Self-assembled squalenoyl-cytarabine nanostructures as a potent nanomedicine for treatment of leukemic diseases.

BACKGROUND: In this investigation, the antileukemic activity of a new nanomedicine based on the conjugation of 1,1',2-tris-nor-squalenic acid with cytarabine (Ara-C) was evaluated. METHODS: Squalenoyl-Ara-C conjugate (Sq-Ara-C) self-assembled nanosystems were obtained by the nanoprecipitation method and characterized in vitro and in vivo. RESULTS: This new nanomedicine, which had a mean diameter of approximately 150 nm, improved the in vitro antitumoral activity of Ara-C in different cancer cell lines (L1210, K562, and MCF-7). Sq-Ara-C nanomedicine allowed reduction of the IC(50) value with respect to the free drug and was also active against drug-resistant leukemic cells (L1210R). A noticeable increase in the survival rate of mice with aggressive metastatic L1210R leukemia was observed after treatment with Sq-Ara-C (50 mg/kg) as compared with the free active compound (100 mg/kg). Finally, evaluation of the biodistribution and pharmacokinetic profiles of the drug demonstrated that these nanoaggregates preferentially localized to the liver and spleen, and protected the drug from physiological metabolism. CONCLUSION: Squalenoylation of cytarabine offers several pharmacological benefits both in vitro and in vivo.

Synthesis of n-squalenoyl cytarabine and evaluation of its affinity with phospholipid bilayers and monolayers.

Cytarabine (1-ß-D-arabinofuranosylcytosine, Ara-C), a pyrimidine nucleoside analogue, is an attractive therapeutic agent for the treatment of both acute and chronic myeloblastic leukemias. 1,1',2-tris-nor-Squalene acid (squaleneCOOH) has been conjugated to cytarabine with the formation of the squalenoyl-cytarabine prodrug, in order to improve the drug lipophilicity and, consequently, the affinity towards the environment of biological membranes, as well as of lipophilic carriers. The interaction of cytarabine and its prodrug with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and monolayers has been studied by the differential scanning calorimetry and the Langmuir-Blodgett techniques. The interaction has been evaluated considering the effect of the compounds on the DMPC MLV and monolayers behaviour. The aim was to have information on the interaction of the drug and the prodrug with the biological membranes and on the possibility to use liposomes as carriers for the prodrug. The results showed an improved affinity of the prodrug with MLV and monolayers with respect to the free drug.

Interaction of acyclovir and its squalenoyl-acyclovir prodrug with DMPC in monolayers at the air/water interface.

Acyclovir has been conjugated to the acyclic isoprenoid chain of squalene to form the squalenoyl-acyclovir prodrug. Its interaction with biomembrane models constituted by dimyristoylphosphatidylcholine (DMPC) monolayers has been studied by employing the Langmuir-Blodgett technique. The aim of the work was to gain information on the interaction of these compounds with phospholipid membranes. DMPC/acyclovir or squalenoyl-acyclovir prodrug mixed monolayers have been prepared at increasing molar fractions of the compound and the isotherm mean molecular area/surface pressure has been registered at 10 and 37 degrees C. Results reveal that the squalenoyl moiety enhances the affinity of acyclovir for the biomembrane model.

Antifungal activity of bis-azasqualenes, inhibitors of oxidosqualene cyclase.

The antifungal activity and in vitro toxicity toward animal cells of two inhibitors of oxidosqualene cyclase, squalene bis-diethylamine (SBD) and squalene bis-diethylmethylammonium iodide (SBDI) were studied. Minimum inhibitory concentration (MIC) against dermatophytes and other fungi involved in cutaneous and systemic infections (12 isolates from seven species) were determined by the broth microdilution method based on the reference documents M38-A and M27-A2 of Clinical and Laboratory Standards Institute (CLSI). Both compounds exerted fungistatic activities, although with different action. SBDI was the more active compound and displayed low MIC values (in the 3.12-12.5 µg ml(-1) range) against Microsporum canis, Trichophyton mentagrophytes and one isolate of Scopulariopsis brevicaulis, while SBD showed MIC values against these species in the 3.12-25 µg ml(-1) range. Toxicity was tested on Madin-Darby canine kidney (MDCK) epithelial cells and human microvascular endothelial cells (HMEC). SBDI proved the less toxic compound: it inhibited M. canis, T. mentagrophytes and S. brevicaulis at concentrations below those found toxic for MDCK cells. HMEC were the more sensitive cells.

Conjugation of squalene to acyclovir improves the affinity for biomembrane models.

