Low toxicity deep eutectic solvent-based ferrofluid for the determination of UV filters in environmental waters by stir bar dispersive liquid microextraction.

In this work, a low toxicity deep eutectic solvent-based ferrofluid is presented for the first time as magnetic fluid to be used as an efficient solvent in liquid-based microextraction techniques. This ferrofluid is made of a hydrophobic deep eutectic solvent, composed by menthol and thymol in a 1:5 molar ratio as carrier solvent, and oleic acid-coated cobalt ferrite (CoFe2O4@oleic acid) magnetic nanoparticles. This material was characterized via magnetism measurement, scanning electron microscopy, infrared spectroscopy and density measurement. The determination of UV filters in environmental water samples was selected as model analytical application to test the extraction performance of this new ferrofluid by employing stir bar dispersive liquid microextraction, prior to liquid chromatography-tandem mass spectrometry analysis. The response surface methodology was used as a multivariate optimization method for extraction step. Under the optimized conditions, good analytical features were obtained, such as low limits of detection between 7 and 83 ng L-1, good repeatability (relative standard deviations, RSD (%) below 15%), enrichment factors between 46 and 101 and relative recoveries between 80 and 117%, proving the good extraction capability of this ferrofluid. Finally, the method was successfully applied to three environmental waters (beach and river waters), finding trace amounts of the target UV filters. The presented low toxicity deep eutectic solvent-based ferrofluid results to be a good alternative to conventional solvents used in liquid-phase microextraction techniques.

Complete inhibition of extranodal dissemination of lymphoma by edelfosine-loaded lipid nanoparticles.

BACKGROUND: Lipid nanoparticles (LNs) made of synthetic lipids Compritol(®) 888 ATO and Precirol(®) ATO 5 were developed with an average size of 110.4 ± 2.1 and 103.1 ± 2.9 nm, and an encapsulation efficiency above 85% for both type of lipids. These LNs decrease the hemolytic toxicity of the drug by 90%. MATERIALS & METHODS: Pharmacokinetic and biodistribution profiles of the drug were studied after intravenous and oral administration of edelfosine-containing LNs. RESULTS: This provided an increase in relative oral bioavailability of 1500% after a single oral administration of drug-loaded LNs, maintaining edelfosine plasma levels over 7 days in contrast to a single oral administration of edelfosine solution, which presented a relative oral bioavailability of 10%. Moreover, edelfosine-loaded LNs showed a high accumulation of the drug in lymph nodes and resulted in slower tumor growth than the free drug in a murine lymphoma xenograft model, as well as potent extranodal dissemination inhibition.

In vitro and In vivo selective antitumor activity of Edelfosine against mantle cell lymphoma and chronic lymphocytic leukemia involving lipid rafts.

PURPOSE: Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) remain B-cell malignancies with limited therapeutic options. The present study investigates the in vitro and in vivo effect of the phospholipid ether edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) in MCL and CLL. EXPERIMENTAL DESIGN: Several cell lines, patient-derived tumor cells, and xenografts in severe combined immunodeficient mice were used to examine the anti-MCL and anti-CLL activity of edelfosine. Furthermore, we analyzed the mechanism of action and drug biodistribution of edelfosine in MCL and CLL tumor-bearing severe combined immunodeficient mice. RESULTS: Here, we have found that the phospholipid ether edelfosine was the most potent alkyl-lysophospholipid analogue in killing MCL and CLL cells, including patient-derived primary cells, while sparing normal resting lymphocytes. Alkyl-lysophospholipid analogues ranked edelfosine > perifosine >> erucylphosphocholine > or = miltefosine in their capacity to elicit apoptosis in MCL and CLL cells. Edelfosine induced coclustering of Fas/CD95 death receptor and rafts in MCL and CLL cells. Edelfosine was taken up by malignant cells, whereas normal resting lymphocytes hardly incorporated the drug. Raft disruption by cholesterol depletion inhibited drug uptake, Fas/CD95 clustering, and edelfosine-induced apoptosis. Edelfosine oral administration showed a potent in vivo anticancer activity in MCL and CLL xenograft mouse models, and the drug accumulated dramatically and preferentially in the tumor. CONCLUSIONS: Our data indicate that edelfosine accumulates and kills MCL and CLL cells in a rather selective way, and set coclustering of Fas/CD95 and lipid rafts as a new framework in MCL and CLL therapy. Our data support a selective antitumor action of edelfosine.

