, R. Koganty, T. Wong, J. Samuel, M.B. Yacyshyn, A. Meikle, P.Y.S. Fung, J. Taylor-Papadimitriou, B.M. Longenecker, Cancer Immunol. Immunother. 36 (1993) 9--17). GZHI cells seed into the lungs of Balb/c mice following intravenous injection. The latter used the 4T1-MUC1 cell line, a MUC-1 transfectant of the mouse mammary carcinoma cell line 4T1, which metastasizes from a primary mammary fatpad (mfp) implant to the lungs (C.J. Aslakson, F.R. Miller, Cancer Res. 52 (1992) 1399--1405). B27.29, a monoclonal antibody against the MUC-1 antigen, was used to target sterically stabilized immunoliposomes (SIL[B27.29]) to tumour cells. In vitro, SIL[B27.29] showed high specific binding to both GZHI and 4T1-MUC1 cells. The IC(50) of DXR-loaded SIL[B27.29] was similar to that of free drug for GZHI cells. In the pseudometastatic model, mice treated with a single injection of 6 mg DXR/kg in DXR-SIL[B27.29] at 24 h after cell implantation had longer survival times than those injected with non-targeted liposomal drug. In the metastatic model, severe combined immune deficiency mice given weekly injectionsx3 of 2.5 mg DXR/kg encapsulated in either targeted or non-targeted liposomes were almost equally effective in slowing growth of the primary tumour and reducing development of lung tumours. Surgical removal of the primary tumour from mfp, followed by various chemotherapy regimens, was attempted, but removal of the primary tumour was generally incomplete; tumour regrowth occurred and metastases developed in the lungs in all treatment groups. DXR-SL reduced the occurrence of regrowth of the primary tumour, whereas neither targeted liposomal drug or free drug prevented regrowth. We conclude that monoclonal antibody-targeted liposomal DXR is effective in treating early lesions in both the pseudometastatic and metastatic models, but limitations to the access of the targeted liposomes to tumour cells in the primary tumour compromised their therapeutic efficacy in treating the more advanced lesions.

Adventures in targeting.

ABSTRACT 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.

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.

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.

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.

Stealth™ Liposomes for the Targeting of Drugs in Cancer Therapy

Anticancer drugs have a variety of dose-limiting side effects as a consequence of their distribution to normal, sensitive tissues as well as to cancerous tissues. The goal for the targeting of liposomal drugs in cancer therapy is to increase the localization of the anticancer drugs to the diseased tissue, while at the same time decreasing their localization to normal tissue, i.e. to increase the selective toxicity of the drugs. Put another way, by the entrapment of anticancer drugs in liposomes we are aiming to increase the efficacy of the drugs, while reducing their side effects (toxicity), resulting in an increase in the therapeutic indices for the drugs.