Luigi Cattel

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.

Design of Folic Acid‐Conjugated Nanoparticles for Drug Targeting

The new concept developed in this study is the design of poly(ethylene glycol) (PEG)-coated biodegradable nanoparticles coupled to folic acid to target the folate-binding protein; this molecule is the soluble form of the folate receptor that is overexpressed on the surface of many tumoral cells. For this purpose, a novel copolymer, the poly[aminopoly(ethylene glycol)cyanoacrylate-co-hexadecyl cyanoacrylate] [poly(H(2)NPEGCA-co-HDCA)] was synthesized and characterized. Then nanoparticles were prepared by nanoprecipitation of the obtained copolymer, and their size, zeta potential, and surface hydrophobicity were investigated. Nanoparticles were then conjugated to the activated folic acid via PEG terminal amino groups and purified from unreacted products. Finally, the specific interaction between the conjugate folate-nanoparticles and the folate-binding protein was evaluated by surface plasmon resonance. This analysis confirmed a specific binding of the folate-nanoparticles to the folate-binding protein. This interaction did not occur with nonconjugated nanoparticles used as control. Thus, folate-linked nanoparticles represent a potential new drug carrier for tumor cell-selective targeting.

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.

Systematic analysis of yeast strains with possible defects in lipid metabolism

Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomyces cerevisiae involved in these processes. These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism. During these investigations it was demonstrated that different wild‐type strains, among them FY1679, CEN.PK2‐1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism. Copyright

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.

Lipoplexes Targeting the CD44 Hyaluronic Acid Receptor for Efficient Transfection of Breast Cancer Cells

Lipoplexes containing a hyaluronic acid-dioleoylphosphatidylethanolamine (HA-DOPE) conjugate were designed to target the CD44 receptor on breast cancer cells. Cationic liposomes composed of a mixture of [2-(2,3-didodecyloxypropyl)hydroxyethyl]ammonium bromide (DE) and dioleoylphosphatidylethanolamine (DOPE) with or without HA-DOPE were prepared, characterized, and used to form a complex with plasmid DNA pCMV-luc. Lipoplexes displayed a negative zeta potential and a mean diameter between 250-300 nm. Cytotoxicity and transfection efficiency of the lipoplexes were determined on the MDA-MB-231and MCF-7 breast cancer cell lines. Cytotoxicity was not modified by the presence of HA-DOPE. However HA-DOPE increased the level of transfection on CD44-expressing MDA-MB-231 cells compared to the MCF-7 line, which expresses very low levels of CD44. The transfection on the MDA-MB-231 cells was highly inhibited by anti-CD44 Hermes-1 antibody but not by the nonspecific anti-ErbB2 antibody. In conclusion, cationic liposomes containing the HA-DOPE conjugate mediated good transfection on CD44 expressing cell lines in culture.

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.

In vitro inhibition of animal and higher plants 2,3-oxidosqualene-sterol cyclases by 2-aza-2,3- dihydrosqualene and derivatives, and by other ammonium-containing molecules

2-Aza-2,3-dihydrosqualene and related molecules, a series of new compounds designed as analogues of the transient carbocationic high energy intermediate, occurring in the oxirane ring opening during the cyclization of 2,3-oxidosqualene, were tested in vitro as inhibitors of the microsomal 2,3-oxidosqualene cyclase of animals (rat liver) and of higher plants (maize, pea). These molecules proved to be good and specific inhibitors for the cyclases of both phyla. The inhibition is due to positively charged species and is sensitive to the steric hindrance around the nitrogen-atom. 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol and 4,10 beta-dimethyl-trans-decal-3 beta-ol, which have previously been described (J.A. Nelson et al., J. Am. chem. Soc. 100, 4900 (1978] as inhibitors of the 2,3-oxidosqualene cyclase of chinese hamster ovary cells, were found to be non-competitive inhibitors of the rat liver microsomal enzyme and presented no activity towards the higher plants cyclases. Aza derivatives of these decalines (A. Rahier et al., Phytochemistry, in press), which were aimed to mimic the C-8 carbocationic intermediate occurring during later steps of the 2,3-oxidosqualene cyclization did not inhibit the cyclases. This result underlines the theoretical limitations of the high energy analogues concept in designing enzyme inhibitors. Amongst other molecules tested, 2,3-epiminosqualene was found to be a reversible, non-competitive inhibitor of the cyclases; similarly U18666A was a very potent inhibitor of the microsomal cyclases. In contrast AMO 1618, a known anticholesterolemic agent reported previously to act at the level of the 2,3-oxidosqualene cyclization step, was not found per se to act on the cyclases.

The squalene-2,3-epoxide cyclase as a model for the development of new drugs

The 2,3-oxido squalene (SO) cyclases represent a group of enzymes which convert SO into polycyclic triterpenoids such as lanosterol, cycloartenol, cucurbitadienol and β-amyrin. Taking into account the postulated model of the enzymatic cyclization of SO, we have investigated the possibility of designing compounds that would be selective and potent inhibitors of SO cyclases. Due to the fundamental role of sterols in animal, higher plant and fungal tissues, these inhibitors might behave as very selective (ipocholesterolemic, antifungal or phytotoxic) drugs.Our first approach was the synthesis and biological evaluation of 2-aza-2,3-dihydrosqualene and its derivatives which, being protonated at physiological pH, would present some similarities to the C-2 carbon ion generated by the opening of the oxirane ring of SO. Microsomes from different sources (germinated pea cotyledons, maize seedlings, rat liver and yeasts) were utilized to determine the inhibition values (I50: concentration of inhibitor producing 50% inhibition at a given substrate concentration).From the results obtained so far we conclude that 2-aza-2-dihydrosqualene and its derivatives strongly inhibited the cyclases, the site of the enzyme responsible for binding to the inhibitor is quite sensitive to the steric hindrance, and the degree of the inhibitory activity is greater in higher plants than in rat liver or fungi.