Josbert M. Metselaar

Role of complement activation in hypersensitivity reactions to doxil and hynic PEG liposomes: experimental and clinical studies.

ABSTRACT Pegylated liposomal doxorubicin (Doxil) and 99mTc-HYNIC PEG liposomes (HPL) were reported earlier to cause hypersensitivity reactions (HSRs) in a substantial percentage of patients treated i.v. with these formulations. Here we report that (1) Doxil, HPL, pegylated phosphatidylethanolamine (PEG-PE)-containing empty liposomes matched with Doxil and HPL in size and lipid composition, and phosphatidylglycerol (PG)-containing negatively charged vesicles were potent C activators in human serum in vitro, whereas small neutral liposomes caused no C activation. (2) Doxil and other size-matched PEG-PE and/or PG-containing liposomes also caused massive cardiopulmonary distress with anaphylactoid shock in pigs via C activation, whereas equivalent neutral liposomes caused no hemodynamic changes. (3) A clinical study showed more frequent and greater C activation in patients displaying HSR than in non-reactive patients. These data suggest that liposome-induced HSRs in susceptible individuals may be due to C activation, which, in turn, is due to the presence of negatively charged PEG-PE in these vesicles.

Improving solubility and chemical stability of natural compounds for medicinal use by incorporation into liposomes

Natural bioactive compounds have been studied for a long time for their chemopreventive and therapeutic potential in several chronic inflammatory diseases, including cancer. However, their physicochemical properties generally result in poor chemical stability and lack of in vivo bioavailability. Very few human clinical trials have addressed absorption, distribution, metabolism, and excretion of these compounds in relation to efficacy. This limits the use of these valuable natural compounds in the clinic. In this study, we examined caffeic acid (derivatives), carvacrol (derivatives), thymol, pterostilbene (derivatives), and N-(3-oxo-dodecanoyl)-l-homoserine lactone. These are natural compounds with strong anti-inflammatory properties derived from plants and bacteria. However, these compounds have poor water solubility or are chemically unstable. To overcome these limitations we have prepared liposomal formulations. Our results show that lipophilic 3-oxo-C(12)-homoserine lactone and stilbene derivatives can be loaded into liposomal lipid bilayer with efficiencies of 50-70%. Thereby, the liposomes solubilize these compounds, allowing intravenous administration without use of solvents. When compounds could not be loaded into the lipid bilayer (carvacrol and thymol) or are rapidly extracted from the liposomes in the presence of serum albumin (3-oxo-C(12)-homoserine lactone and pterostilbene derivatives), derivatization of the compound into a water-soluble prodrug was shown to improve loading efficiency and encapsulation stability. The phosphate forms of carvacrol and pterostilbene were loaded into the aqueous interior of the liposomes and encapsulation was unaffected by the presence of serum albumin. Chemical instability of resveratrol was improved by liposome-encapsulation, preventing inactivating cis-trans isomerization. For caffeic acid, liposomal encapsulation did not prevent oxidation into a variety of products. Still, by derivatization into a phenyl ester, the compound could be stably encapsulated without chemical degradation. Despite the instability of liposome-association of 3-oxo-C(12)-homoserine lactone and resveratrol, intravenous administration of these compounds inhibited tumor growth for approximately 70% in a murine tumor model, showing that simple solubilization can have important therapeutic benefits.

Multimodal clinical imaging to longitudinally assess a nanomedical anti-inflammatory treatment in experimental atherosclerosis.

Atherosclerosis is an inflammatory disease causing great morbidity and mortality in the Western world. To increase the anti-inflammatory action and decrease adverse effects of glucocorticoids (PLP), a nanomedicinal liposomal formulation of this drug (L-PLP) was developed and intravenously applied at a dose of 15 mg/kg PLP to a rabbit model of atherosclerosis. Since atherosclerosis is a systemic disease, emerging imaging modalities for assessing atherosclerotic plaque are being developed. (18)F-Fluoro-deoxy-glucose positron emission tomography and dynamic contrast enhanced magnetic resonance imaging, methods commonly used in oncology, were applied to longitudinally assess therapeutic efficacy. Significant anti-inflammatory effects were observed as early as 2 days that lasted up to at least 7 days after administration of a single dose of L-PLP. No significant changes were found for the free PLP treated animals. These findings were corroborated by immunohistochemical analysis of macrophage density in the vessel wall. In conclusion, this study evaluates a powerful two-pronged strategy for efficient treatment of atherosclerosis that includes nanomedical therapy of atherosclerotic plaques and the application of noninvasive and clinically approved imaging techniques to monitor delivery and therapeutic responses. Importantly, we demonstrate unprecedented rapid anti-inflammatory effects in atherosclerotic lesions after the nanomedical therapy.

Liposomal Drug Formulations in the Treatment of Rheumatoid Arthritis

Liposomes have been extensively investigated as drug delivery systems in the treatment of rheumatoid arthritis (RA). Low bioavailability, high clearance rates and limited selectivity of several important drugs used for RA treatment require high and frequent dosing to achieve sufficient therapeutic efficacy. However, high doses also increase the risk for systemic side effects. The use of liposomes as drug carriers may increase the therapeutic index of these antirheumatic drugs. Liposomal physicochemical properties can be changed to optimize penetration through biological barriers and retention at the site of administration, and to prevent premature degradation and toxicity to nontarget tissues. Optimal liposomal properties depend on the administration route: large-sized liposomes show good retention upon local injection, small-sized liposomes are better suited to achieve passive targeting. PEGylation reduces the uptake of the liposomes by liver and spleen, and increases the circulation time, resulting in increased localization at the inflamed site due to the enhanced permeability and retention (EPR) effect. Additionally liposomal surfaces can be modified to achieve selective delivery of the encapsulated drug to specific target cells in RA. This review gives an overview of liposomal drug formulations studied in a preclinical setting as well as in clinical practice. It covers the use of liposomes for existing antirheumatic drugs as well as for new possible treatment strategies for RA. Both local administration of liposomal depot formulations and intravenous administration of passively and actively targeted liposomes are reviewed.