Influence of the chitosan nature on the transfection efficacy of DNA-loaded nanoparticles after hydrodynamic administration in mice.

A commercially available chitosan with a degree of deacetylation (DD) of 85% and a molecular weight (Mw) of 400 kDa was modified by acetylation with acetic anhydride to obtain a chitosan with a DD of 75%. Both polysaccharides were used to prepare DNA-chitosan nanoparticles by charge interactions with pDNA (coacervation process). Both resulting nanoparticles showed an almost total DNA loading efficiency (96%) and displayed similar physico-chemical properties with a size of approximately 200 nm and a zeta potential close to +20 mV. In order to study the effect of the DD on the properties of DNA-chitosan nanoparticles as gene delivery systems, the hydrodynamics-based procedure was used. The transgene expression was observed using either the green fluorescent protein (GFP) or the luciferase (Luc) as reporter genes. After the hydrodynamic injection, the DNA-chitosan nanoparticles were accumulated in the liver, where the transgene expression was mostly localized. Interestingly, the decrease of the DD affected the transgene expression, improving the initial burst effect and accelerating the DNA release. Both combined effects led to an increase in the transgene expression levels. In addition, the emitted bioluminescence could be detected over 105 days for all the formulations injected. The calculation of the kinetic parameters (C(max), AUC, Ke, t(1/2) Ke and MET) gave some interesting information regarding the abilities to control the DNA release of the two DNA-chitosan formulations tested and allowed narrower comparisons.

Antitumor alkyl ether lipid edelfosine: tissue distribution and pharmacokinetic behavior in healthy and tumor-bearing immunosuppressed mice.

PURPOSE: The present study investigates and compares the dose-dependent pharmacokinetics and oral bioavailability of edelfosine in healthy, immunodeficient, and tumor-bearing immunosuppressed mouse animal models, as well as edelfosine uptake and apoptotic activity in the Z-138 mantle cell lymphoma (MCL) cell line. EXPERIMENTAL DESIGN: Biodistribution study of edelfosine was done in both BALB/c and severe combined immune deficiency (SCID) mice, and then the in vivo behavior of the drug after i.v. and oral administration was monitored. RESULTS: We found that edelfosine is incorporated and induces apoptosis in the Z-138 human mantle cell lymphoma cell line, whereas normal resting peripheral blood human lymphocytes were not affected. In vivo biodistribution studies revealed that accumulation of edelfosine in the tumor of a MCL-bearing mouse animal model was considerably higher (P < 0.01) than in the other organs analyzed. Besides, no statistical differences were observed between the pharmacokinetic parameters of BALB/c and SCID mice. Edelfosine presented slow elimination and high distribution to tissues. Bioavailability for a single oral dose of edelfosine was <10%, but a multiple-dose oral administration increased this value up to 64%. CONCLUSION: Our results show that edelfosine is widely scattered across different organs, but it is preferentially internalized by the tumor both in vitro and in vivo. Our data, together with the apoptotic action of the drug on cancer cells, support a rather selective action of edelfosine in cancer treatment, and that multiple oral administration is required to increase oral bioavailability.

Lipid nanomedicines for anticancer drug therapy.

More than half of all people diagnosed with cancer receive chemotherapy. Unfortunately, most chemotherapy drugs cannot tell the difference between a cancer cell and a healthy cell. In this sense, some other drawbacks often encountered with antineoplastic compounds, such as poor stability and specificity and a high occurrence of drug-resistant tumor cells may be overcome to some degree by incorporating them into drug delivery systems. Solid Lipid Nanoparticles (SLN) have arisen considerable interest in recent years. These are particles of submicron size made from a lipid matrix that is solid at room and body temperature. Moreover, the biodegradable and biocompatible nature of SLN makes them less toxic than other nanoparticulate systems. SLN are capable of encapsulating hydrophobic and hydrophilic drugs, and they also provide protection against chemical, photochemical or oxidative degradation of drugs, as well as the possibility of a sustained release of the incorporated drugs. Along with these last issues, the feasibility of scaling up for large scale production and the low cost of lipids as compared to biodegradable polymers or phospholipids have favoured their use as potential drug delivery systems. This review focuses on the recent advances in SLN as carriers for chemotherapeutic agent delivery.

