Franco Dosio

Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential

Among several promising new drug-delivery systems, liposomes represent an advanced technology to deliver active molecules to the site of action, and at present several formulations are in clinical use. Research on liposome technology has progressed from conventional vesicles (“first-generation liposomes”) to “second-generation liposomes”, in which long-circulating liposomes are obtained by modulating the lipid composition, size, and charge of the vesicle. Liposomes with modified surfaces have also been developed using several molecules, such as glycolipids or sialic acid. A significant step in the development of long-circulating liposomes came with inclusion of the synthetic polymer poly-(ethylene glycol) (PEG) in liposome composition. The presence of PEG on the surface of the liposomal carrier has been shown to extend blood-circulation time while reducing mononuclear phagocyte system uptake (stealth liposomes). This technology has resulted in a large number of liposome formulations encapsulating active molecules, with high target efficiency and activity. Further, by synthetic modification of the terminal PEG molecule, stealth liposomes can be actively targeted with monoclonal antibodies or ligands. This review focuses on stealth technology and summarizes pre-clinical and clinical data relating to the principal liposome formulations; it also discusses emerging trends of this promising technology.

Preparation, characterization and properties of sterically stabilized paclitaxel-containing liposomes.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, approved by the FDA for the treatment of ovarian and breast cancers. Due to its low solubility in water, it is clinically administered dissolved in Cremophor EL, (polyethoxylated castor oil) and ethanol, which cause serious side effects. Inclusion of paclitaxel in liposomal formulations has proved to be a good approach to eliminating this vehicle and improving the drugs antitumor efficacy. We prepared different conventional and PEGylated liposomes containing paclitaxel and determined encapsulation efficiency, physical stability and drug leakage in human plasma. The best conventional liposome formulation was composed of ePC/PG 9:1, while for PEGylated liposomes the best composition was ePC/PG/CHOL/PEG(5000)-DPPE 9:1:2:0.7. PEGylated liposomes were found to be less stable during storage than the corresponding conventional liposomes and to have lower drug release in human plasma at 37 degrees C. In vitro cytotoxic activities were evaluated on HT-29 human colon adenocarcinoma and MeWo melanoma cell lines. After 2 and 48 h, conventional liposomes had the same cytotoxicity as free paclitaxel, while PEGylated liposomes were as active as free drug, only after 48 h. Pharmacokinetics and biodistribution were evaluated in Balb/c mice after i.v. injection of paclitaxel, formulated in Cremophor EL or in conventional or in PEGylated liposomes. Encapsulation of paclitaxel in conventional liposomes produced marked differences over the free drug pharmacokinetics. PEGylated liposomes were long-circulating liposomes, with an increased t(1/2) beta 48.6 h, against t(1/2) beta 9.27 h of conventional liposomes. Biodistribution studies showed a considerable decrease in drug uptake in MPS-containing organs (liver and spleen) at 0.5 and 3 h after injection with PEGylated compared to conventional liposomes.

Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing docetaxel.

The taxanes, paclitaxel and docetaxel, are anticancer agents used in clinical trials against ovarian carcinoma, breast, lung and head/neck cancer. Paclitaxel, very insoluble in water, is generally formulated using Cremophor EL. Docetaxel, more soluble in water, is formulated using Tween 80 and ethanol. Tween 80, albeit less toxic than Cremophor EL, may be responsible of some toxic effects. To eliminate these vehicles and improve the drugs antitumor efficacy, taxanes have been incorporated in liposomes. We compared formulation, stability, biodistribution and pharmacokinetics of docetaxel in conventional and PEGylated liposomes. Of the several formulations examined, docetaxel-liposomes composed of ePC/PG/CHOL 9:1:2 and ePC/PG/DSPE-PEG2000/CHOL 9:1:2:0.7 were the most effective. Both conventional and PEGylated docetaxel-liposomes were stable at 4 degrees C after 15 days, whereas in the presence of serum at 37 degrees C they were less stable. The IC50 values of docetaxel-liposomes, evaluated on HT-29 and Igrov1 cell lines, remained very high. Pharmacokinetics and biodistribution were evaluated in Balb/c mice after i.v. injection of [14C]docetaxel, formulated in Tween 80 or in 3H-labeled conventional or PEGylated liposomes. The t(1/2)beta, which was low for docetaxel (52.3 min), rose to 260 min for conventional docetaxel-liposomes and to 665 min for PEGylated docetaxel liposomes. Biodistribution studies confirmed the pharmacokinetics.

From conventional to stealth liposomes: a new frontier in cancer chemotherapy.

