Down-regulation of carboxylesterases 1 and 2 plays an important role in prodrug metabolism in immunological liver injury rats

Liver plays a central role in xenobiotics metabolism, thus affecting the in vivo disposition and therapeutic effects of drugs. Carboxylesterases (CESs), with the main isoforms CES1 and CES2, are important in the metabolism of ester-type prodrugs. However, influences of immunological liver injury on the activity of CES remain undefined. In the present study, we demonstrated treatment with lipopolysaccharide (LPS) suppressed the activities of CES1 and CES2. The decreased activities of CES1 and CES2 were preliminarily assessed by the hydrolysis assay for their common substrate p-nitrophenyl acetate (PNPA) with rat hepatic microsomal enzyme. Subsequently, RT-PCR results showed that the levels of CES1 mRNA and mRNA of CES2 (AB010635) and CES2 (AY034877) in the model group were significantly lower than those of the normal control group (P < 0.05). Western blot results showed that the expressions of CES1 and CES2 proteins were decreased (P < 0.05). To further clarify the effects of LPS on the metabolic activities of CESs, pharmacokinetic studies were performed in rats by utilizing imidapril and irinotecan (CPT-11) as the specific substrates for CES1 and CES2, respectively. After treatment with LPS, AUC_0 -∞ and C_max of imidaprilat were decreased from 2084.86 ± 340.66 ng · h^− 1 · mL^− 1 and 234.66 ± 68.85 ng · mL^− 1 to 983.87 ± 315.34 ng · h^− 1 · mL^− 1 and 113.1 ± 19.69 ng · mL^− 1 (P < 0.05), respectively. Moreover, AUC_0 -∞ and C_max of SN-38 were decreased from 8100 ± 918.6 ng · h^− 1 · mL^− 1 and 144.67 ± 20.28 ng · mL^− 1 to 3270 ± 500.5 ng · h^− 1 · mL^− 1 and 56.19 ± 10.38 ng · mL^− 1 (P < 0.05), respectively. In summary, immunological liver injury remarkably attenuated the expressions and metabolic activities of CES1 and CES2, which may be associated with the regulatory effects of cytokines under inflammation.     

Determination of lamivudine and zidovudine permeability using a different ex vivo method in Franz cells

IntroductionThe major processes that control the absorption of orally administered drugs are dissolution and gastrointestinal permeation. These processes depend on two main properties: solubility and permeability. Based on these characteristics, the Biopharmaceutical Classification System (BCS) was proposed as a tool to assist in biowaiver and bioavailability prediction of drugs.MethodsThe purpose of the present study was to evaluate the permeability of lamivudine (3TC) and zidovudine (AZT) using a different ex vivo method in Franz cells. A segment of jejunum was inserted in a Franz cells apparatus, in order to assess drug permeability in the apical–basolateral (A–B) and basolateral–apical (B–A) directions. Each drug was added to the donor chamber, collected from the acceptor chamber and analyzed by HPLC. Fluorescein (FLU) and metoprolol (METO) were used as low and high permeability markers, respectively.ResultsThe apparent permeability (P_app) results for the A–B direction were: P_{app FLU A–B} = 0.54 × 10^? 4 cm·s^? 1, P_{app METO A–B} = 7.99 × 10^? 4 cm·s^? 1, P_{app 3TC A–B} = 4.58 × 10^? 4 cm·s^? 1 and P_{app AZT A–B} = 5.34 × 10^? 4 cm·s^? 1. For the B–A direction, the P_app results were: P_{app FLU B–A} = 0.56 × 10^? 4 cm·s^? 1, P_{app METO B–A} = 0.25 × 10^? 4 cm·s^? 1, P_{app 3TC B–A} = 0.24 × 10^? 4 cm·s^? 1 and P_{app AZT B–A} = 0.19 × 10^? 4 cm·s^? 1.DiscussionFor the A–B direction, the P_app results of fluorescein and metoprolol show low and high permeability, respectively, indicating that the membranes were appropriate for permeability studies. For the A–B direction, the P_app results of 3TC and AZT suggest that these antiretroviral drugs have permeability values close to metoprolol. Nevertheless, for the B–A direction the P_app results do not suggest efflux mechanism for any of the drugs. Thereby, the different ex vivo methods using Franz cells can be successfully applied in drug permeability studies, in particular for drug biopharmaceutical classification.     

Intracellular disposition of arabinogalactan and asialofetuin in HepG2 cells

The binding and uptake of arabinogalactan and asialofetuin in HepG2 cells was kinetically characterized using ^I25I-labeled ligands. The number of binding sites (n) and the association constant (K) of arabinogalactan was 1.9 × 10⁵ ± 1.2 × 10⁵ sites/cell and 5.0 × 10⁶ ± 3.9 × 10⁶ M^? 1, respectively, whereas the n and K_a of asialofetuin was 2.7 × 10⁵ ± 1.1 × 10⁵ sites/cell and 1.1 × 10⁷ ± 0.7 × 10⁷ M^? 1, respectively. These results suggest that the binding capacity of HepG2 cells for arabinogalactan is lower than that for asialofetuin. Moreover, the amount of arabinogalactan uptake by HepG2 cells was lower than that of asialofetuin. Thus, asialofetuin was preferentially bound and internalized by hepatoma cells compared to arabinogalactan.     

