Transdermal Absorption Enhancement of N-Terminal Tat–GFP Fusion Protein (TG) Loaded in Novel Low-Toxic Elastic Anionic Niosomes

Elastic anionic niosomes (Tween 61/cholesterol/dicetyl phosphate at 1:1:0.05 molar ratio of 20 mM) with various concentrations of ethanol and edge activators sodium cholate (NaC) and sodium deoxycholate (NaDC) showed larger vesicular size (171.94 ± 63.52 – 683.17 ± 331.47 nm) and higher negative zeta potential (?6.45 ± 2.76 to ? 17.40 ± 2.51 mV) than the nonelastic anionic niosomes. The elasticity (deformability index) and entrapment efficiency of all elastic vesicles except the NaDC vesicles were higher than the nonelastic vesicles. The morphology, under transmission electron microscope, of elastic and nonelastic niosomes loaded and not loaded with Tat–green fluorescent protein fusion protein (TG) were in large unilamellar structure. TG loaded in elastic (1 mol% NaC) anionic niosomes gave the highest cell viability both in HT-29 (92.32 ± 3.82%) and KB cells (96.62 ± 5.96%), the highest cumulative amounts (62.75 ± 2.68 μg/cm²) and fluxes (10.46 ± 3.45 μg/cm²h) in receiving chamber in rat skin transdermal study by Franz diffusion cells. This study has not only indicated the synergistic enhancement effects of the Tat peptide and the niosomal delivery system on the cellular uptake and transdermal absorption of TG but also 1 mol% NaC as an edge activator to obtain a novel low-toxic elastic anionic niosomes for topical use of therapeutic macromolecules such as proteins, as well.     

Enhancement of gene expression and melanin production of human tyrosinase gene loaded in elastic cationic niosomes

Objectives Disturbance in the synthesis of tyrosinase might be one of the major causes of vitiligo. The enhancement of tyrosinase gene expression and melanin production by loading the plasmid in elastic cationic niosomes was investigated in tyrosinase gene knocked out human melanoma (M5) cells and in tyrosine‐producing mouse melanoma (B₁₆F₁₀) cells. Methods Niosomes composed of Tween 61/cholesterol/dimethyl dioctadecyl ammonium bromide at 1 : 1 : 0.5 molar ratio were prepared by the freeze‐dried empty liposomes method. The thin lipid film was redissolved in distilled water or 25% ethanol to obtain the non‐elastic or elastic cationic niosomes, respectively. Key findings The maximum loading of the plasmid in non‐elastic and elastic niosomes was 130 and 100 µg per 16 mg of the niosomal contents, respectively. The plasmid‐loaded elastic cationic niosomes exhibited high specific tyrosinase activity of 1.66 and 1.50 fold in M5 cells and 6.81 and 4.37 fold in B₁₆F₁₀ cells compared with the free plasmid and the plasmid‐loaded non‐elastic cationic niosomes, respectively. Conclusions This study has demonstrated not only the enhancement of the expression of human tyrosinase gene by loading in elastic cationic niosomes, but also the potential application of this gene delivery system for the further development of vitiligo gene therapy.     

Occurrence and exposure to polycyclic aromatic hydrocarbons in kindling-free-charcoal grilled meat products in Taiwan

This study aimed to determine the contents of 16 PAHs in kindling-free-charcoal grilled meat and seafood products by GC–MS coupled with a QuEChERS method, and estimate the potential risk associated with consumption of those products in Taiwan. Results showed that the total PAHs contents ranged from 6.3 ± 0.9 to 238.8 ± 8.3 ng/g in poultry meat, 0.1 ± 0.0–547.5 ± 12.2 ng/g in red meat, and 6.6 ± 1.4–249.7 ± 6.4 ng/g in seafood products. Among various PAHs, the highly carcinogenic benzo[a]pyrene was detected in chicken breast grilled at 84 °C (30 min), chicken heart at 100 °C (26 min), chicken drumstick at 74 °C (20 min), duck drumstick at 85 °C (40 min), and lamb steak at 88 °C (12 min), with its level amounting to 1.3 ± 0.0, 2.4 ± 0.1, 4.0 ± 1.3, 3.1 ± 0.0, and 5.8 ± 0.5 ng/g, respectively. The generation of PAHs was associated with grilling time, temperature and fat content. Risk assessment of dietary exposure to PAHs revealed toxicity equivalent to range from ND – 6.174 ± 0.505 μg/g and margin of exposure was >10,000, which agreed with the EFSA’s definition of low public health concern. The lifelong average daily PAHs intake was higher for adults than for elderly people in Taiwan, however, consumption of kindling-free-charcoal grilled meat should not be a public health concern based on cancer risk potency.     

