Harnessing Nanomedicine to Potentiate the Chemo-Immunotherapeutic Effects of Doxorubicin and Alendronate Co-Encapsulated in Pegylated Liposomes.

Encapsulation of Doxorubicin (Dox), a potent cytotoxic agent and immunogenic cell death inducer, in pegylated (Stealth) liposomes, is well known to have major pharmacologic advantages over treatment with free Dox. Reformulation of alendronate (Ald), a potent amino-bisphosphonate, by encapsulation in pegylated liposomes, results in significant immune modulatory effects through interaction with tumor-associated macrophages and activation of a subset of gamma-delta T lymphocytes. We present here recent findings of our research work with a formulation of Dox and Ald co-encapsulated in pegylated liposomes (PLAD) and discuss its pharmacological properties vis-à-vis free Dox and the current clinical formulation of pegylated liposomal Dox.

Pegylated Liposomal Alendronate Biodistribution, Immune Modulation, and Tumor Growth Inhibition in a Murine Melanoma Model.

While tumor-associated macrophages (TAM) have pro-tumoral activity, the ablation of macrophages in cancer may be undesirable since they also have anti-tumoral functions, including T cell priming and activation against tumor antigens. Alendronate is a potent amino-bisphosphonate that modulates the function of macrophages in vitro, with potential as an immunotherapy if its low systemic bioavailability can be addressed. We repurposed alendronate in a non-leaky and long-circulating liposomal carrier similar to that of the clinically approved pegylated liposomal doxorubicin to facilitate rapid clinical translation.

Comparative effects of free doxorubicin, liposome encapsulated doxorubicin and liposome co-encapsulated alendronate and doxorubicin (PLAD) on the tumor immunologic milieu in a mouse fibrosarcoma model.

We have previously shown that alendronate, an amino-bisphosphonate, when reformulated in liposomes, can significantly enhance the efficacy of cytotoxic chemotherapies and help remodel the immunosuppressive tumor microenvironment towards an immune-permissive milieu resulting in increased anticancer efficacy. In addition, we have previously shown that the strong metal-chelating properties of alendronate can be exploited for nuclear imaging of liposomal biodistribution. To further improve anticancer efficacy, a pegylated liposome formulation co-encapsulating alendronate and doxorubicin (PLAD) has been developed.

Ex-vivo activation of a liposomal prodrug of mitomycin C by human tumors.

Purpose: To examine the ex- vivo ability of explanted human tumors and normal tissue to activate liposomal mitomycin C lipidic prodrug (MLP) by releasing the active free drug form, mitomycin C (MMC).

Methods: We tested conversion of MLP to MMC in an ex vivo assay using explanted tissues obtained during routine surgery to remove primary tumors or metastases. Tumor and adjacent normal tissue were obtained from freshly explanted tumors and were immediately deep frozen at - 70 °C.

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

: 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. : 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.

Development of Promitil®, a lipidic prodrug of mitomycin c in PEGylated liposomes: From bench to bedside.

Several liposome products have been approved for the treatment of cancer. In all of them, the active agents are encapsulated in the liposome water phase passively or by transmembrane ion gradients. An alternative approach in liposomal drug delivery consists of chemically modifying drugs to form lipophilic prodrugs with strong association to the liposomal bilayer.

Pharmacokinetics of mitomycin-c lipidic prodrug entrapped in liposomes and clinical correlations in metastatic colorectal cancer patients.

Background Pegylated liposomal (PL) mitomycin-c lipidic prodrug MLP) may be a useful agent in patients with metastatic colo-rectal carcinoma (CRC). We report here on the pharmacokinetics and clinical observations in a phase 1A/B study with PL-MLP. Methods Plasma levels of MLP were examined in 53 CRC patients, who received PL-MLP either as single agent or in combination with capecitabine and/or bevacizumab.

Repurposing amino-bisphosphonates by liposome formulation for a new role in cancer treatment.

Amino-bisphosphonates (N-BPs) have been commercially available for over four decades and are used for the treatment of osteoporosis, Paget's disease, hypercalcemia of malignancy, and bone metastases derived from various cancer types. Zoledronate and alendronate, two of the most potent N-BPs, have demonstrated direct tumoricidal activity on tumor cells and immune modulatory effects on myeloid cells and T cells in vitro and in animal models of cancer. However, the rapid renal clearance and sequestration in mineral bone of these drugs in free form severely limit their systemic exposure and applications in cancer patients.

Folate receptor targeting of radiolabeled liposomes reduces intratumoral liposome accumulation in human KB carcinoma xenografts.

Background: Active, ligand-mediated, targeting of functionalized liposomes to folate receptors (FRs) overexpressed on cancer cells could potentially improve drug delivery and specificity. Studies on folate-targeting liposomes (FTLs) have, however, yielded varying results and generally fail to display a clear benefit of FR targeting.

Method: Tumor accumulating potential of FTLs and NTLs were investigated in a FR overex-pressing xenograft model by positron emission tomography/computed tomography imaging.

In Vivo PET Tracking of Zr-Labeled Vγ9Vδ2 T Cells to Mouse Xenograft Breast Tumors Activated with Liposomal Alendronate.

Gammadelta T (γδ-T) cells are strong candidates for adoptive immunotherapy in oncology due to their cytotoxicity, ease of expansion, and favorable safety profile. The development of γδ-T cell therapies would benefit from non-invasive cell-tracking methods and increased targeting to tumor sites. Here we report the use of [Zr]Zr(oxinate) to track Vγ9Vδ2 T cells in vivo by positron emission tomography (PET).

