Lipid Nanosystems Enhance the Bioavailability and the Therapeutic Efficacy of FTY720 in Acute Myeloid Leukemia.

Protein phosphatase 2A (PP2A) is a serin-threonin phosphatase that regulates many proteins critical for malignant cell behavior; therefore, PP2A is considered to be a human tumor suppressor. In this study, we assessed the pharmacokinetic profile and the antileukemic effects of the PP2A activator FTY720, free or encapsulated in lipid nanoparticles, in in vitro and in vivo models of acute myeloid leukemia. FTY720 lipid nanoparticles presented diameters around 210 nm, with an encapsulation efficiency up to 75% and significantly increased FTY720 oral bioavailability. In addition, FTY720 restores PP2A phosphatase activity and decreases phosphorylation of PP2A and its targets Akt, ERK1/2 and STAT5, all implicated in the pathogenesis of acute myeloid leukemia. Moreover, FTY720 exerts an additive anti-leukemic effect in combination with drugs used in standard induction therapy. Importantly, FTY720 lipid nanoparticles were more efficient at inducing cell growth arrest and apoptosis than FTY720 solution. Finally, oral administration of FTY720 lipid nanoparticles to mice every three days was as effective in reducing acute myeloid leukemia xenograft tumor growth as daily oral administration of FTY720. These results provide the first evidence for the potential use of FTY720 lipid nanoparticles as an oral therapeutic agent in acute myeloid leukemia.

Ultra high performance liquid chromatography-tandem mass spectrometry method for cyclosporine a quantification in biological samples and lipid nanosystems.

Cyclosporine A (CyA) is an immunosuppressant cyclic undecapeptide used for the prevention of organ transplant rejection and in the treatment of several autoimmune disorders. An ultra high performance liquid chromatography-tandem mass spectrometry method (UHPLC-MS/MS) to quantify CyA in lipid nanosystems and mouse biological matrices (whole blood, kidneys, lungs, spleen, liver, heart, brain, stomach and intestine) was developed and fully validated. Chromatographic separation was performed on an Acquity UPLC(®) BEH C18 column with a gradient elution consisting of methanol and 2mM ammonium acetate aqueous solution containing 0.1% formic acid at a flow rate of 0.6mL/min. Amiodarone was used as internal standard (IS). Retention times of IS and CyA were 0.69min and 1.09min, respectively. Mass spectrometer operated in electrospray ionization positive mode (ESI+) and multiple reaction monitoring (MRM) transitions were detected, m/z 1220.69→1203.7 for CyA and m/z 646→58 for IS. The extraction method from biological samples consisted of a simple protein precipitation with 10% trichloroacetic acid aqueous solution and acetonitrile and 5µL of supernatant were directly injected into the UHPLC-MS/MS system. Linearity was observed between 0.001µg/mL-2.5µg/mL (r≥0.99) in all matrices. The precision expressed in coefficient of variation (CV) was below 11.44% and accuracy in bias ranged from -12.78% to 7.99% including methanol and biological matrices. Recovery in all cases was above 70.54% and some matrix effect was observed. CyA was found to be stable in post-extraction whole blood and liver homogenate samples exposed for 6h at room temperature and 72h at 4°C. The present method was successfully applied for quality control of lipid nanocarriers as well as in vivo studies in BALB/c mice.

Comparison of pharmacokinetic profiles of PM02734 loaded lipid nanoparticles and cyclodextrins: in vitro and in vivo characterization.

PM02734 is a chemically synthesized depsipeptide derived from the marine kahalalides family with a broad spectrum of activity against solid tumors in vitro and in vivo, but presenting low bioavailability. In this work, solid lipid nanoparticles made of Precirol ATO 5 have been developed using a hot homogenization method followed by high shear homogenization and ultrasonication. These solid lipid nanoparticles show suitable size (around 150 nm) and encapsulation efficiency (nearly 70%) for the oral administration of the compound PM02734. A physical-chemical stability study was performed after 6 months of storage at different thermical conditions, concluding that solid lipid nanoparticles stored at 4 degrees C were more stable than solid lipid nanoparticles stored at 25 degrees C. The pharmacokinetic profile of drug-loaded solid lipid nanoparticles was also evaluated in Beagle dogs and compared with that of a cyclodextrin-based delivery system by means of AUC, C(max) and T(max) parameter estimation. Solid lipid nanoparticle based formulation provided a sustained release of the drug for a longer period of time than the cyclodextrins.