A pragmatic approach for engineering porous mannitol and mechanistic evaluation of particle performance

The importance of mannitol has increased recently as an emerging diluent for orodispersible dosage forms. The study aims to prepare spray dried mannitol retaining high porosity and mechanical strength for the development of orally disintegrating tablets (ODTs).Aqueous feed of d-mannitol (10% w/v) comprising ammonium bicarbonate, NH₄HCO₃ (5% w/v) as pore former was spray dried at inlet temperature of 110–170 °C. Compacts were prepared at 151 MPa and characterized for porosity, hardness and disintegration time. Particle morphology and drying mechanisms were studied using thermal (HSM, DSC and TGA) and polymorphic (XRD) methods.Tablet porosity increased from 0.20 ± 0.002 for pure mannitol to 0.53 ± 0.03 using fabricated porous mannitol. Disintegration time dropped by 50–77% from 135 ± 5.29 s for pure mannitol to 75.33 ± 2.52–31.67 ± 1.53 s for mannitol 110–170 °C. Hardness increased by 150% at 110 °C (258.67 ± 28.89 N) and 30% at 150 °C (152.70 ± 10.58 N) compared to pure mannitol tablets (104.17 ± 1.70 N). Increasing inlet temperature resulted in reducing tablet hardness due to generation of ‘micro-sponge’-like particles exhibiting significant elastic recovery. Impact of mannitol polymorphism on plasticity/elasticity cannot be ruled out as a mixture of α and β polymorphs formed upon spray drying.     

Tissue accumulation and toxicity of isothiazolinone in Ctenopharyngodon idellus (grass carp): Association with P-glycoprotein expression and location within tissues

Isothiazolinone is widely used as a broad-spectrum fungicide in various industries, such as oil, paper, pesticide, dyes, tanning and cosmetics. There is an increasing concern over protection of the aquatic environment due to its large-scale use. The acute toxicity (LC₅₀) of isothiazolinone in Ctenopharyngodon idellus was investigated. The residual time and accumulation in tissues, P-glycoprotein mRNA level and localization of P-glycoprotein in the liver and kidney were also analyzed. The LC₅₀ (48 h) values of isothiazolinone to C. idellus were 0.53 ± 0.17 mg/L and 0.41 ± 0.08 mg/L at 15 °C and 25 °C, respectively. The LC₅₀ values decreased as the temperature increased. The accumulation of isothiazolinone in livers and kidneys in the high temperature group (25 °C) was significantly greater than that of the low temperature group (15 °C). Prolonged tissue residual time of isothiazolinone was seen in all the groups. There were significant differences in P-glycoprotein mRNA expression between isothiazolinone-treated groups and control samples (P < 0.05–0.01). Temperature affected accumulation and toxicity of isothiazolinone.     

Surface functionalization of doxorubicin-loaded liposomes with octa-arginine for enhanced anticancer activity

Doxorubicin-loaded PEGylated liposomes (commercially available as DOXIL® or Lipodox®) were surface functionalized with a cell-penetrating peptide, octa-arginine (R8). For this purpose, R8-peptide was conjugated to the polyethylene glycol–dioleoyl phosphatidylethanolamine (PEG–DOPE) amphiphilic co-polymer. The resultant R8–PEG–PE conjugate was introduced into the lipid bilayer of liposomes at 2 mol% of total lipid amount via spontaneous micelle-transfer technique. The liposomal modification did not alter the particle size distribution, as measured by Particle Size Analyzer and transmission electron microscopy (TEM). However, surface-associated cationic peptide increased zeta potential of the modified liposomes. R8-functionalized liposomes (R8-Dox-L) markedly increased the intracellular and intratumoral delivery of doxorubicin as measured by flow cytometry and visualizing by confocal laser scanning microscopy (CLSM) compared to unmodified Doxorubicin-loaded PEGylated liposomes (Dox-L). R8-Dox-L delivered loaded Doxorubicin to the nucleus, being released from the endosomes at higher efficiency compared to unmodified liposomes, which had marked entrapment in the endosomes at tested time point of 1 h. The significantly higher accumulation of loaded drug to its site of action for R8-Dox-L resulted in improved cytotoxic activity in vitro (cell viability of 58.5 ± 7% for R8-Dox-L compared to 90.6 ± 2% for Dox-L at Dox dose of 50 μg/mL for 4 h followed by 24 h incubation) and enhanced suppression of tumor growth (348 ± 53 mm³ for R8-Dox-L, compared to 504 ± 54 mm³ for Dox-L treatment) in vivo compared to Dox-L. R8-modification has the potential for broadening the therapeutic window of pegylated liposomal doxorubicin treatment, which could lead to lower non-specific toxicity.     