New methodologies to characterize the effectiveness of the gene transfer mediated by DNA-chitosan nanoparticles.

In this work three DNA-chitosan nanoparticle formulations (Np), differing in the molecular weight (MW; 150 kDa, 400 kDa, and 600 kDa) of the polysaccharide, were prepared and administered by two different administration routes: the hydrodynamics-based procedure and the intraduodenal injection. After the hydrodynamic injection, DNA-chitosan nanoparticles were predominantly accumulated in the liver, where the transgene was expressed during at least 105 days. No significant influence of MW was observed on the levels of luciferase expression. The curves of bioluminescence versus time obtained using the charge-coupled device (CCD) camera were described and divided in three phases: (i) the initial phase, (ii) the sustained release step and (iii) the decline phase (promotor inactivation, immunological and physiological processes). From these curves, which describe the transgene expression profile, the behavior of the different formulations as gene delivery systems was characterized. Therefore, the following parameters such as C(max) (maximum level of detected bioluminescence), AUC (area under the bioluminescence-time curve) and MET (mean time of the transgene expression) were calculated. This approach offers the possibility of studying and comparing transgene expression kinetics among a wide variety of gene delivery systems. Finally, the intraduodenal administration of naked DNA permitted the gene transfer in a dose dependent manner quantifiable with the CCD camera within 3 days. Nevertheless, the same administration procedure of the three formulations did not improve the levels of transgene expression obtained with naked DNA. This fact could be explained by the rapid physiological turn-over of enterocytes and by the ability of chitosan nanoparticles to control the DNA release.

Randomized crossover pharmacokinetic evaluation of subcutaneous versus intravenous granisetron in cancer patients treated with platinum-based chemotherapy.

BACKGROUND: 5-HT3-receptor antagonists are one of the mainstays of antiemetic treatment, and they are administered either i.v. or orally. Nevertheless, sometimes neither administration route is feasible, such as in patients unable to admit oral intake managed in an outpatient setting. Our objective was to evaluate the bioavailability of s.c. granisetron. PATIENTS AND METHODS: Patients receiving platinum-based chemotherapy were randomized to receive 3 mg of granisetron either s.c. or i.v. in a crossover manner during two cycles. Blood and urine samples were collected after each cycle. Pharmacokinetic parameters observed with each administration route were compared by analysis of variance. RESULTS: From May to November 2005, 31 patients were included and 25 were evaluable. Subcutaneous granisetron resulted in a 27% higher area under the concentration-time curve for 0-12 hours (AUC(0-12h)) and higher levels at 12 hours, with similar values for AUC(0-24h). The maximum concentration was lower with the s.c. than with the i.v. route and was observed 30 minutes following s.c. administration. CONCLUSION: Granisetron administered s.c. achieves complete bioavailability. This is the first study that shows that s.c. granisetron might be a valid alternative to i.v. delivery. Further trials to confirm clinical equivalence are warranted. This new route of administration might be especially relevant for outpatient management of emesis in cancer patients.

Bioadhesive mannosylated nanoparticles for oral drug delivery.

The aim of this work was to design mannosylated Gantrez AN nanoparticles (M-NP) and to describe their gut bioadhesive properties in order to develop a promising carrier for future applications in oral drug delivery. For that purpose, the process of the nanoparticles coating with mannosamine was optimized by the incubation of Gantrez AN nanoparticles with different volumes of mannosamine aqueous solutions at different times. Then, the nanoparticles were characterized by measuring the size, zeta potential, mannosamine content, and concanavalin A (Con A) binding. Furthermore, in vivo quantitative bioadhesion study and kinetic analysis of the bioadhesion curves were performed after oral administration to rats of fluorescently labelled nanoparticles. The selected mannosylated nanoparticles (M-NP1 and M-NP10) were of homogenous sizes (about 300 and 200 nm), negatively charged and successfully coated with 36 and 18 microg mannosamine/mg NP, respectively. In vitro agglutination assay using Con A confirmed the successful coating of nanoparticles with mannosamine. The gut distribution profile of M-NP1 indicated a stronger bioadhesive capacity than M-NP10 and non-mannosylated ones, 1 h post-administration. Interestingly, M-NP1 showed an important ileum tropism where around 20% of the given dose remained adhered. Besides, the kinetic parameters of the bioadhesion profile of M-NP1 indicated their higher bioadhesive capacity with Q(max) and AUC(adh) about 2-times higher than control ones. Moreover, fluorescence microscopy corroborated the stronger interactions of M-NP1 with the normal mucosa and demonstrated a strong uptake of these carriers by Peyer's patches. In conclusion, we propose that mannosylated nanoparticles could be a promising non-live vector for oral delivery strategies.

