Investigation of the micellar effect of pluronic P85 on P-glycoprotein inhibition: Cell accumulation and equilibrium dialysis studies

The objective of this study was: (1) to characterize the P-gp inhibitory effect of different concentrations of Pluronic P85 on anti-HIV-1 drug cellular accumulation, and (2) to investigate the relationship between cellular accumulation and free fraction of drug. Cellular accumulation studies in MDCKII-WT and MDCKII-MDR1 cell monolayers showed a biphasic dose response characterized by decline in accumulation at Pluronic concentrations greater than the CMC. This phenomenon was independent of the inhibition of P-gp efflux by Pluronic. Cell-free equilibrium dialysis was used to determine the effect of Pluronic P85 on drug free fraction and the affinity of Pluronic micelles for drug was modeled. Nelfinavir and saquinavir associated extensively with micelles and equilibrium free fractions were low at P85 concentrations above the CMC, with association constants being in the order nelfinavir > saquinavir ? abacavir. Abacavir, a P-gp substrate, showed no association with micelles yet showed a biphasic response in cellular accumulation. These data suggest that, above the CMC, inhibition of P-gp is not affected but rather factors such as micellar trapping could contribute to decreased accumulation. Therefore, the in vitro evaluation of the effect of Pluronic formulations on active transport should take into account both the physicochemical properties of drug and the composition of Pluronic. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4170–4190, 2009     

Spray-dried casein-based micelles as a vehicle for solubilization and controlled delivery of flutamide: Formulation,characterization, and in vivo pharmacokinetics

Novel casein (CAS)-based micelles loaded with the poorly soluble anti-cancer drug, flutamide (FLT), were successfully developed in a powdered form via spray-drying technique. Genipin (GNP) was used to crosslink CAS micelles as demonstrated by color variation of the micelles. Drug solubilization was enhanced by incorporation within the hydrophobic micellar core which was confirmed by solubility study and UV spectra. Spherical core–shell micelles were obtained with a particle size below 100 nm and zeta potential around −30 mV. At low drug loading, FLT was totally incorporated within micellar core as revealed by thermal analysis. However, at higher loading, excess non-incorporated drug at micelle surface caused a significant reduction in the surface charge density. Turbidity measurements demonstrated the high physical stability of micelles for 2 weeks dependent on GNP-crosslinking degree. In a dry powdered form, the micelles were stable for 6 months with no significant changes in drug content or particle size. A sustained drug release from CAS micelles up to 5 days was observed. After i.v. administration into rats, CAS micelles exhibited a prolonged plasma circulation of FLT compared to drug solution. Furthermore, a more prolonged drug systemic circulation was observed for GNP-crosslinked micelles. Overall, this study reports the application of spray-dried natural protein-based micelles for i.v. delivery of hydrophobic anti-cancer drugs such as FLT.     

Enhanced Oral Absorption of Paclitaxel in N-Deoxycholic Acid-N,O-Hydroxyethyl Chitosan Micellar System

The overall goal of this study was to develop a micellar system of paclitaxel (PTX) to enhance its oral absorption. An amphiphilic chitosan derivative, N-deoxycholic acid-N, O-hydroxyethyl chitosan (DHC), was synthesized and characterized by FTIR, 1H NMR, elemental analysis, and X-ray diffraction (XRD) techniques. The degree of substitution (DS) of hydroxyethyl group and deoxycholic acid group ranged from 89.5–114.5% and 1.11-8.17%, respectively. The critical micelle concentration (CMC) values of DHC decreased from 0.26 to 0.16 mg/mL as the DS of deoxycholic acid group increased. PTX was successfully loaded in DHC micelles with a high drug loading (31.68 ± 0.14%) and entrapment efficiency (77.57 ± 0.51%). The particle size of PTX-loaded DHC micelles ranged from 203.35 ± 2.19 to 236.70 ± 3.40 nm as the DS of deoxycholic acid group increased. After orally administration of PTX-loaded DHC micelles, the bioavailability was threefold compared with that of an orally dosed Taxol®. The single-pass intestinal perfusion studies (SPIP) showed that the intestinal absorption of micelles was via endocytosis involving a saturable process and a p-glycoprotein (P-gp)-inde-pendent way. All these indicated that the DHC micelles might be a promising tool for oral delivery of poorly water-soluble drugs. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:4543–4553, 2010     