Novel semi-interpenetrating hydrogel networks with enhanced mechanical properties and thermoresponsive engineered drug delivery,designed as bioactive endotracheal tube biomaterials

Thermoresponsive polymeric platforms are used to optimise drug delivery in pharmaceutical systems and bioactive medical devices. However, the practical application of these systems is compromised by their poor mechanical properties. This study describes the design of thermoresponsive semi-interpenetrating polymer networks (s-IPNs) based on cross-linked p(NIPAA) or p(NIPAA-co-HEMA) hydrogels containing poly(ε-caprolactone) designed to address this issue. Using DSC, the lower critical solution temperature of the co-polymer and p(NIPAA) matrices were circa 34 °C and 32 °C, respectively. PCL was physically dispersed within the hydrogel matrices as confirmed using confocal scanning laser microscopy and DSC and resulted in marked changes in the mechanical properties (ultimate tensile strength, Young’s modulus) without adversely compromising the elongation properties. P(NIPAA) networks containing dispersed PCL exhibited thermoresponsive swelling properties following immersion in buffer (pH 7), with the equilibrium-swelling ratio being greater at 20 °C than 37 °C and greatest for p(NIPAA)/PCL systems at 20 °C. The incorporation of PCL significantly lowered the equilibrium swelling ratio of the various networks but this was not deemed practically significant for s-IPNs based on p(NIPAA). Thermoresponsive release of metronidazole was observed from s-IPN composed of p(NIPAA)/PCL at 37 °C but not from p(NIPAA-co-HEMA)/PCL at this temperature. In all other platforms, drug release at 20 °C was significantly similar to that at 37 °C and was diffusion controlled. This study has uniquely described a strategy by which thermoresponsive drug release may be performed from polymeric platforms with highly elastic properties. It is proposed that these materials may be used clinically as bioactive endotracheal tubes, designed to offer enhanced resistance to ventilator associated pneumonia, a clinical condition associated with the use of endotracheal tubes where stimulus responsive drug release from biomaterials of significant mechanical properties would be advantageous.     

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.     

Potent Melanin Production Enhancement of Human Tyrosinase Gene by Tat and an Entrapment in Elastic Cationic Niosomes: Potential Application in Vitiligo Gene Therapy

Potent melanin production enhancement of human tyrosinase plasmid (pAH7/Tyr, P) in mouse melanoma cells (B₁₆F₁₀) by Tat peptide (T) and an entrapment in elastic cationic niosomes (E) was described. The E composed of Tween 61/cholesterol/dodecyl dimethyl ammonium bromide at 1:1:0.5 molar ratio was prepared by freeze‐dried emptying liposomes method. PE at P/E ratio of 1:160 w/w and TPE at T/P/E ratio of 0.125:1:160, 0.25:1:160, and 0.5:1:160 w/w/w were prepared. The final concentration of the plasmid in the study was 4 ng/μL. By sulforhodamine B assay, PE and TPE complexes showed slight or no cytotoxic effect. The cells transfected with TPE (0.5:1:160) exhibited the highest enhancement of tyrosinase enzyme activity of 11.82‐, 7.67‐, 5.07‐, and 6.29‐folds of control, P, PE, and TP (0.5:1) and melanin production of 13.03‐, 8.46‐, 5.36‐, and 6.58‐folds of control, P, PE, and TP (0.5:1), respectively. The elastic cationic niosomes demonstrated an increase in thermal stability of P at 4 ± 2, 25 ± 2, and 45 ± 2 °C. The vesicular size and the zeta potential values of PE and TPE complexes were slightly increased but still in the range of stable dispersion (out of ±30 mV). These results indicated the high potential application of the TPE complexes for further investigation for vitiligo gene therapy.     