A comparative study of the pharmacokinetics of ibuprofen arginate versus dexibuprofen in healthy volunteers.

OBJECTIVE: Ibuprofen arginate is a salt formulation of ibuprofen designed to reach target concentrations rapidly. The primary objective of this study was to compare the 12-h pharmacokinetic profile of S(+)-ibuprofen following administration of single doses of ibuprofen arginate (600 mg) and dexibuprofen (400 mg) in healthy volunteers. METHODS: Twenty-four volunteers were recruited into an open-label, randomised, two-period, single-centre study with crossover design. RESULTS: Both treatments were well tolerated. Ibuprofen arginate and dexibuprofen showed similar bioavailability for S(+)-ibuprofen. Compared with dexibuprofen, ibuprofen arginate demonstrated a 45% higher maximum concentration (C(max)), and a time to peak concentration (T(max)) 2 h sooner. CONCLUSION: Ibuprofen arginate approaches maximum concentrations of S(+)-ibuprofen faster and higher than dexibuprofen.

Determination of gentamicin in different matrices by a new sensitive high-performance liquid chromatography-mass spectrometric method.

OBJECTIVES: The aim of this work was to develop and validate an HPLC method for gentamicin quantification in different types of biological samples such as animal tissues and cellular material and also in pharmaceuticals. METHODS: Poly(lactide-co-glycolide) microparticles (MP) of gentamicin (PLGA 502H MP), THP-1 cells, and plasma and tissue samples of mice treated with the antibiotic either free or loaded into PLGA 502H MP were processed by a simple preparation procedure, subjected to chromatography on a reversed-phase column and measured by mass spectrometry detection. The developed method was compared with bioassay and fluorimetric assay methods previously used for gentamicin determination. RESULTS: The HPLC method was linear over the ranges 40-800 ng/mL and 0.1-100 microg/mL and showed good accuracy (average accuracy < 5.59%) and reproducibility (CV < 6.13%). Encapsulation of gentamicin in PLGA 502H MP was determined by the three methods. Good correlation was observed between bioassay (reference method) and HPLC. Extra- and intracellular in vitro antibiotic accumulation was determined by bioassay and chromatography. Both methods gave similar extracellular concentrations but the HPLC-MS technique demonstrated an improved accuracy (5.59% versus 14%) and precision (6.13% versus 15%) compared with bioassay. However, only the HPLC-MS method was sensitive enough to detect the drug, intracellularly and in tissues. CONCLUSIONS: All these data favour the use of chromatography-mass spectrometry as a versatile technique not only suitable for gentamicin quantification loaded in drug delivery systems, but also sensitive and specific enough for in vivo and intracellular studies.

Effects of a 250-mL enema containing sodium phosphate on electrolyte concentrations in healthy volunteers: An open-label, randomized, controlled, two-period, crossover clinical trial.