The pharmacokinetics of orally administered S-carboxymethyl-l-cysteine in the dog,calf and sheep

The aim of this study was to investigate the feasibility of employing S-carboxymethyl-l-cysteine as a treatment of chronic obstructive pulmonary disease in dogs. To this end the pharmacokinetic parameters of orally administered S-carboxymethyl-l-cysteine were determined in the dog, cow and sheep. Six healthy beagle dogs, six endogenous Greek sheep and four Holstein Fresian calves were orally dosed with 10 mg/kg body weight of S-carboxymethyl-l-cysteine. No significant differences in T_max and T_1/2 were reported between the species. However, significantly higher AUC_(0–last), 21.56 ± 6.67 μg h ml^?1 and AUC_(0–∞), 21.63 ± 6.68 μg h ml^?1 were seen in the dogs compared to the sheep and calves. The calculated V_D was significantly higher in the sheep (10.4 ± 2.7 L kg^?1) and the calves (3.8 ± 0.7 L kg^?1) compared to the dogs (1.0 ± 0.6 L kg^?1). The rank order of increasing C_L was sheep (3.4 ± 2.7 L h^?1 kg^?1) > calves (2.7 ± 0.4 L h^?1 kg^?1) > dogs (0.5 ± 0.2 L h^?1 kg^?1). The result for the dogs was significantly lower that the calculated C_L for the sheep and calves.All these results indicate that the oral administration of S-carboxymethyl-l-cysteine may be useful during the therapeutic management of chronic obstructive pulmonary disease in dogs.     

High Shear Rheology and Anisotropy in Concentrated Solutions of Monoclonal Antibodies

The high shear rheology of three concentrated solutions of immunoglobulin G1 monoclonal antibodies (mAbl, mAb2, and mAb3), differing only in their complementarity determining regions, was characterized using rotary and capillary rheometry. The more viscous solutions (mAb1 and mAb3) showed non-Newtonian behavior at high shear rates exhibiting both shear thinning and appreciable normal stress differences (NSDs) in the shear rate range γ = 10 to 10⁴ s^? 1. The rheograms were retraced after γ is increased and decreased, suggesting reversible self-associations under shear. In contrast, mAb2 solutions showed Newtonian behavior up to γ = 6 × 10⁴ s^? 1. The critical shear stress τ_c, corresponding to the onset of the reduction in the viscosity η, is a measure of mAb equilibrium cluster strength and increased rapidly with concentration for the high viscosity mAb solutions above 100 mg/mL. In addition, decreasing the temperature from 20 °C to 5 °C increased η at low γ, but shear-thinning was enhanced and its onset occurred at a lower γ_c. Using an Arrhenius model η = A exp(E_a/kT), the activation energy for viscous flow E_a was found to decrease for mAb1 solutions as γ was increased from 10 to 10⁴ s^? 1, suggesting mAb cluster disruption or rearrangement under shear. In contrast, for mAb2, this E_a remained constant in the γ range. Finally, mAb1 and mAb3 solutions showed appreciable NSDs, with their N₁ > 0 scaling linearly with γ in the range 10³ to 10⁴ s^? 1, whereas their |N₂/N₁| was less than 0.25 in this region. These suggest anisotropy and deformation of their solution microstructure toward the extensional quadrant of the flow at high γ. In contrast, the NSDs for mAb2 were close to zero indicating that the solution microstructure under shear is practically isotropic. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2538–2549, 2013     

Contractile effects of endothelins on isolated ampullar segment of human oviduct in luteal phase of menstrual cycle

Endothelin-1 induces contractions of human oviduct ampullar segment in follicular phase of menstrual cycle, acting on ET_A receptors. The aim of our study was to investigate effects of endothelin-1, endothelin-2 and endothelin-3 on isolated ampullar segment of human oviducts, taken from the patients in luteal phase of menstrual cycle. Fallopian tubes were taken from 20 female patients (one tube from each patient) during abdominal hysterectomy with adnexectomy, due to extensive uterine fibroids. The oviduct ampulla was mounted in an organ bath longitudinally, and the tension of the isolated preparation was recorded with the isometric transducer. Endothelin-1 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC₅₀ = 6.80 ± 1.2 × 10^?10 M), and concentration-dependent inhibition of its rhythmic contractions (EC₅₀ = 7.86 ± 2.3 × 10^?10 M). Endothelin-2 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC₅₀ = 4.56 ± 0.3 × 10^?10 M), without affecting its rhythmic contractions. Endothelin-3 did not affect either tone or rhythmic contractions of the isolated preparations. Selective antagonist for ET_A receptor subtype, BQ 123, produced inhibition of endothelin-1 effects on both tone (pA₂ = 9.50) and spontaneous rhythmic contractions (pA₂ = 10.73), while selective antagonist for ET_B receptor subtype, BQ 788, produced only inhibition of endothelin-1 effects on tone (pA₂ = 9.61), while the effect of endothelin-1 on spontaneous rhythmic contractions remained unaffected. The results of our study suggest that in the luteal phase both ET_A and ET_B receptors regulate tone, and only ET_A receptors regulate rhythmic activity of human oviduct's ampullar segment.     