Development and statistical optimization of nefopam hydrochloride loaded nanospheres for neuropathic pain using Box–Behnken design

Nefopam hydrochloride (NFH) is a non-opioid centrally acting analgesic drug used to treat chronic condition such as neuropathic pain. In current research, sustained release nefopam hydrochloride loaded nanospheres (NFH-NS) were auspiciously synthesized using binary mixture of eudragit RL 100 and RS 100 with sorbitan monooleate as surfactant by quasi solvent diffusion technique and optimized by 3⁵ Box–Behnken designs to evaluate the effects of process and formulation variables. Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetric (DSC) and X-ray diffraction (XRD) affirmed absence of drug–polymer incompatibility and confirmed formation of nanospheres. Desirability function scrutinized by design-expert software for optimized formulation was 0.920. Optimized batch of NFH-NS had mean particle size 328.36 nm ± 2.23, % entrapment efficiency (% EE) 84.97 ± 1.23, % process yield 83.60 ± 1.31 and % drug loading (% DL) 21.41 ± 0.89. Dynamic light scattering (DLS), zeta potential analysis and scanning electron microscopy (SEM) validated size, charge and shape of nanospheres, respectively. In-vitro drug release study revealed biphasic release pattern from optimized nanospheres. Korsmeyer Peppas found excellent kinetics model with release exponent less than 0.45. Chronic constricted injury (CCI) model of optimized NFH-NS in Wistar rats produced significant difference in neuropathic pain behavior (p < 0.05) as compared to free NFH over 10 h indicating sustained action. Long term and accelerated stability testing of optimized NFH-NS revealed degradation rate constant 1.695 × 10⁻⁴ and shelf-life 621 days at 25 ± 2 °C/60% ± 5% RH.     

Demonstration of the Terms Enantiotropy and Monotropy in Polymorphism Research Exemplified by Flurbiprofen

The thermodynamic terms enantiotropy and monotropy are demonstrated by means of solid‐state analytical results of polymorphous flurbiprofen (FBP). Vibrational spectra, differential scanning calorimetry (DSC), and thermomicroscopy investigations as well as X‐ray powder patterns for three modifications of FBP are described. The melting points are mod. I 113–114 °C (enthalpy of fusion 27.9 ± 0.2 kJ mol⁻¹) for modification I (mod. I), 92 °C for mod. II, and 87 °C for mod. III. The true densities of mod. I (1.279 ± 0.001 g cm⁻³) and mod. II (1.231 ± 0.002 g cm⁻³) were measured at 25 °C. Modification I (commercial product) is the thermodynamically stable crystal form from absolute zero to its melting point. Modification II was crystallized on a gram scale from a warm saturated solution of FBP in n‐heptane and rapid cooling of the solution to −18 °C. Modification I is monotropically related to mod. II and mod. III, due to application of the density rule and the entropy‐of‐fusion rule. The thermodynamic relationships between the three modifications are demonstrated by a semischematic energy/temperature diagram. Theoretical vapor pressure/temperature diagrams and energy/temperature diagrams are compared and briefly discussed.     

Preparation of a fast dissolving oral thin film containing dexamethasone: A possible application to antiemesis during cancer chemotherapy

We prepared fast dissolving oral thin film that contains dexamethasone and base materials, including microcrystalline cellulose, polyethylene glycol, hydroxypropylmethyl cellulose, polysorbate 80 and low-substituted hydroxypropyl cellulose. This preparation showed excellent uniformity and stability, when stored at 40 °C and 75% in humidity for up to 24 weeks. The film was disintegrated within 15 s after immersion into distilled water. The dissolution test showed that approximately 90% of dexamethasone was dissolved within 5 min. Subsequently, pharmacokinetic properties of dexamethasone were compared in rats with oral administration of 4 mg dexamethasone suspension or topical application of the film preparation containing 4 mg dexamethasone to the oral cavity. Pharmacokinetic parameters were similar between the two groups in which C_max (h), T_max (μg/mL), AUC (μg/mL/h) and half-life (h) were 12.7 ± 6.6 (mean ± SD, N = 10), 3.4 ± 1.4, 93.6 ± 37.8 and 1.66 ± 0.07, respectively, for oral suspension and 13.3 ± 4.0, 3.2 ± 1.0, 98.0 ± 22.3 and 1.65 ± 0.06, respectively, for film preparation. These findings suggest that the fast dissolving oral thin film containing dexamethasone is likely to become one of choices of dexamethasone preparations for antiemesis during cancer chemotherapy.     