Many attempts have been made to achieve good selectivity to targeted tumor cells by preparing specialized carrier agents that are therapeutically profitable for anticancer therapy. Among these, liposomes are the most studied colloidal particles thus far applied in medicine and in particular in antitumor therapy. Although they were first described in the 1960s, only at the beginning of 1990s did the first therapeutic liposomes appear on the market. The first-generation liposomes (conventional liposomes) comprised a liposome-containing amphotericin B, Ambisome (Nexstar, Boulder, CO, USA), used as an antifungal drug, and Myocet (Elan Pharma Int, Princeton, NJ, USA), a doxorubicin-containing liposome, used in clinical trials to treat metastatic breast cancer. The second-generation liposomes (“pure lipid approach”) were long-circulating liposomes, such as Daunoxome, a daunorubicin-containing liposome approved in the US and Europe to treat AIDS-related Kaposis sarcoma. The third-generation liposomes were surface-modified liposomes with gangliosides or sialic acid, which can evade the immune system responsible for removing liposomes from circulation. The fourth-generation liposomes, pegylated liposomal doxorubicin, were called “stealth liposomes” because of their ability to evade interception by the immune system, in the same way as the stealth bomber was able to evade radar. Actually, the only stealth liposome on the market is Caelyx/Doxil (Schering-Plough, Madison NJ, USA), used to cure AIDS-related Kaposis sarcoma, resistant ovarian cancer and metastatic breast cancer. Pegylated liposomal doxorubicin is characterized by a very long-circulation half-life, favorable pharmacokinetic behavior and specific accumulation in tumor tissues. These features account for the much lower toxicity shown by Caelyx in comparison to free doxorubicin, in terms of cardiotoxicity, vesicant effects, nausea, vomiting and alopecia. Pegylated liposomal doxorubicin also appeared to be less myelotoxic than doxorubicin. Typical forms of toxicity associated to it are acute infusion reaction, mucositis and palmar plantar erythrodysesthesia, which occur especially at high doses or short dosing intervals. Active and cell targeted liposomes can be obtained by attaching some antigen-directed monoclonal antibodies (Moab or Moab fragments) or small proteins and molecules (folate, epidermal growth factor, transferrin) to the distal end of polyethylene glycol in pegylated liposomal doxorubicin. The most promising therapeutic application of liposomes is as non-viral vector agents in gene therapy, characterized by the use of cationic phospholipids complexed with the negatively charged DNA plasmid. The use of liposome formulations in local-regional anticancer therapy is also discussed. Finally, pegylated liposomal doxorubicin containing radionuclides are used in clinical trials as tumor-imaging agents or in positron emission tomography.

Preparation, characterization and properties in vitro and in vivo of a paclitaxel-albumin conjugate

Paclitaxel (taxol) is in routine clinical use for treatment of a variety of cancers. Because of its low aqueous solubility, it requires Cremophor EL (polyethoxylated castor oil) and ethanol as a vehicle (Diluent 12). These agents cause severe allergic reactions upon intravenous administration. In this study paclitaxel was covalently attached to human serum albumin. The 2′-hydroxyl group of the drug was esterefied with succinic anhydride and then derivatized to give the N-hydroxy-3-sulfo-succinimide active ester, highly reactive to lysyl amino groups of the protein. Two different conjugate populations (with 6 or 30 average molecules of drug linked to each albumin molecule) were prepared, purified and characterized. The conjugates were stable in physiological solution and in serum whereas the presence of proteases or liver extract released the drug in a linear fashion. The antitumor activity of free drug and conjugates was tested on three different tumor cell lines. The conjugates maintained high cytotoxicity with efficient cell binding and internalization followed by release of the drug inside the cell. The pharmacokinetics of the conjugate (after iv administration) was evaluated and compared to that of the free drug. Both followed a bicompartmental model but elimination of the conjugate from the plasma was much slower than the free drug, giving a relevant rise in AUC and MRT values. The conjugate also released of parent drug continuously to the plasma over prolonged periods, thus providing a depot effect. The acute toxicity noted with the standard formulation of taxol was strongly reduced in our albumin-conjugated preparation.

Hyaluronic acid for anticancer drug and nucleic acid delivery.

Hyaluronic acid (HA) is widely used in anticancer drug delivery, since it is biocompatible, biodegradable, non-toxic, and non-immunogenic; moreover, HA receptors are overexpressed on many tumor cells. Exploiting this ligand-receptor interaction, the use of HA is now a rapidly-growing platform for targeting CD44-overexpressing cells, to improve anticancer therapies. The rationale underlying approaches, chemical strategies, and recent advances in the use of HA to design drug carriers for delivering anticancer agents, are reviewed. Comprehensive descriptions are given of HA-based drug conjugates, particulate carriers (micelles, liposomes, nanoparticles, microparticles), inorganic nanostructures, and hydrogels, with particular emphasis on reports of preclinical/clinical results.