BACKGROUND: Enemas are used by individuals with constipation and are often required before certain medical diagnostic procedures and surgical interventions. However, abnormalities in serum electrolyte concentrations have been associated with enema use. OBJECTIVE: The aim of this study was to determine the changes in serum electrolyte concentrations (phosphorus, calcium, sodium, and potassium) and urinary phosphorus elimination after the administration of a sodium phosphate enema. METHODS: Healthy volunteers aged 35 to 70 years were eligible for this open-label, randomized, controlled, 2-period, crossover clinical trial at the Clinical Research Unit of the University Hospital of Navarra, Pamplona, Spain. The study comprised 2 one-day periods separated by a 7-day washout. All subjects were randomly assigned in a 1:1 ratio to 1 of 2 study sequences: (1) a single dose of Enema Casen® 250 mL in the first period followed by no treatment (control) in the second period, or (2) no treatment in the first period followed by a single dose of the study drug in the second period. The sequence of treatment was assigned using a randomization table that was prepared before the beginning of the study. Serum concentrations of phosphorus, sodium, potassium, and calcium were measured in both periods. Urinary phosphorus elimination was measured for 12 hours after enema administration (Ae0-12) in a subset of the subjects in the second period. Adverse events (AEs) were monitored by the investigators throughout the study. Normal ranges for the electrolytes were as follows: phosphorus, 2.5 to 5 mg/dL; calcium, 8.5 to 10.5 mg/dL; sodium, 135 to 145 mEq/L; and potassium, 3.5 to 5 mEq/L. RESULTS: Twenty-four subjects (12 men, 12 women; mean [SD] age, 47.8 [9.6] years [range, 36-68 years]) participated in the study. All of the subjects were white and none were smokers. Twelve hours after enema administration, mean serum phosphorus and sodium concentrations increased by a mean of 1.18 mg/dL and 1.32 mEq/L, respectively (both, P < 0.001). Mean serum phosphorus concentrations were above the upper limit of normal (5 mg/dL) at 30 and 60 minutes after enema administration. In all subjects the values returned to normal within 4 hours after enema administration; a meal was provided after a 3-hour fast. Four subjects (16.7%) had ≥1 serum phosphorus concentration measurement ≥7 mg/dL, a value that is considered serious hyperphosphatemia. A statistically significant correlation was found between phosphorus Cmax and enema retention time (r (2) = 0.452; P < 0.001). No abnormal serum concentrations were obtained for the other electrolytes measured. Phosphorus Ae0-12 was increased after enema administration by 86% (P < 0.001). No serious AEs were observed, although 13 AEs were reported in 9 subjects. None of the changes in serum electrolyte concentrations were associated with clinical symptoms. CONCLUSIONS: Administration of an enema containing 250 mL of sodium phosphate was associated with serum phosphorus concentrations of ≥7 mg/dL in 16.7% of the healthy subjects who participated in the study; however, none of those subjects experienced hypocalcemia. Enema retention time was significantly correlated with the degree of phosphatemia.

Salmonella-like bioadhesive nanoparticles.

The aim of this work was to evaluate the bioadhesive potential of a polymeric vector obtained by the association between Gantrez AN nanoparticles and flagella-enriched Salmonella enteritidis extract. Fluorescently labelled nanoparticles (SE-NP) were prepared, after incubation between the polymer and the extract, by a solvent displacement method and cross-linkage with 1,3-diaminopropane. SE-NP displayed a size close to 280 nm and the amount of associated bacterial extract was 18 mug/mg nanoparticle. Flagellin represents more than 80% of the total proteins associated with SE-NP, which was identified by SDS-PAGE and confirmed by Western blotting. Concerning the bioadhesive properties, SE-NP shows an important tropism for the ileum. In fact, about 50% of the given dose of SE-NP was found in this gut region for at least 3 h. Interestingly, the bioadhesive ability of SE-NP correlated well with the described colonisation profile for Salmonella enteritidis. This fact was corroborated by competitive tissue distribution studies. Thus, when SE-NP and Salmonella cells were administered together by the oral route, both the bacteria and the nanoparticles displayed a similar distribution within the intestinal mucosa. However, the ability of SE-NP to be taken up by Peyer's patches appeared to be negatively affected by the presence of the bacteria. Similarly, when SE-NP was administered 30 min before cells, SE-NP were found broadly distributed in Peyer's patches, whereas the bacteria were neither able to adhere to nor penetrate this lymphoid tissue. In summary, SE-NP demonstrated their Salmonella-like gut colonization, which can be a useful vector for oral targeting strategies.

Quantitative determination of the antitumor alkyl ether phospholipid edelfosine by reversed-phase liquid chromatography-electrospray mass spectrometry: application to cell uptake studies and characterization of drug delivery systems.