In Vivo Efficacy of Enabling Formulations Based on Hydroxypropyl-β-Cyclodextrins,Micellar Preparation,and Liposomes for the Lipophilic Cannabinoid CB2 Agonist,MDA7

Enabling formulations based on hydroxypropyl-β-cyclodextrins (HPβCD), micellar preparation, and liposomes have been designed to deliver the racemic mixture of a lipophilic cannabinoid type 2 agonist, MDA7. The antiallodynic effects of MDA7 formulated in these three different systems were compared after intravenous (i.v.) administration in rats. Stoichiometry of the inclusion complex formed by MDA7 in HPβCD was determined by continuous variation plot, electrospray ionization–mass spectrometry (ESI–MS) analysis, phase solubility, and nuclear magnetic resonance studies and indicate formation of exclusively 1:1 adduct. Morphology and particle sizes determined by dynamic light scattering and transmission electron microscopy show the presence of a homogeneous population of closed round-shaped oligolamellar MDA7 containing liposomes, with an average size of 117 nm [polydispersity index (PDI) <0.1]. Monodisperse micelles exhibited an average size of 15 nm (PDI 0.1). HPβCD-based formulation administrated in vivo was composed of two discrete particles populations with a narrow size distribution of 3 nm (PDI <0.1) and 510 nm (PDI <0.1). HPβCD-based formulation dramatically improved antiallodynic effect of MDA7 in comparison with the liposomes preparation. Through inclusion complexation and possibly formation of aggregates, HPβCD can enhance the aqueous solubility of lipophilic drugs, thereby improving their bioavailability for i.v. administration.     

Investigation of PEG Crystallization in Frozen PEG-Sucrose-Water Solutions. I. Characterization of the Nonequilibrium Behavior during Freeze-Thawing

Our objective was to characterize the nonequilibrium thermal behavior of frozen aqueous solutions containing PEG and sucrose. Aqueous solutions of (i) sucrose (10%, w/v) with different concentrations of PEG (1–20%, w/v), and (ii) PEG (10%, w/v) with different concentrations of sucrose (2–20%, w/v), were cooled to ? 70°C at 5°C/min and heated to 25°C at 2°C/min in a differential scanning calorimeter. Annealing was performed at temperatures ranging from ? 50 to ? 20°C for 2 or 6 h. Similar experiments were also performed in the low-temperature stage of a powder X-ray diffractometer. A limited number of additional DSC experiments were performed wherein the samples were cooled to ? 100°C. In unannealed systems with a fixed sucrose concentration (10%, w/v), the T′_g decreased from ? 35 to ? 48°C when PEG concentration was increased from 1% to 20% (w/v). On annealing at ? 25°C, PEG crystallized. This was evident from the increase in T′_g and the appearance of a secondary melting endotherm in the DSC. Low- temperature XRD provided direct evidence of PEG crystallization. Annealing at temperatures ≤?40°C did not result in crystallization and a devitrification event was observed above the T′_g. In unannealed systems with a fixed PEG concentration (10%, w/v), the T′_g increased from ? 50 to ? 40°C when sucrose concentration was increased from 5% to 50%, w/v. As the annealing time increased (at ? 25°C), the T′_g approached that of a sucrose-water system, reflecting progressive PEG crystallization. A second glass transition at ~? 65°C was evident in unannealed systems [10%, w/v sucrose and 10 (or 20%), w/v PEG] cooled to ? 100°C. Investigation of the nonequilibrium behavior of frozen PEG-sucrose-water ternary system revealed phase separation in the freeze-concentrate. Annealing facilitated PEG crystallization.     

Through-vial impedance spectroscopy of critical events during the freezing stage of the lyophilization cycle: The example of the impact of sucrose on the crystallization of mannitol

The aim of this work was to evaluate the application of through-vial impedance spectroscopy in the measurement of eutectic crystallization during the freezing stage of the lyophilisation cycle.Impedance measurements of various sugar solutions (mannitol 5%, 10% and 15% w/v, sucrose 5% w/v and mannitol 5% w/v, and sucrose 5% w/v solutions) were taken during a freeze–thaw cycle, over a frequency range 10–10⁶ Hz with a scan interval of 1.5 min, using measurement vials with externally attached electrodes connected to a high resolution impedance analyzer.Estimates for the electrical resistance of the mannitol solutions record the exothermic crystallization of mannitol at a temperature of −24 °C during the temperature ramp down stage of the freezing cycle, which is in close agreement with the off-line DSC measurement of −22 °C. The freezing profile of a 5% mannitol solution with 5% sucrose (a component that does not crystallize in the frozen solution) demonstrated the inhibition of mannitol crystallization (with the implication that the product will then require sub-T_g′ freezing and drying).The work suggests a role for through-vial impedance spectroscopy in the concurrent development of the product formulation and freeze drying cycle without the uncertainty introduced when using off-line date to define the critical process parameters.     