Development and validation of ultra high performance liquid chromatography-mass spectrometry method for LBH589 in mouse plasma and tissues.

An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed and validated for the quantitation of LBH589, a novel histone deacetylase inhibitor (HDACi), in mouse plasma and tissues (liver, spleen, kidney and lung). Tobramycin was employed as the internal standard. Separation was performed on an Acquity UPLC™ BEH column, with a mobile phase consisting of 10% water (with 0.1% of trifluoroacetic acid) and 90% methanol (with 0.1% trifluoroacetic acid). LBH589 and tobramycin were determined using an electrospray ionization (ESI) interface. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of LBH589 at m/z 349.42→157.95 and of tobramycin at 468.2→163. Calibration curves for the UHPLC method (0.0025-1 µg/mL for plasma and tissue homogenates, equivalent to 0.0357-14.2857 µg/g for tissue samples) showed a linear range of detector responses (r>0.998). Intra-batch and inter-batch precision expressed as coefficient of variation (CV) ranged from 0.92 to 8.40%. Accuracy expressed as bias, ranged from -2.41 to 2.62%. The lower limit of quantitation (LLOQ) was 0.0025 µg/mL for both plasma and tissue homogenate samples, equivalent to 0.0357 µg/g tissue. This method was successfully applied to quantify LBH589 in plasma and tissue samples obtained after the intraperitoneal administration of a single dose of 20 mg/kg of LBH589 in BALB/c mice.

Lipid raft-targeted therapy in multiple myeloma.

Despite recent advances in treatment, multiple myeloma (MM) remains an incurable malignancy. By using in vitro, ex vivo and in vivo approaches, we have identified here that lipid rafts constitute a new target in MM. We have found that the phospholipid ether edelfosine targets and accumulates in MM cell membrane rafts, inducing apoptosis through co-clustering of rafts and death receptors. Raft disruption by cholesterol depletion inhibited drug uptake by tumor cells as well as cell killing. Cholesterol replenishment restored MM cell ability to take up edelfosine and to undergo drug-induced apoptosis. Ceramide addition displaced cholesterol from rafts, and inhibited edelfosine-induced apoptosis. In an MM animal model, edelfosine oral administration showed a potent in vivo antimyeloma activity, and the drug accumulated preferentially and dramatically in the tumor. A decrease in tumor cell cholesterol, a major raft component, inhibited the in vivo antimyeloma action of edelfosine and reduced drug uptake by the tumor. The results reported here provide the proof-of-principle and rationale for further clinical evaluation of edelfosine and for this raft-targeted therapy to improve patient outcome in MM. Our data reveal cholesterol-containing lipid rafts as a novel and efficient therapeutic target in MM, opening a new avenue in cancer treatment.

Comparative study of A HPLC-MS assay versus an UHPLC-MS/MS for anti-tumoral alkyl lysophospholipid edelfosine determination in both biological samples and in lipid nanoparticulate systems.

The anti-tumor agent edelfosine represents a promising option in the treatment of cancer due to its capacity of promoting apoptosis in tumor cells selectively, while sparing healthy ones. In the present study, a novel ultra high performance liquid chromatography-tandem mass spectrometry method (UHPLC-MS/MS) was developed to quantify edelfosine concentrations in biological matrices (plasma, tissues or tumor) and in lipid nanoparticles, and compared with a conventional high performance liquid chromatography-mass spectrometry method (HPLC-MS). Compared with the HPLC method, the UHPLC method offered a threefold decrease in retention time, and a twofold decrease in asymmetry USP factor. Both methods were validated. Calibration curves for the HPLC method (0.1-1 and 1-75 microg/mL range in the plasma samples, 1-75 microg/mL range in lipid nanoparticle samples and 0.2-31.75 microg/mL range in tissue homogenate samples), and UHPLC method (0.0075-75 microg/mL for all kind of samples) showed a linear range of detector response (r>0.999). Intra-batch and inter-batch precision ranged from 1.66% to 7.77% for the HPLC method and from 3.72% to 12.23% for the UHPLC method. Accuracy of the HPLC and UHPLC assays, expressed as bias, ranged from -5.83% to 7.13% and from -6.84% to 6.49%, respectively. Matrix effects on edelfosine were similar in the HPLC and UHPLC methods. The assay methods developed were successfully applied to the quality control procedure of the manufacture of edelfosine lipid nanoparticles, and to evaluate the pharmacokinetic and in vivo tissue distribution in mice after oral administration of edelfosine-loaded lipid nanoparticles. A good correlation between both techniques was found (r=0.953) when tissue samples were analyzed with both methods.