Targeting of folate-conjugated liposomes with co-entrapped drugs to prostate cancer cells via prostate-specific membrane antigen (PSMA).

Folate-targeted liposomes (FTL) were tested as drug delivery vehicles to PSMA-positive cancer cells. We used FL with co-entrapped mitomycin C lipophilic prodrug (MLP) and doxorubicin (DOX), and the LNCaP prostate cancer cell line which expresses PSMA but is negative for folate receptor. A major increase in cell drug levels was observed when LNCaP cells were incubated with FTL as compared to non-targeted liposomes (NTL).

Liposome-induced immunosuppression and tumor growth is mediated by macrophages and mitigated by liposome-encapsulated alendronate.

Liposomal nanoparticles are the most commonly used drug nano-delivery platforms. However, recent reports show that certain pegylated liposomal nanoparticles (PLNs) and polymeric nanoparticles have the potential to enhance tumor growth and inhibit antitumor immunity in murine cancer models. We sought herein to identify the mechanisms and determine whether PLN-associated immunosuppression and tumor growth can be reversed using alendronate, an immune modulatory drug.

Characterization of Pegylated Liposomal Mitomycin C Lipid-Based Prodrug (Promitil) by High Sensitivity Differential Scanning Calorimetry and Cryogenic Transmission Electron Microscopy.

The effect of a lipidated prodrug of mitomycin C (MLP) on the membrane of a pegylated liposome formulation (PL-MLP), also known as Promitil, was characterized through high-sensitivity differential scanning calorimetry (DSC) and cryo-TEM. The thermodynamic analysis demonstrated that MLP led to the formation of heterogeneous domains in the membrane plane of PL-MLP. MLP concentrated in prodrug-rich domains, arranged in high-ordered crystal-like structures, as suggested by the sharp and high enthalpy endotherm in the first heating scanning.

Exploiting the Metal-Chelating Properties of the Drug Cargo for In Vivo Positron Emission Tomography Imaging of Liposomal Nanomedicines.

The clinical value of current and future nanomedicines can be improved by introducing patient selection strategies based on noninvasive sensitive whole-body imaging techniques such as positron emission tomography (PET). Thus, a broad method to radiolabel and track preformed nanomedicines such as liposomal drugs with PET radionuclides will have a wide impact in nanomedicine. Here, we introduce a simple and efficient PET radiolabeling method that exploits the metal-chelating properties of certain drugs (e.

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.

Targeting of pegylated liposomal mitomycin-C prodrug to the folate receptor of cancer cells: Intracellular activation and enhanced cytotoxicity.

Mitomycin C (MMC) is a powerful anti-bacterial, anti-fungal and anti-tumor antibiotic, often active against multidrug resistant cells. Despite a broad spectrum of antitumor activity, MMC clinical use is relatively limited due to its fast clearance and dose-limiting toxicity. To exploit the potential antitumor activity of MMC and reduce its toxicity we have previously developed a formulation of pegylated liposomes with a lipophilic prodrug of MMC (PL-MLP), activated by endogenous reducing agents which are abundant in the tumor cell environment in the form of different thiols.

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.

Pegylated liposomal mitomycin C prodrug enhances tolerance of mitomycin C: a phase 1 study in advanced solid tumor patients.

Mitomycin C (MMC) has potent cytotoxicity but cumulative toxicity limits widespread use. In animals, pegylated liposomal mitomycin C lipid-based prodrug (PL-MLP) was well tolerated and more effective than free MMC. We evaluated PL-MLP in patients with advanced cancer.

A comparative study of folate receptor-targeted doxorubicin delivery systems: dosing regimens and therapeutic index.

Ligand-receptor mediated targeting may affect differently the performance of supramolecular drug carriers depending on the nature of the nanocarrier. In this study, we compare the selectivity, safety and activity of doxorubicin (Dox) entrapped in liposomes versus Dox conjugated to polymeric nanocarriers in the presence or absence of a folic acid (FA)-targeting ligand to cancer cells that overexpress the folate receptor (FR). Two pullulan (Pull)-based conjugates of Dox were synthesized, (FA-PEG)-Pull-(Cyst-Dox) and (NH2-PEG)-Pull-(Cyst-Dox).

Adoptive immunotherapy of epithelial ovarian cancer with Vγ9Vδ2 T cells, potentiated by liposomal alendronic acid.

Adoptive immunotherapy using γδ T cells harnesses their natural role in tumor immunosurveillance. The efficacy of this approach is enhanced by aminobisphosphonates such as zoledronic acid and alendronic acid, both of which promote the accumulation of stimulatory phosphoantigens in target cells. However, the inefficient and nonselective uptake of these agents by tumor cells compromises the effective clinical exploitation of this principle.

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.

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.

Pharmacological basis of pegylated liposomal doxorubicin: impact on cancer therapy.

We review here various pharmacological aspects of pegylated liposomal doxorubicin (PLD) which have important implications on the safety and efficacy profile of this important agent. Particularly, the formulation properties of PLD and its long circulation time and the relationship between the high microvascular permeability of tumors and the selective accumulation of PLD in tumors are addressed. Emphasis is given to the correlation of pharmacokinetic parameters with pharmacodynamic effects of PLD.

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.

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).