PEGylated chitosan-based polymer micelle as an intracellular delivery carrier for anti-tumor targeting therapy

Stearic acid-grafted chitosan oligosaccharide (CSO-SA) micelles presented a potential candidate for intracellular drug delivery carrier due to its special spatial structure. In this article, CSO-SA was further modified by polyethylene glycol (PEG). The physicochemical properties of PEGylated CSO-SA (PEG-CSO-SA) micelles were characterized. After PEGylation, the critical micelle concentration (CMC) of PEG-CSO-SA had no significant change; the micelle size increased; and the zeta potential decreased. The cellular uptake of CSO-SA micelles before and after PEGylation in macrophage RAW264.7, immortalized rat liver cells BRL-3A and human liver tumor cells HepG2 was studied. About 58.4 ± 0.63% of CSO-SA micelles were uptaked by RAW264.7 in 24 h, however, only 17.7 ± 0.94% of PEG-CSO-SA micelles were internalized into RAW264.7 after the CSO-SA was modified with PEG in five molar times. Meanwhile, there were no changes in the uptake after PEGylation of CSO-SA in BRL-3A and HepG2. Using mitomycin C as a model drug, the in vitro anti-tumor activities of the drug loaded in the micelles were investigated. The 50% cellular growth inhibition (IC₅₀) of the drug decreased from 1.97 ± 0.2 to 0.13 ± 0.02 μg/mL after mitomycin C was loaded into CSO-SA micelles, and the IC₅₀ value of the drug had no obvious change when the CSO-SA was modified by PEG.     

Investigation of PEG Crystallization in Frozen PEG–Sucrose–Water Solutions: II. Characterization of the Equilibrium Behavior During Freeze-Thawing

Our objective was to characterize, by DSC and XRD, the equilibrium thermal behavior of frozen aqueous solutions containing polyethylene glycol (PEG) and sucrose. Aqueous solutions of (i) PEG (2.5–50% w/w), (ii) sucrose (10% w/v) with different concentrations of PEG (1–20% w/v), and (iii) PEG (2% or 10% w/v) with different concentrations of sucrose (2–20% w/v), were cooled to ? 70°C at 5°C/min and heated to 25°C at 2°C/min in a DSC. Annealing was performed for 2 or 6 h at temperatures, ranging from ? 50 to ? 20°C. Experiments under similar conditions, on select compositions, were also performed in a powder X-ray diffractometer. Two endotherms, observed during heating of a frozen PEG solution (10% w/v), were attributed to PEG–ice eutectic melting and ice melting, and were confirmed by XRD. At higher PEG concentrations (> 37.5% w/w), only the endotherm attributed to the PEG–ice eutectic melting was observed. Inclusion of sucrose decreased both PEG–ice melting and ice melting temperatures. In unannealed systems with a fixed sucrose concentration (10% w/v), the PEG–ice melting event was not observed at PEG concentration < 5% w/v. Annealing for 2–6 h facilitated PEG crystallization. In unannealed systems with a fixed PEG concentration (10% w/v), an increase in the sucrose concentration increased the devitrification but decreased the PEG–ice melting temperature. The PEG–ice melting temperatures obtained by DSC and XRD were in good agreement. In ternary systems at a fixed PEG to sucrose ratio, the T_g^′ as well as the PEG–ice melting temperature were unaffected by the total solute concentration. XRD confirmed the absence of a PEG–sucrose–ice ternary eutectic. When the PEG to sucrose ratio was systematically varied, the PEG–ice and ice melting temperatures decreased with an increase in the sucrose concentration. However, at a fixed PEG to sucrose ratio, the PEG–ice melting temperature, was unaffected by the total solute concentration.     