Preferential accumulation of severe variants of Citrus tristeza virus in plants co-inoculated with mild and severe variants.

The viral population in sweet orange plants, either healthy or pre-inoculated with the asymptomatic isolate of Citrus tristeza virus (CTV) T32, and then graft- or aphid-inoculated with the stem-pitting isolate T318, was characterized with respect to symptom expression, reaction with monoclonal antibody MCA13, single-strand conformation polymorphism (SSCP) of genes p18 and p20, bi-directional RT-PCR, and dot-blot hybridisation. All plants inoculated with T318, with or without pre-inoculation, showed stem pitting, reacted with MCA13, had the SSCP profile characteristic of this isolate, and in bi-directional RT-PCR yielded a 450-bp DNA product associated with severe isolates, indicating that T32 afforded no protection against T318. The latter isolate had two main sequence variants, the minor one of which was indistinguishable from the main T32 sequence, and both were detected in most plants that were graft-inoculated with T318. However, the T32 variant was not detected in plants that were aphid-inoculated only with T318 and also showed stem pitting. This suggested an association of symptoms with the major T318 sequence and preferential transmission of this variant by aphids. The T318-specific variant accumulated more than the T32 variant in plants in which both were replicating, suggesting a higher fitness of the former. Our results clearly emphasize the potential threat of severe CTV variants in areas where mild isolates are presently predominant.

Intervention thresholds for Aphis spiraecola (Hemiptera: Aphididae) on Citrus clementina.

The production of clementine fruit was affected by varying densities of Aphis spiraecola Patch (Hemiptera: Aphididae) maintained on four groups of clementine, Citrus clementina Hort. ex Tan., trees individually isolated in mesh cages. A formula relating the number of aphids per square meter of canopy per group of trees to yield loss in that group of trees was obtained, permitting us to calculate the economic injury levels (EILs) and economic thresholds (ETs) for treatment against A. spiraecola, i.e., EIL, ET, environmental economic injury level (EEIL), and environmental economic threshold (EET). In an example case with current values, EIL = 370, ET = 322, EEIL = 614, and EET = 533 aphids per m2 of canopy. Formulae designed for easier use in the field were obtained to express each of these thresholds. The formulae are compared with those obtained for Aphis gossypii Glover in a previous study; the intervention thresholds for A. spiraecola are slightly higher than those found for A. gossypii.

Economic thresholds for Aphis gossypii (Hemiptera: Aphididae) on Citrus clementina.

Different levels of Aphis gossypii Glover were maintained on clementine trees by means of isolation inside mesh cages. Clementine yield loss was correlated with the number of aphids per square meter of canopy, obtaining the corresponding formula. Starting from this formula, the economic injury level (EIL) was calculated as follows: [218,333C + 11,130.6 VP0K]/[57.08 VP0K - 100C] = EIL (aphids/m2), where C = total insecticide cost per hectare, V = fruit price per kilogram, P0 = yield per hectare of a minimum pest level orchard, and K = reduction of injury due to treatment. Starting from the EIL, the economic threshold, the environmental EIL, and the environmental economic threshold were obtained. Formulae relating the number of aphids per square meter with other simpler indices expressing these thresholds (percentage of infested shoots, infested shoots per 0.25 m2 ring, index of infestation, percentage of occupied rings, and number of weekly captured aphids in a yellow trap) also were calculated.