Association between chemotherapy and plasma adipokines in patients with colorectal cancer

BackgroundA link between chemotherapy, the serum level of selected adipokines and clinical outcome in colorectal patients was investigated.MethodsLeptin, adiponectin, resistin, visfatin and insulin were measured by ELISA in colorectal cancer patients before and 3 months after the administration of cancer therapy. From August 2012 to August 2013, 34 patients with pathologically documented advanced colorectal cancer (T3/T4 with metastases or nodal status up to N3) and measurable metastatic disease, who required palliative chemotherapy based on the combination of 5-fluorouracil, oxaliplatin and irinotecan, were prospectively recruited in this study. Patients previously underwent curative surgical tumour resection, but the disease was disseminated (metastases in the liver and/or lungs) at the time of admission to the hospital.ResultsOf the 34 patients in this study, 5 accomplished a chemotherapy course with partial response (PR), 23 with SD (stabilisation) and 6 with progression (PD). For further study, only patients with good prognostic outcomes (i.e., PR and SD patients) were included. The mean level of leptin before chemotherapy was 26.39 ± 19.53 ng/ml. After six courses of cancer treatment, the leptin level increased by 118–57.44 ± 27.72 ng/ml (p < 0.001). Additionally, the adiponectin level increased considerably (47%) from 9.89 ± 3.96 ng/ml to 14.51 ± 7.79 ng/ml (p < 0.001). In contrast to leptin and adiponectin, the resistin and visfatin levels decreased significantly from 7.24 ± 1.17 and 1.98 ± 0.44 to 6.36 ± 1.36 and 1.48 ± 0.34 ng/ml (p < 0.001), respectively. Insulin also declined remarkably from 16.20 ± 1.96 to 12.87 ± 1.80 (p < 0.001). There were no significant differences the between male and female patients regarding age, BMI, and leptin, adiponectin, resistin, visfatin and insulin serum levels.ConclusionsThe results of the present study are relevant because we found that chemotherapy in colorectal cancer patients, in addition to its beneficial clinical impact on the course of disease, positively affects cytokine production and release (increases the anti-inflammatory adiponectin and decreases visfatin and resistin, which are proangiogenic and promote cancer cell proliferation). The restoration of adequate adipose tissue function is essential for patients to achieve a good survival prognosis.     

Combination therapy with lercanidipine and enalapril reduced central blood pressure augmentation in hypertensive patients with metabolic syndrome

Arterial stiffness and blood pressure (BP) augmentation are independent predictors of cardiovascular events. In a randomized, open, parallel group study we compared the effect on these parameters of combination therapy with an ACE-inhibitor plus calcium channel blocker or thiazide diuretic in 76 hypertensive patients with metabolic syndrome uncontrolled by ACE-inhibitor monotherapy.After 4 weeks run-in with enalapril (ENA, 20 mg), patients were randomized to a combination therapy with lercanidipine (LER, 10–20 mg) or hydrochlorothiazide (HCT, 12.5–25 mg) for 24 weeks. Aortic stiffness (carotid to femoral pulse wave velocity, PWV), central BP values and augmentation (augmentation index, AIx) were measured by applanation tonometry.The two groups showed similar office and central BP after run-in. Office (ENA/LER: from 149.1 ± 4.9/94.5 ± 1.5 to 131.7 ± 8.1/82.2 ± 5.3; ENA/HCT: from 150.3 ± 4.7/94.7 ± 2.1 to 133.1 ± 7.1/82.8 ± 5.3 mm Hg) and central BP (ENA/LER 127.4 ± 17.1/85.2 ± 12.1 to 120.5 ± 13.5/80.0 ± 9.5 mm Hg; ENA/HCT 121.6 ± 13.4/79.3 ± 9.5 mm Hg) were similarly reduced after 24 weeks. PWV was comparable after run-in and not differently reduced by the two treatments (ENA/LER from 8.6 ± 1.5 to 8.1 ± 1.3 m/s, p < 0.05; ENA/HCT from 8.5 ± 1.2 to 8.2 ± 1.0 m/s, p < 0.05). Finally, both combinations reduced AIx, but its reduction was significantly greater (p < 0.05) in ENA/LER (from 26.8 ± 10.9 to 20.6 ± 9.1%) than in ENA/HCT arm (from 28.2 ± 9.0 to 24.7 ± 8.7%).In conclusion, the combination with LER caused a similar PWV reduction as compared to HCT, but a greater reduction in AIx in hypertensive patients with metabolic syndrome not controlled by ENA alone. These results indicate a positive effect of the combination of ENA/LER on central BP augmentation, suggesting a potential additive role for cardiovascular protection.     