Differential scanning calorimetry was used to study the interaction of acyclovir and its prodrug squalenoyl-acyclovir (obtained by conjugation of 1,1',2-tris-nor-squalene acid (squaleneCOOH) with acyclovir) with biomembrane models made of DMPC multilamellar vesicles with the aim to verify whether a stronger interaction of the prodrug with respect to the free drug can be obtained. Multilamellar vesicles were prepared in the presence of increasing molar fractions of acyclovir, squaleneCOOH or prodrug and the effect of the compounds on the thermotropic behavior of vesicles was researched, revealing no effect of acyclovir but a strong effect of squaleneCOOH and prodrug. To evaluate if acyclovir, squaleneCOOH and prodrug can be absorbed by the biomembrane model, an experiment was carried out in which the considered compounds were left in contact with the biomembrane model and their eventual uptake was evaluated analyzing the effect on the thermotropic behavior of the biomembrane model. A very small uptake was revealed for all the compounds. To check the potential use of liposomes as a delivery system for the prodrug, the biomembrane models were incubated with liposomes loaded with the compounds and the compounds transferring from the loaded liposomes to the unloaded biomembrane model was followed. The results suggest that liposomes could be used to deliver the squalenoyl-acyclovir to the biomembrane model.

Interaction of lipophilic gemcitabine prodrugs with biomembrane models studied by Langmuir-Blodgett technique.

The stability and bioavailability of anticancer agents, such as gemcitabine, can be increased by forming prodrugs. Gemcitabine is rapidly deaminated to the inactive metabolite (2('),2(')-difluorodeoxyuridine), thus to improve its stability a series of increasingly lipophilic gemcitabine prodrugs linked through the 4-amino group to valeroyl, lauroyl, and stearoyl acyl chains were synthesized. Studies of monolayer properties are important to improve understanding of biological phenomena involving lipid/gemcitabine or lipid/gemcitabine derivative interactions. The interfacial behavior of monolayers constituted by DMPC plus gemcitabine or lipophilic gemcitabine prodrugs at increasing molar fractions was studied at the air/water interface at temperatures below (10 degrees C) and above (37 degrees C) the lipid phase transition. The effect of the hydrophobic chain length of gemcitabine derivatives on the isotherm of pure DMPC was investigated by surface tension measurement, and the results are reported as molar fractions as a function of mean molecular area per molecule. The results show that the compounds interact with DMPC producing mixed monolayers that are subject to an expansion effect, depending on the prodrug chain length. The results give useful hints of the interaction of these prodrugs with biological membranes and increase knowledge on the incorporation site of such compounds, as a function of their lipophilicity, in a lipid carrier; they may lead to improved liposomal formulation design.

Characterization of lipophilic gemcitabine prodrug-liposomal membrane interaction by differential scanning calorimetry.

Gemcitabine is an anticancer agent rapidly deaminated to the inactive metabolite 2',2'-difluorodeoxyuridine. Its stability as well as bioavailability can be increased by making prodrugs. A series of lipophilic prodrugs of gemcitabine were synthesized by linking the 4-amino group with valeroyl, lauroyl, and stearoyl linear acyl derivatives. We studied, by the differential scanning calorimetry technique, and compared the interaction of pure gemcitabine and its prodrugs with dimyristoylphosphatidylcholine and distearoylphosphatidylcholine vesicles with the aim of demonstrating if the gemcitabine prodrug is more able than the pure gemcitabine to interact with lipid vesicles employed both as model biomembranes and as carriers in the transport of antitumor drugs. These studies, carried out by static and kinetic calorimetric measurements, give evidence that the increase of the prodrug's lipophilic character improves the interaction with lipid bilayers, favoring the absorption through the lipid barriers and allowing the liposomes to work (when the prodrug is inserted inside the vesicles) as a lipophilic carrier which is able to deliver the drug near the cell surface. The use of different prodrugs modified in their lipophilic character, of different kinds of vesicles (multilamellar and unilamellar), and of different kinds of vesicles forming phospholipids permitted us to determine the better equilibrium between in-vesicle solubility and through-vesicle diffusion of the drug, important in the preformulative studies of antitumor carriers based on phospholipid formulations. Such studies suggest that the prodrug lipophilic tail should modulate the transport and the release of gemcitabine inside the cellular compartments, and the efficiency of the liposomal system is related to the length of the prodrug's acyl chain which has to match the phospholipid acyl chain allowing or retarding the migration through the lipid release device.

Synthesis and biological activity of new iodoacetamide derivatives on mutants of squalene-hopene cyclase.

New iodoacetamide derivatives, containing a dodecyl or a squalenyl moiety, were synthesized. The effect of these new thiol-reacting molecules was studied on two mutants of Alicyclobacillus acidocaldarius squalene-hopene cyclase constructed especially for this purpose. In the quintuple mutant, all five cysteine residues of the enzyme are substituted with serine; in the sextuple mutant, this quintuple substitution is accompanied by the substitution of aspartate D376, located at the enzyme's active site, with a cysteine. N-Dodecyliodoacetamide had little activity toward either mutant, whereas N-squalenyliodoacetamide showed a stronger effect on the sextuple than on the quintuple mutant, as expected.