Liposomes in the treatment of inflammatory disorders.

This review focuses on the therapeutic utility of liposomes in the treatment of inflammatory disorders, and aims to offer the reader an overview of the in vivo results obtained with liposomally encapsulated anti-inflammatory and immune suppressive drugs. The past 30 years has clearly indicated the added value of liposomes in the search for solutions for the delivery problems encountered. However, only a few liposomal anti-inflammatory therapeutics have entered the clinic. Reasons for the hurdles existing in the translation of promising preclinical findings to clinical studies are discussed.

Nanomedicines for Inflammatory Arthritis: Head-to-Head Comparison of Glucocorticoid-Containing Polymers, Micelles, and Liposomes

As an emerging research direction, nanomedicine has been increasingly utilized to treat inflammatory diseases. In this head-to-head comparison study, four established nanomedicine formulations of dexamethasone, including liposomes (L-Dex), core-cross-linked micelles (M-Dex), slow releasing polymeric prodrugs (P-Dex-slow), and fast releasing polymeric prodrugs (P-Dex-fast), were evaluated in an adjuvant-induced arthritis rat model with an equivalent dose treatment design. It was found that after a single i.v. injection, the formulations with the slower drug release kinetics (i.e., M-Dex and P-Dex-slow) maintained longer duration of therapeutic activity than those with relatively faster drug release kinetics, resulting in better joint protection. This finding will be instructional in the future development and optimization of nanomedicines for the clinical management of rheumatoid arthritis. The outcome of this study also illustrates the value of such head-to-head comparison studies in translational nanomedicine research.

Therapeutic Application of Long-Circulating Liposomal Glucocorticoids in Auto-Immune Diseases and Cancer

Glucorticoids are potent drugs that have a multitude of pharmacological actions both at genomic and non-genomic levels. Many of the diseases in which glucorticoids are routinely administered are featured by angiogenesis and enhanced capillary permeability, permitting targeted delivery using long-circulating drug delivery systems. By encapsulation of glucorticoids in long-circulating liposomes, drug levels at the site of the pathology are markedly higher, increasing and prolonging therapeutic efficacy in models of rheumatoid arthritis, multiple sclerosis, and cancer.

Liposomes for intravenous drug targeting: design and applications.

Drug targeting with liposomes has been studied for over 25 years and has demonstrated its value in clinical practice. This mini review offers an overview of the design and application of liposomes for i.v. drug targeting. Two approaches are outlined: passive and active targeting. The former approach is based on liposomes with prolonged circulation and selective target localization properties, while in the latter approach specific targeting ligands are coupled to the liposome surface in order to achieve enhanced interaction with target cell membranes.

Liposomal Encapsulation of Glucocorticoids Alters Their Mode of Action in the Treatment of Experimental Autoimmune Encephalomyelitis

Glucocorticoids (GCs) are widely used to treat acute relapses of multiple sclerosis (MS). In this study, we demonstrate that liposomal encapsulation augments the therapeutic potency of GCs as they ameliorate experimental autoimmune encephalomyelitis (EAE) to the same extent as free GC, but at strongly reduced dosage and application frequency. Importantly, this is accompanied by an altered mode of action. Unlike free GCs, which mainly target T lymphocytes during EAE therapy, liposomal GCs only marginally affect T cell apoptosis and function. In contrast, liposomal GCs efficiently repress proinflammatory macrophage functions and upregulate anti-inflammatory genes associated with the alternatively activated M2 phenotype. The GC receptor (GR) per se is indispensable for the therapeutic efficacy of liposomal GC. In contrast to free GCs, however, the individual deletion of the GR either in T cells or myeloid cells has little effect on the efficacy of liposomal GCs in the treatment of EAE. Only the combined deletion of the GR in both cellular compartments markedly compromises the therapeutic effect of liposomal GCs on disease progression. In conclusion, encapsulation of GC does not only enhance their efficacy in the treatment of EAE but also alters their target cell specificity and their mode of action compared with free GCs.

Liposomal glucocorticoids as tumor-targeted anti-angiogenic nanomedicine in B16 melanoma-bearing mice.

This study evaluates whether the inhibitory effects of prednisolone phosphate (PLP) encapsulated in long-circulating liposomes (LCL-PLP) on tumor growth and tumor angiogenesis described previously can be generalized to other types of glucocorticoids (GC) encapsulated in LCL (LCL-GC). Four types of synthetic GC, i.e. budesonide disodium phosphate (BUP), dexamethasone disodium phosphate (DXP), methylprednisolone disodium phosphate (MPLP), and PLP, were selected based on the difference in their potency to activate the human glucocorticoid receptor. The effects of all LCL-GC on the production of angiogenic/inflammatory factors in vivo in the B16.F10 murine melanoma model as well as on the viability and proliferation of tumor cells and endothelial cells in vitro were investigated. Our results show that all four selected LCL-GC formulations inhibit tumor growth, albeit to different degrees. The differences in antitumor activity of LCL-GC correlate with their efficacy to suppress tumor angiogenesis and inflammation. The strongest antitumor effect is achieved by LCL-encapsulated BUP (LCL-BUP), due to the highest potency of BUP versus the other three GC types. The in vitro results presented herein suggest that LCL-BUP has strong cytotoxic effects on B16.F10 melanoma cells and the anti-proliferative effects of all LCL-GC towards angiogenic endothelial cells play a role in their antitumor activity.