Development of Respirable Nanomicelle Carriers for Delivery of Amphotericin B by Jet Nebulization

The aim of the present work was to prepare and characterize chitosan-stearic acid conjugate nanomicelles for encapsulation of amphotericin B (AmB) and to evaluate the in vitro nebulization of the formulations. Water soluble chitosan was grafted to stearic acid (SA) chains via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide mediated coupling reaction. The chemical structure of depolymerized chitosan (DC)-SA copolymers and degree of amino substitution was determined by ¹H NMR. AmB was loaded in nanomicelles with a maximal encapsulation efficiency of 97%. The physicochemical properties and formation of polymeric micelles were studied by dynamic light scattering and fluorescence spectroscopy method. Nanomicelles possessed positive charges with mean particle sizes of 101–248 nm. AmB-loaded micelles were also characterized for their antifungal activity, aggregation state of the drug, nebulization efficiency and retention of AmB in the micelles after nebulization. The results indicated that encapsulation of AmB in DC-SA micelles could improve the antifungal activity of the drug in some of the cases. The nebulization efficiency was up to 56% and the fine particle fraction (FPF) varied from 40% to 52%. Since there was only a little change in encapsulation of the drug in micelles after nebulization, DC-SA micellar formulations can be a suitable choice for pulmonary delivery of AmB.     

Evaluation of bioimpedance spectroscopy for the measurement of body fluid compartment volumes in rats

IntroductionBioimpedance spectroscopy (BIS) has been used in human and large animal research to assess body fluid compartment volumes (BFC) such as total body water (TBW), extracellular fluid volume (ECFV), and intracellular fluid volume (ICFV). To date, the application of BIS for determination of BFC in small research animals has been limited.MethodsWe sought to evaluate the sensitivity and consistency of BIS for the determination of BFC in male SD rats. Thus, in separate series of experiments, we a) compared BFC values determined using BIS to BFC values obtained using radioisotope indicator dilution methods; b) examined day-to-day intra- and inter-rat BFC variability in small (267.8 ± 5.4 g) and large (372.6 ± 5.6 g) rats (n = 8/group) as compared to empirical normative mammalian values; c) evaluated the sensitivity of BIS to detect time-dependent responses to repeated administration of a potent diuretic; and d) compared empirically generated BFC data to predicted osmotically-induced ECFV and ICFV shifts in response to i.v. administration of hypotonic (0.3%), isotonic (0.9%) or hypertonic (3.0%) saline (n = 6/concentration).ResultsBFC values generated using radioisotope dilution agreed with those generated using BIS. BIS reliably detected differences between small and large rats (p < 0.001), and was associated with low (< 3.5%) day-to-day, intra-animal coefficient of variation (% = Standard Deviation/mean). BIS detected small reductions (~ 10%) in ECFV induced by as few as 2 days of the loop diuretic, furosemide, relative to vehicle treatment (70.8 ± 1.5 ml vs. 84.0 ± 1.5 ml; respectively, p < 0.05). BIS rapidly detected shifts between ECFV and ICFV in response to osmotic saline challenge, and these responses were similar to physiologically predicted responses.DiscussionThe current studies support using BIS as a means of sensitively and reliably performing repeated measurements of BFC in rats of a) differing sizes, b) in response to therapeutic agents known to influence renal sodium handling and c) in response to osmotic challenge.     

Spinning Disc Processing Technology: Potential for Large-Scale Manufacture of Chitosan Nanoparticles

Mass production of nanoparticles using a reliable cost-effective approach is a challenge in the pharmaceutical industry. In this study, the spinning disc processing (SDP) technology was used to fabricate chitosan nanoparticles, with a view to commercially produce chitosan nanoparticle-based drug delivery platforms. Chitosan solution (0.25%, w/v, in dilute acid, 27.5 mL, 1.5 mL/s) was intensely mixed with sodium tripolyphosphate solution (0.10%, w/v, in water, 20mL, 1.1mL/s) on the spinning disc (1000rpm). Transmission electron microscopy and dynamic light scattering data confirmed that the nanoparticles (20 ± 3 nm) were comparable in size and shape to those synthesised using a beaker and magnetic stirrer (31 ± 13 nm). Larger nanoparticles (131 ± 5 nm) were produced by increasing the chitosan and TPP feed concentrations to 0.5% and 0.125%, respectively. Drug loading further increased the size of the nanoparticles, with N-acetyl cysteine (NAC) having a greater effect (403 ± 4 nm) than paracetamol (165 ± 4 nm). Co-loading of both drugs increased the size of the particles to the micron range. In conclusion, the SDP is a robust technology capable of expanding the production of blank and drug-loaded chitosan nanoparticles for the biomedical and pharmaceutical industries.     