Protective effect of extract of Crataegus pinnatifida pollen on DNA damage response to oxidative stress

The protective effect of extract of Crataegus pinnatifida (Rosaceae) pollen (ECPP) on the DNA damage response to oxidative stress was investigated and assessed with an alkaline single-cell gel electrophoresis (SCGE) assay and pBR322 plasmid DNA breaks in site-specific and non-site-specific systems. Total phenolic content, total flavonoid content, individual phenolic compounds, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), radical scavenging activity, FRAP, and chelating activity) were also determined. The results showed that ECPP possessed a strong ability to protect DNA from being damaged by hydroxyl radicals in both the site-specific system and the non-site-specific system. It also exhibited a cytoprotection effect in mouse lymphocytes against H₂O₂-induced DNA damage. These protective effects may be related to its high total phenolic content (17.65 ± 0.97 mg GAE/g), total flavonoid content (8.04 ± 0.97 mg rutin/g), strong free radical scavenging activity and considerable ferrous ion chelating ability (14.48 ± 0.21 mg Na₂EDTA/g).     

The direct effects of streptozotocin and alloxan on contractile function in rat heart

Streptozotocin (STZ) and alloxan (ALX) are widely used to induce diabetes mellitus in experimental animals. The direct effects of STZ and ALX on the amplitude and time course of ventricular myocyte shortening and on cardiac action potentials were investigated. STZ and ALX (10^?5 M) were dissolved in normal Tyrode (NT), maintained at pH 7.4 and 37 °C and stored for either 15 or 60–120 min. Both compounds reduced the amplitude of myocyte shortening. Compared to NT the amplitude of shortening was 34.7 ± 5.0% and 35.2 ± 6.8% with STZ and 41.0 ± 5.5% and 37.3 ± 5.7% with ALX stored for 15 and 60–120 min, respectively. During a 10 min NT washout STZ myocytes recovered to 56.2 ± 8.3% and 60.5 ± 8.2% and ALX myocytes recovered to 88.9 ± 10.0% and 83.7 ± 9.9% after storage of compounds for 15 and 60–120 min, respectively. Perfusion of the whole heart with ALX induced bradycardia but had no effects on the duration of action potential repolarization at 50% and 70% from peak action potential. The negative inotropic effects of STZ and ALX were not altered by storage. The results suggest that some of the effects on heart reported in STZ- and ALX-induced diabetes may be partly attributed to direct action of these compounds.     

Trophic transfer of Cd from larval chironomids (Chironomus riparius) exposed via sediment or waterborne routes,to zebrafish (Danio rerio): Tissue-specific and subcellular comparisons

Zebrafish were fed chironomid larvae (8% wet weight daily ration) for 7 days, followed by 3 days of gut clearance in a static-renewal system. Regardless of whether the chironomids had been loaded with Cd via a waterborne exposure or sediment exposure, they had similar subcellular distributions of Cd, with the largest areas of storage being metal rich granules (MRG) > organelles (ORG) > enzymes (ENZ) except that sediment-exposed chironomids had significantly more Cd in the metallothionein-like protein (MTLP) fraction, and significantly less Cd in the cellular debris (CD) fraction. When zebrafish fed sediment-exposed chironomids (153 ± 11 μg Cd/g dry weight) were compared directly to zebrafish fed waterborne exposed chironomids (288 ± 12 μg Cd/g dry weight), identical whole-body Cd levels were observed, despite the difference in the concentration in the food source. Thus trophic transfer efficiency (TTE) of Cd was significantly greater from sediment-exposed chironomids (2.0 ± 0.5%) than from waterborne-exposed chironomids (0.7 ± 0.2%). Subsequent tests with waterborne exposed chironomids loaded to comparable Cd concentrations, as well as with Cd-spiked manufactured pellets, demonstrated that TTEs were concentration-independent. In all treatments, zebrafish exhibited similar subcellular storage of Cd, with the greatest uptake occurring in the ORG fraction followed by the ENZ fraction. However, neither trophically available metal (TAM) nor metabolically available fractions (MAF) were good predictors for the TTEs found in this study. Tissue Cd concentrations were highest in the kidney and gut tissue, then liver, but lower in the gill, and carcass. Overall, the gut and carcass contributed ≥71% to total body burdens on a mass-weighted basis. This study presents evidence that Cd may be acquired by fish from natural diets at levels of environmental relevance for contaminated sites, and that the exposure route of the prey influences the TTE.     