Structure and reactivity of the dammarenyl cation: configurational transmission in triterpene synthesis.

[reaction: see text] The dammarenyl cation (13) is the last common intermediate in the cyclization of oxidosqualene to a diverse array of secondary triterpene metabolites in plants. We studied the structure and reactivity of 13 to understand the factors governing the regio- and stereospecificity of triterpene synthesis. First, we demonstrated that 13 has a 17beta side chain in Arabidopsis thaliana lupeol synthase (LUP1) by incubating the substrate analogue (18E)-22,23-dihydro-20-oxaoxidosqualene (21) with LUP1 from a recombinant yeast strain devoid of other cyclases and showing that the sole product of 21 was 3beta-hydroxy-22,23,24,25,26,27-hexanor-17beta-dammaran-20-one. Quantum mechanical calculations were carried out on gas-phase models to show that the 20-oxa substitution has negligible effect on substrate binding and on the activation energies of reactions leading to either C17 epimer of 13. Further molecular modeling indicated that, because of limited rotational freedom in the cyclase active site cavity, the C17 configuration of the tetracyclic intermediate 13 can be deduced from the angular methyl configuration of the pentacyclic or 6-6-6-6 tetracyclic product. This rule of configurational transmission aided in elucidating the mechanistic pathway accessed by individual cyclases. Grouping of cyclases according to mechanistic and taxonomic criteria suggested that the transition between pathways involving 17alpha and 17beta intermediates occurred rarely in evolutionary history. Two other mechanistic changes were also rare, whereas variations on cation rearrangements evolved readily. This perspective furnished insights into the phylogenetic relationships of triterpene synthases.

1,5-Benzodiazepines XIV. Synthesis of new substituted 9H-bis-[1,2,4]triazolo[4,3-a:3',4'-d] [1,5]benzodiazepines and relate compounds endowed with in vitro cytotoxic properties.

A series of 11 new 9H-bis-[1,2,4]triazolo[4,3-a:3',4'-d] [1,5]benzodiazepine derivatives 8e-o was synthesized. Ten of these compounds (8e-m,o), along with four analogues (8a-d) (previously synthesized by us) were tested in vitro in order to evaluate their cytotoxic and anti-HIV-1 properties. In this connection other six original compounds, i.e., five 9-substituted compounds prepared starting from the 6,12-diphenylderivative 8c (compounds 10, 11, 12, 13a,b) and the bis-triazolone derivative 14, were synthesized and tested for the same purpose. While none of the 20 compounds tested exhibited any appreciable anti-HIV-1 activity, some of them exhibited interesting cytotoxic properties, the best results being shown by compounds 8c,d,k and 11 (CC(50) range=3-12 microM). Therefore, these four compounds were further evaluated for their antiproliferative activity against a panel of human tumor cell lines; actually, compounds 8d, 8k and 11 showed antiproliferative properties against either or both leukemia- and lymphoma-derived cell lines in the low micromolar range.

Analogs of squalene and oxidosqualene inhibit oxidosqualene cyclase of Trypanosoma cruzi expressed in Saccharomyces cerevisiae.

Recently, a number of inhibitors of the enzyme oxidosqualene cyclase (OSC; EC 5.4.99.7), a key enzyme in sterol biosynthesis, were shown to inhibit in mammalian cells the multiplication of Trypanosoma cruzi, the parasite agent of Chagas' disease. The gene coding for the OSC of T. cruzi has been cloned and expressed in Saccharomyces cerevisiae. The expression in yeast cells could be a safe and easy model for studying the activity and the selectivity of the potential inhibitors of T. cruzi OSC. Using a homogenate of S. cerevisiae cells expressing T. cruzi OSC, we have tested 19 inhibitors: aza, methylidene, vinyl sulfide, and conjugated vinyl sulfide derivatives of oxidosqualene and squalene, selected as representative of different classes of substrate analog inhibitors of OSC. The IC50 values of inhibition (the compound concentration at which the enzyme is inhibited by 50%) are compared with the values obtained using OSC of pig liver and S. cerevisiae. Many inhibitors of pig liver and S. cerevisiae OSC show comparable IC50 for T. cruzi OSC, but some phenylthiovinyl derivatives are 10-100 times more effective on the T. cruzi enzyme than on the pig or S. cerevisiae enzymes. The expression of proteins of pathogenic organisms in yeast seems very promising for preliminary screening of compounds that have potential therapeutic activity.

Synthesis, characterization and transfection activity of new saturated and unsaturated cationic lipids.