A randomised, double-blind, placebo controlled, duloxetine-referenced, fixed-dose study of three dosages of Lu AA21004 in acute treatment of major depressive disorder (MDD)

The efficacy, safety, and tolerability of Lu AA21004 versus placebo, using duloxetine as active reference, in patients with DSM-IV-TR diagnosed major depressive disorder (MDD) were evaluated in this 8-week, multi-site study. Patients (n = 766) had a baseline Montgomery–Åsberg Depression Rating Scale (MADRS) total score ≥ 26 and were randomly assigned (1:1:1:1:1) to 2.5, 5 or 10 mg Lu AA21004, placebo, or 60 mg duloxetine. The 5 mg and 10 mg doses of Lu AA21004 were tested separately versus placebo at p ≤ 0.025 in a pre-specified order. In the pre-defined primary efficacy analysis [mean change from baseline in MADRS total score at Week 8, full analysis set, ANCOVA, last observation carried forward (LOCF)], the differences to placebo (n = 145) of ? 1.7 (Lu AA21004 5 mg, n = 155) and ? 1.5 points (Lu AA21004 10 mg, n = 151) were not statistically significant; nor were those for Lu AA21004 2.5 mg (? 1.4 points, n = 155) or duloxetine (? 2.0 points, n = 149). Using mixed model, repeated measures (MMRM) analyses of the primary endpoint and most secondary endpoints were supportive of likely efficacy for Lu AA21004 5 mg and 10 mg and duloxetine. Treatment-emergent adverse events led to the withdrawal of 72 patients: 8% (placebo), 12% (duloxetine), and 6%, 11% and 9% in the Lu AA21004 groups (2.5 mg, 5 mg and 10 mg, respectively). The most common adverse events were nausea, headache, dizziness, and dry mouth. No clinically relevant changes were seen in vital signs, weight, ECG, or laboratory results. In summary, none of the active treatment groups, including duloxetine, separated from placebo in the primary analysis in this 'failed' study. Findings on secondary outcome measures, using MMRM instead of LOCF, were supportive of likely efficacy for Lu AA21004 5 mg and 10 mg and duloxetine. Lu AA21004 (2.5, 5 and 10 mg) was well tolerated.     

Self-Assembled Methoxy Poly(Ethylene Glycol)-Cholesterol Micelles for Hydrophobic Drug Delivery

To promote the application of methoxy poly(ethylene glycol)-cholesterol (mPEG—Chol), mPEG–Chol was used to prepare core-shell micelles encapsulating poorly water-soluble docetaxel (DTX-PM) by modified cosolvent evaporation method. Approaches to enhance DTX entrapment efficiency (EE) and minimize particle size were investigated in detail, including organic and aqueous phase composition, organic/aqueous phase ratio, and polymer concentration. In optimal formulation, micelles had higher EE (97.6%) and drug loading (4.76%) with the diameter of 13.76 ± 0.68 nm and polydispersity index of 0.213 ± 0.006. Transmission electron microscopy (TEM) showed that the micelles were spherical, and differential scanning calorimetry (DSC) analysis proved that DTX was successfully entrapped into mPEG–Chol micelles. The in vitro cytotoxicity experiments displayed that blank micelles had no effect on the growth of SKOV-3, BXPC-3, A549, and HepG-2 cells, demonstrating that mPEG–Chol was one of the biocompatible biomaterials. The half inhibition concentration of DTX-PM on SKOV-3, BXPC-3, A549, and HepG-2 cells were 10.08, 7.6, 28.37, and 125.75 ng/mL, respectively. DTX-PM had the similar antitumor activity to free DTX, indicating that mPEG–Chol was a promising micellar vector for hydrophobic drug delivery. In addition, this work provided a new and facile approach to prepare drug-loaded micelles with controllable performances. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1054–1062, 2013     