Edelfosine is a synthetic alkyl ether phospholipid that represents a promising class of antitumor agents. However, analytical methods to measure these type compounds are scarce. The lack of a reliable methodology to quantify edelfosine is a major problem in ongoing and scheduled preclinical and clinical trials with this drug. We evaluated the applicability of high-performance liquid chromatography-mass spectrometry to determine edelfosine in biological samples and polymeric delivery systems. Sample pre-treatment involved polymer precipitation or cell lysis with methanol. HPLC separation was performed on an Alltima RPC(18) narrow-bore column and edelfosine quantification was done by electrospray ionization mass spectrometry (ESI-MS) using positive ion mode and selected ion monitoring. Assays were linear in the tested range of 0.3-10 microg/ml. The limit of quantification was 0.3 ng/sample in both matrices, namely biological samples and polymeric delivery systems. The interassay precision ranging from 0.79 to 1.49%, with relative errors of -6.7 and 12.8%. Mean extraction recovery was 95.6%. HPLC-ESI-MS is a reliable system for edelfosine analysis and quantification in samples from different sources, combining advantages of full automation (rapidity, ease of use, no need of extensive extraction procedures) with high analytical performance and throughput.

Importance of single or blended polymer types for controlled in vitro release and plasma levels of a somatostatin analogue entrapped in PLA/PLGA microspheres.

The aim of the work was to develop biodegradable microspheres for controlled delivery of the somatostatin analogue vapreotide and maintenance of sustained plasma levels over 2-4 weeks after a single injection in rats. Vapreotide was microencapsulated into end-group capped and uncapped low molecular weight poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) by spray-drying and coacervation. Microspheres were prepared from single and blended (1:1) polymer types. The microparticles were characterized for peptide loading, in vitro release and pharmocokinetics in rats. Spray-drying and coacervation produced microspheres in the size range of 1-15 and 10-70 microm, respectively, and with encapsulation efficiencies varying between 46% and 87%. In vitro release of vapreotide followed a regular pattern and lasted more than 4 weeks, time at which 40-80% of the total dose were released. Microspheres made of 14-kDa end-group uncapped PLGA50:50 or 1:1 blends of this polymer with 35 kDa end-group uncapped PLGA50:50 gave the best release profiles and yielded the most sustained plasma levels above a pre-defined 1 ng/ml over approximately 14 days. In vitro/in vivo correlation analyses showed for several microsphere formulations a linear correlation between the mean residence time in vivo and the mean dissolution time (r=0.958) and also between the amount released between 6 h and 14 days and the AUC(6h-14d) (r=0.932). For several other parameters or time periods, no in vitro/in vivo correlation was found. This study demonstrates that controlled release of the vapreotide is possible in vivo for a duration of a least 2 weeks when administered i.m. to rats. These results constitute a step forward towards a twice-a-month or once-a-month microsphere-formulation for the treatment of acromegaly and neuroendocrine tumors.

Intestinal absorption of penclomedine from lipid vehicles in the conscious rat: contribution of emulsification versus digestibility.

While the inclusion of highly lipophilic compounds in self-emulsifying drug delivery systems (SEDDS) is often reported to result in strongly enhanced oral absorption, it is still controversial whether further lipolysis of the dispersed lipidic material is required for final transfer to the enterocyte membranes. In order to assess the relative roles of lipid vehicle dispersion and vehicle digestibility in the oral absorption of penclomedine (Pcm), a series of formulations of Pcm in medium chain triglyceride (MCT)/tocophersolan (TPGS) was developed having three sizes (160 nm, 720 nm, and mm-sized ('crude' oil)); with or without the inclusion of tetrahydrolipstatin (THL), a known lipase-inhibitor. Oral absorption of Pcm was studied after administration of small volumes of these formulations in the conscious rat. Kinetic evaluation was performed using population analysis. Formulations with particle size 160 nm had the highest relative bioavailability (set at F=1), whereas administration in particle size 720 nm had slightly lower bioavailability (F=0.79). Co-inclusion of THL yielded similar bioavailability for these two SEDDS. 'Crude' oil formulations had F=0.62 (without THL) and 0.25 (with THL). The data in the current investigation emphasize the prominent role of increased vehicle dispersion relative to digestibility in the absorption of Pcm from MCT-TPGS in submicron emulsions. Only with Pcm administered as undispersed MCT, absorption was more dependent on the action of lipase as bioavailability was inhibited two-fold by the co-incorporation of THL.