We synthesized new cationic lipids, analogue to N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), in order to compare those containing a dodecyl chain with those having a relatively long chain with two or five double bonds, such as squalenyl and dihydrofarnesyl derivatives, or complex saturated structures, such as squalane derivatives. The fusogenic helper lipid dioleoylphosphatidylethanolamine (DOPE) was added to cationic lipids to form a stable complex. Liposomes composed of 50:50 w/w cationic lipid/DOPE were prepared and incubated with plasmidic DNA at various charge ratios and the diameter and zeta potential of the complexes were measured. The surface charge of the DNA/lipid complexes can be controlled by adjusting the cationic lipid/DNA ratio. Finally, we tested the in vitro transfection efficiency of the cationic lipid/DNA complexes using different cell lines. The transfection efficiency was highest for the dodecyloxy derivative containing a single hydroxyethyl group in the head, followed by the dodecyloxy and the farnesyloxy trimethylammonium derivatives. Instead the C27 squalenyl and C27 squalanyl derivatives resulted inactive.

Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing lipophilic gemcitabine prodrugs.

Gemcitabine is a known anticancer agent rapidly deaminated to the inactive metabolite 2',2'-difluorodeoxyuridine; it must therefore be administered at very high dose. Many different approaches have been tried to improve the metabolic stability; we synthesized a series of increasingly lipophilic prodrugs of gemcitabine by linking the 4-amino group with valeroyl, heptanoyl, lauroyl and stearoyl linear acyl derivatives. We studied their stability at storage, in plasma and with the lysosomal intracellular enzyme cathepsins. We studied incorporation of these lipophilic prodrugs in liposomes, where their encapsulation efficiency (EE) closely depends on the length of the saturated 4-(N)-acyl chain, the phospholipids chosen and the presence of cholesterol. A maximum EE of 98% was determined for 4-(N)-stearoyl-gemcitabine incorporated in DSPC/DSPG 9:1. This formulation was correlated with the highest stability in vitro and in vivo. Cytotoxicity of gemcitabine prodrugs, free or encapsulated in liposomes, was between two- and sevenfold that of free gemcitabine. Encapsulation of long-chain lipophilic prodrugs of gemcitabine in liposomes protected the drug from degradation in plasma, assuring a long plasma half-time and intracellular release of the free drug.

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 Kaposi's 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 Kaposi's 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.

Conjugated methyl sulfide and phenyl sulfide derivatives of oxidosqualene as inhibitors of oxidosqualene and squalene-hopene cyclases.

Various (1E,3E)- and (1Z,3E)-conjugated methylthio derivatives of oxidosqualene (OS) and conjugated and non-conjugated phenylthio derivatives of OS were obtained. These compounds, designed as inhibitors of pig liver and Saccharomyces cerevisiae 2,3-oxidosqualene-lanosterol cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene-hopene cyclase (SHC) (EC 5.4.99.-), contain the reactive function adjacent to carbons involved in the formation of the third and the fourth cycle during OS cyclization. All the new compounds are inhibitors of OSC and SHC, with various degrees of selectivity. The conjugated methylthio derivatives behaved as potent inhibitors of S. cerevisiae OSC, whereas most of the phenylthio derivatives were especially active toward SHC.

Novel poly(ethylene glycol) derivatives for preparation of ribosome-inactivating protein conjugates.

This study describes the synthesis, characterization, and reactivity of new methoxypoly(ethylene glycol) (mPEG) derivatives containing a thioimidoester reactive group. These activated polymers are able to react with the lysyl epsilon-amino groups of suitable proteins, generating an amidinated linkage and thereby preserving the protein's positive charge. mPEG derivatives of molecular weight 2000 and 5000 Da were used, and two spacer arms were prepared, introducing chains of different lengths between the hydroxyl group of the polymer and the thioimidate group. These mPEG derivatives were used to modify gelonin, a cytotoxic single-chain glycoprotein widely used in preparation of antitumoral conjugates, whose biological activity is strongly influenced by charge modification. The reactivity of mPEG thioimidates toward lysil epsilon-amino groups of gelonin was evaluated, and the results showed an increased degree of derivatization in proportion to the molar excesses of the polymer used and to the length of the alkyl spacer. Further studies showed that the thioimidate reactive is able to maintain gelonin's significant biological activity and immunogenicity. On the contrary, modification of the protein with N-hydroxysuccinimide derivative of mPEG strongly reduces the protein's cytotoxic activity. Evaluation of the pharmacokinetic behavior of native and PEG-grafted gelonin showed a marked increase in plasma half-life after protein PEGylation; in particular, the circulating life of the conjugates increased with increased molecular weight of the polymer used. The biodistribution test showed lower organ uptake after PEGylation, in particular by the liver and spleen.