Distribution of Doxorubicin in Rats Undergoing Ultrasonic Drug Delivery

Ultrasound (US) increases efficacy of drugs delivered from micelles, but the pharmacokinetics have not been studied previously. In this study, US was used to deliver doxorubicin (Dox) sequestered in micelles in an in vivo rat model with bilateral leg tumors. One of two frequencies with identical mechanical index and intensity was delivered for 15 min to one tumor immediately after systemic injection of micellar Dox. Pharmacokinetics in myocardium, liver, skeletal muscle, and tumors were measured for 1 week. When applied in combination with micellar Dox, the ultrasoincated tumor had higher Dox concentrations at 30 min, compared to bilateral noninsonated controls. Initially, concentrations were highest in heart and liver, but within 24 h they decreased significantly. From 24 h to 7 days, concentrations remained highest in tumors, regardless of whether they received US or not. Comparison of insonated and noninsonated tumors showed 50% more Dox in the insonated tumor at 30 min posttreatment. Four weekly treatment produced additional Dox accumulation in the myocardium but not in liver, skeletal leg muscle, or tumors compared to single treatment. Controls showed that neither US nor the empty carrier impacted tumor growth. This study shows that US causes more release of drug at the targeted tumor. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3122–3131, 2010     

Street-level policing in the Downtown Eastside of Vancouver, Canada, during the 2010 Winter Olympics

BackgroundPolice presence within street-based drug scenes has the potential to disrupt injection drug users’ (IDUs) access to health services and prompt increased injection-related risk behaviour. We examined street-level policing in the Downtown Eastside (DTES) of Vancouver during the Olympic Winter games, to assess the potential impact on access to harm reduction services and injection-related risk behaviour.MethodsWe analysed data from observational activities documenting police and drug user behaviour, unstructured interviews with drug users in street settings (n = 15), expert interviews with legal and health professionals (n = 6), as well as utilisation statistics from a local supervised injection facility (SIF).ResultsAlthough police presence was elevated within the DTES during the Olympics, there was little evidence to suggest that police activities influenced IDUs’ access to health services or injection-related risk behaviour. SIF attendance during the Olympics was consistent with regular monthly patterns.ConclusionPolice presence during the Olympics did not reduce access to health services amongst local IDUs or prompt increased injection-related risk behaviour. Increased cooperation between local law enforcement and public health bodies likely offset the potential for negative health consequences resulting from police activity.     

A Nonionic Surfactant/Chitosan Micelle System in an Innovative Eye Drop Formulation

Micelle systems composed of the polyoxyethylated nonionic surfactant Pluronic® F127 (F127) and cationic polyelectrolyte chitosan (CH) were prepared with dexamethasone (DEX) as a hydrophobic model drug. The F127/CH micelles were characterised by their hydrodynamic diameter and a zeta-potential ranging between 25.4 and 28.9 nm and + 9.3 and + 17.6 mV, respectively. The DEX loading was between 0.48% and 0.56%, and no significant influence of CH on DEX loading was observed. All micelle systems were characterised by prolonged release profiles. The addition of CH significantly enhanced the in vitro DEX release rate and transport across Caco-2 cell monolayers, as compared to the CH-free F127 micelle system. This colloidal carrier was well tolerated in rabbit eyes, and no clinically abnormal signs in various ocular structures were observed. The increase in intraocular pressure (IOP) in rabbits was used to evaluate DEX ocular bioavailability. The AUC values showed a 1.7- and 2.4-fold increase in bioavailability with F127 and F127/0.015 (w/v) % CH micelle systems, respectively, as compared to a standard DEX suspension. These data indicate improved intraocular DEX absorption from the micelle systems, which can be ascribed to both F127 and CH corneal permeability enhancement.     

Deposition of ethyl glucuronide in WHP rat hair after chronic ethanol intake

BackgroundThis study investigated the relationship between ethanol intake in rats and the resulting level of ethyl glucuronide (EtG) in rat hair.MethodsRats (n = 50) consumed a 10% ethanol solution for 4 weeks, then EtG was extracted from samples of their hair using a novel extraction procedure involving freezing and thawing. The EtG concentration was measured using gas chromatography and mass spectrometry. The animals voluntarily drank ethanol, with daily consumption in most rats exceeding 5 g/kg b.w. The silylated EtG was stable for at least 28 h. The limit of detection was 0.03 ng/mg, and the limit of quantification was 0.1 ng/mg.ResultsHair samples from rats that consumed ethanol had EtG levels ranging from 0.17–20.72 ng/mg in female rats and 0.15–13.72 ng/mg in males. There was a correlation between the amount of alcohol consumed and the EtG levels in hair from female (p < 0.01), but not male, rats.ConclusionThe method presented allows detection and quantification of EtG in rat hair. We also observed differences in EtG deposition in male and female rats.     