Pre-clinical pharmacokinetics evaluation of an anticonvulsant candidate benzaldehyde semicarbazone free and included in β-cyclodextrin

The study aimed to investigate the pharmacokinetics and tissue distribution of the benzaldehyde semicarbazone (BS) a potential antiepileptic drug, administered as a free drug or complexed β-cyclodextrin (BS/β-CD). Free BS and BS/β-CD were administered to male Wistar rats as a 10 mg/kg intravenous bolus dose. For the oral route, 50 mg/kg and 100 mg/kg doses of the free drug and 50 mg/kg of the complex were administrated and plasma concentrations were determinated by a validated HPLC-UV method. Individual profiles were evaluated by non-compartmental and compartmental analysis using Excel^® and Scientist^®, respectively. Free BS plasma protein binding was 34 ± 5%. A one-compartmental model adequately described all the plasma profiles for both formulations. After intravenous (10 mg/kg) and oral (50 mg/kg) administration, the V_d (1.6 ± 0.5 and 2.2 ± 0.8 L/kg, respectively) and the Cl_tot (1.4 ± 0.5 and 1.8 ± 0.5 L/h kg, respectively) determinated for the BS/β-CD complex were higher than those obtained for the free drug, but the t_1/2 (0.8 ± 0.1 h) was similar (p < 0.05). The oral bioavailability of the BS/β-CD complex (～37%) was approximately 2-fold of the free BS (～20%). The higher drug brain penetration (2.8) after BS/β-CD dosing and the longer mean residence time in this organ, regardless of the administration route, reveals that the complex may be a potential drug carrier for the central nervous system delivery of BS.     

Xenopus laevis oocytes expressing human P-glycoprotein: Probing trans- and cis-inhibitory effects on [3H]vinblastine and [3H]digoxin efflux

P-glycoprotein (P-gp; MDR1) recognizes and actively transports many structurally diverse compounds (hydrophobic neutral and cationic). We studied MDR1-mediated drug transport using a high-throughput (96-well) oocyte expression system. MDR1-expressing oocytes contained sufficient ATP levels to conduct fundamental efflux studies; the optimal experimental temperature was 25 °C. [³H]Vinblastine efflux by MDR1-expressing oocytes was detectable and afforded a K_m of 145.5 ± 25.4 μM. [³H]Vinblastine (5.6 ± 0.3 μM) and [³H]digoxin (1.0 ± 0.1 μM) were individually injected into MDR1-expressing oocytes and their efflux monitored. Quinidine and verapamil, known MDR1 substrates/inhibitors, showed trans-inhibition on MDR1-mediated [³H]vinblastine and [³H]digoxin efflux. Conversely, doxorubicin demonstrated cis-inhibition without trans-inhibition on MDR1-mediated [³H]vinblastine efflux. The MDR1-expressing oocyte system offers researchers with an alternative in vitro method to screen compounds and may allow one to probe P-gp drug–drug and/or drug–inhibitor interactions.     

Toxic effects of juvenile sablefish,Anoplopoma fimbria by ammonia exposure at different water temperature

Juvenile sablefish, Anoplopoma fimbria (mean length 17.1 ± 2.4 cm, and mean weight 75.6 ± 5.7 g) were used to evaluate toxic effects on antioxidant systems, immune responses, and stress indicators by ammonia exposure (0, 0.25, 0.75, and 1.25 mg/L) at different water temperature (12 and 17 °C) in 1 and 2 months. In antioxidant responses, superoxide dismutase (SOD) and catalase (CAT) were significantly increased by ammonia exposure, whereas glutathione (GSH) was decreased. In immune responses, lysozyme and phagocytosis activity were significantly increased by ammonia exposure. In stress indicators, plasma glucose, heat shock protein 70 (HSP 70), and cortisol were significantly increased. At high water temperature (17 °C), alterations by ammonia exposure were more distinctly. The results of this study indicated that ammonia exposure can induce toxic effects in the sablefish, and high water temperature can affect the ammonia exposure toxicity.