Squalene based nanocomposites: a new platform for the design of multifunctional pharmaceutical theragnostics.

This study reports the design of a novel theragnostic nanomedicine which combines (i) the ability to target a prodrug of gemcitabine to an experimental solid tumor under the influence of a magnetic field with (ii) the imaging of the targeted tumoral nodule. This concept is based on the inclusion of magnetite nanocrystals into nanoparticles (NPs) constructed by self-assembling molecules of the squalenoyl gemcitabine (SQgem) bioconjugate. The nanocomposites are characterized by an unusually high drug loading, a significant magnetic susceptibility, and a low burst release. When injected to the L1210 subcutaneous mice tumor model, these magnetite/SQgem NPs were magnetically guided, and they displayed considerably greater anticancer activity than the other anticancer treatments (magnetite/SQgem NPs nonmagnetically guided, SQgem NPs, or gemcitabine free in solution). The histology and immunohistochemistry investigation of the tumor biopsies clearly evidenced the therapeutic superiority of the magnetically guided nanocomposites, while Prussian blue staining confirmed their accumulation at the tumor periphery. The superior therapeutic activity and enhanced tumor accumulation has been successfully visualized using T(2)-weighted imaging in magnetic resonance imaging (MRI). This concept was further enlarged by (i) the design of squalene-based NPs containing the T(1) Gd(3+) contrast agent instead of magnetite and (ii) the application to other anticancer squalenoyls, such as, cisplatin, doxorubicin, and paclitaxel. Thus, by combining different anticancer medicines as well as contrast imaging agents in NPs, we open the door toward generic conceptual framework for cancer treatment and diagnosis. This new theragnostic nanotechnology platform is expected to have important applications in cancer therapy.

Immunotoxins and Anticancer Drug Conjugate Assemblies: The Role of the Linkage between Components

Immunotoxins and antibody-drug conjugates are protein-based drugs combining a target-specific binding domain with a cytotoxic domain. Such compounds are potentially therapeutic against diseases including cancer, and several clinical trials have shown encouraging results. Although the targeted elimination of malignant cells is an elegant concept, there are numerous practical challenges that limit conjugates’ therapeutic use, including inefficient cellular uptake, low cytotoxicity, and off-target effects. During the preparation of immunoconjugates by chemical synthesis, the choice of the hinge component joining the two building blocks is of paramount importance: the conjugate must remain stable in vivo but must afford efficient release of the toxic moiety when the target is reached. Vast efforts have been made, and the present article reviews strategies employed in developing immunoconjugates, focusing on the evolution of chemical linkers.

Hyaluronic Acid Conjugates as Vectors for the Active Targeting of Drugs, Genes and Nanocomposites in Cancer Treatment

Hyaluronic acid (HA) is a naturally-occurring glycosaminoglycan and a major component of the extracellular matrix. Low levels of the hyaluronic acid receptor CD44 are found on the surface of epithelial, hematopoietic, and neuronal cells; it is overexpressed in many cancer cells, and in particular in tumor-initiating cells. HA has recently attracted considerable interest in the field of developing drug delivery systems, having been used, as such or encapsulated in different types of nanoassembly, as ligand to prepare nano-platforms for actively targeting drugs, genes, and diagnostic agents. This review describes recent progress made with the several chemical strategies adopted to synthesize conjugates and prepare novel delivery systems with improved behaviors.

Poly(ethylene glycol)-human serum albumin-paclitaxel conjugates: preparation, characterization and pharmacokinetics.

Paclitaxel has been found to be very effective against several human cancers, such as ovarian, breast and non-small cell lung cancer and has received marketing approval for metastatic cancers. One of main problems with its use is its poor solubility, which makes irritant solubilitazion agents necessary. In previous research we demonstrated that linkage to human serum albumin (HSA) was useful to increase the in vivo performance of paclitaxel. In this article, in order to improve stability and solubility of paclitaxel conjugate, we linked covalently a monomethoxy poly(ethylene glycol) (mPEG) chain to HSA. New thioimidate mPEG derivatives, highly reactive and stable, were used and two different conjugates (with PEG of molecular mass 2 or 5 kDa) were prepared, purified and characterized. The antitumor activity of the free drug and conjugates was tested on three different tumor cell lines. The PEG grafted conjugates maintained high cytotoxicity, similar to that of ungrafted conjugates, with efficient cell binding and internalization followed by release of the drug inside the cell. The changes in pharmacokinetics and distribution of radio-labelled conjugates were evaluated by i.v. administration to mice and compared with those of the free drug and ungrafted conjugates. The total clearance was reduced (from 3.6 ml/h for free drug to 2.9, 1.97 and 1.41 for ungrafted, 2 and 5 kDa PEG conjugates, respectively). Organ uptake was reduced, in particular by liver and spleen.