Novel folated and non-folated pullulan bioconjugates for anticancer drug delivery

Two new anticancer polymer therapeutics were designed for tumour cell targeting. The bioconjugates were synthesised by pullulan derivatisation with either doxorubicin or doxorubicin and folic acid. Pullulan was activated by periodate oxidation and functionalised by reductive conjugation with cysteamine and 1.9 kDa PEG(NH₂)₂. The cysteamine thiol groups were conjugated to doxorubicin through a pH-sensitive hydrazone spacer while the pending PEG-NH₂ functions of one derivatised pullulan batch were conjugated to folic acid to obtain one of the two polymer therapeutics. The reaction intermediates and the final products were characterised by mass spectrometry, UV–vis analysis and reverse phase and gel permeation chromatography. The folic acid-free derivative [(NH₂ PEG)-Pull-(Cyst-Dox)] contained 6.3% (w/w) doxorubicin while the folic acid-doxorubicin-coupled derivative [(FA-PEG)-Pull-(Cyst-Dox)] contained 6% (w/w) doxorubicin and 4.3% (w/w) folic acid. Photon correlation spectroscopy showed that (NH₂ PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) assembled into particles of about 150 and 100 nm diameter, respectively. The two bioconjugates displayed similar drug release profiles either at pH 7.4 buffer or in plasma, where less than 20% of doxorubicin was released within three days. At pH 5.5, both conjugates underwent complete drug release in about 40 h. In vitro studies carried out with KB tumour cells over-expressing folic acid receptor showed that both free doxorubicin and (FA-PEG)-Pull-(Cyst-Dox) were rapidly taken up by the cells, while the internalisation of the non-folated derivative was significantly slower. Cell viability studies did not show relevant difference between the two bioconjugates. After 72 h of incubation with folic acid receptor non-expressing MCF7 cells, the IC₅₀ values of doxorubicin, (NH₂PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) were 0.3 μM, 1.2 μM and 3.1 μM, respectively. After incubation with KB cells over-expressing folic acid receptor, the IC₅₀ values were 0.4 μM, 1.8 μM and 1.1 μM, respectively. Pharmacokinetic studies showed that 4 h after intravenous administration of the conjugates to Balb/c mice about 40% of the administered drug equivalent dose was present in the bloodstream while in the case of unconjugated doxorubicin, 80% of the drug was cleared within 30 min.These findings suggest that the novel doxorubicin–pullulan bioconjugates possess suitable properties for passive tumour targeting. On the other hand, folic acid conjugation has been found to have limited effect on selective cell up-take.     

Evaluation of Lipopeptide (Daptomycin) Aggregation Using Fluorescence,Light Scattering,and Nuclear Magnetic Resonance Spectroscopy

The aggregation behavior and critical aggregation concentration (CAC) values of daptomycin in aqueous solutions were evaluated under the external factors of pH, temperature, daptomycin concentration, and calcium ions concentration by using the complementary characterization techniques, fluorescence, dynamic and static light scattering, and nuclear magnetic resonance (NMR) spectroscopy. On the basis of the intrinsic fluorescence resonance energy transfer of daptomycin, the CAC values were identified by an upward inflection of the fluorescence emission from Kyn-13 at 460 nm. The pH-dependent CAC values were determined to be 0.14 mM at pH 3.0, 0.12 mM at pH 4.0, and 0.20 mM at pH 2.5 and 5.0. The CAC values obtained by fluorescence spectroscopy were confirmed by dynamic light scattering and NMR spectroscopy.     

Influence of ethacrynic acid on the anticonvulsant activity of conventional antiepileptic drugs in the mouse maximal electroshock seizure model

The aim of this study was to determine whether ethacrynic acid (EA), a loop diuretic with anticonvulsant activity, would affect the protective action of the conventional antiepileptics (AEDs) carbamazepine (CBZ), phenytoin (PHT), valproate (VPA) and phenobarbital (PB) in the mouse maximal electroshock seizure (MES) model. The effects of acute and chronic treatment with EA on these AEDs were examined. At a single dose of 100 mg/kg ip, EA enhanced the antielectroshock activity of VPA, decreasing its ED₅₀ value from 225.6 to 146.6 mg/kg (p < 0.05), but enhancement was not observed following continuous administration of EA (12.5 mg/kg) for seven days. Combined treatment of EA with other AEDs had no effect on their ED₅₀ values. The observed interaction between EA and VPA was pharmacodynamic in nature as EA did not alter free plasma (non-protein-bound) and total brain concentrations of VPA. Taking into consideration the clinical use of both drugs, this interaction between EA and VPA can be important for patients receiving these drugs.