In vitro screening of reversible and time-dependent inhibition on CYP3A by TM208 and TM209 in rat liver microsomes

TM208 and TM209, dithiocarbamate derivatives with potential anti-cancer effects, were evaluated in reversible and time-dependent cytochrome P450 (CYP) 3A inhibition assays in rat liver microsomes using testosterone as probe substrate. Both compounds were found to be weak reversible inhibitors and moderate mechanism-based inhibitors of rat CYP3A. For reversible inhibition on rat CYP3A, the K_i values of competitive inhibition model were 12.10±1.75 and 13.94±1.31 μM, respectively. For time-dependent inhibition, the inactivation constants (K_l) were 31.93±12.64 and 32.91±15.58 μM, respectively, and the maximum inactivation rates (k_inact) were 0.03497±0.0069 and 0.07259±0.0172 min^?1 respectively. These findings would provide useful in vitro information for future in vivo DDI studies on TM208 or TM209.     

The effects of sulfur mustard exposure and freezing on transdermal penetration of tritiated water through ex vivo pig skin

The percutaneous absorption of tritiated water (³H₂O) through sulfur mustard (SM) exposed abdominal pig skin was measured using in vitro Franz-type static diffusion cells. The barrier function to water permeation following exposure to liquid SM for 8 min and excision 3 h later did not change significantly. A small, but statistically significant difference (P < 0.05) in steady state penetration (Jss), permeability coefficient (Kp) and lag time (t_L) of ³H₂O was observed between fresh skin and skin stored frozen (?20 °C) for up to two weeks. Steady-state penetration and Kp values were significantly higher (P < 0.05) in skin stored frozen compared with fresh skin. Fresh naïve skin had an average Kp of 1.65 × 10^?3 cm h^?1, whereas frozen naïve skin was 2.04 × 10^?3 cm h^?1. Fresh SM exposed skin had a mean Kp of 1.72 × 10^?3 cm h^?1, whereas frozen SM exposed skin was 2.31 × 10^?3 cm h^?1. Lag times were also shorter (P < 0.05) in skin that had been stored frozen. Frozen, SM-exposed porcine abdominal skin may be used for in vitro penetration studies, but effects of treatment and storage on the barrier layer should be taken into account.     

M1 and M3 muscarinic receptors are involved in the release of urinary bladder-derived relaxant factor

We have investigated the muscarinic receptor subtype(s) mediating the release of urinary bladder-derived relaxant factor that is demonstrated by a coaxial bioassay system. Acetylcholine-induced relaxation of a precontracted anococcygeus muscle mounted within the bladder was considered as an evidence for the release of this factor. M₁-muscarinic agonist McN-A-343 and the cholinesterase inhibitor physostigmine also elicited relaxation responses in the coaxial bioassay besides acetylcholine. Acetylcholine-induced relaxation was antagonized by the subtype-selective muscarinic antagonists (pK_B): M₃-antagonist darifenacin (9.36 ± 0.11), M₃/M₁-antagonist 4-DAMP (9.30 ± 0.11), M₁-antagonist telenzepine (8.56 ± 0.21), M₄-antagonist tropicamide (6.63 ± 0.17) and M₂-antagonist AF-DX 116 (6.01 ± 0.21). The pK_B values of these antagonists have suggested that stimulation of M₁- and M₃-muscarinic receptors in the bladder wall mediates the release of urinary bladder-derived relaxant factor. In addition, McN-A-343, by activating the facilitatory M₁ receptors and physostigmine by inhibiting the acetylcholinesterase may induce the release of this factor through endogenous acetylcholine in the coaxial bioassay system.     

Comprehensive Study of the Interaction Between a Potential Antiprion Cationic Porphyrin and Human Prion Protein at Different pH by Using Multiple Spectroscopic Methods

Cationic porphyrins are potential antiprion drugs; however, the action mechanisms remain poorly understood. Herein, the interaction between a cationic porphyrin and recombinant human prion protein (rPrP^C) was comprehensively studied by using surface plasmon resonance (SPR), fluorescence, resonance light scattering (RLS), and circular dichroism (CD) spectroscopy. The experimental results showed that the interaction between the cationic porphyrin and rPrP^C was pH dependent. The equilibrium association constants obtained from SPR spectroscopy were 4.12 × 10³ M^? 1 at pH 4.0, 1.74 × 10⁵ M^? 1 at pH 6.0, and 5.98 × 10⁵ M^? 1 at pH 7.0. The binding constants at 298 K obtained from the fluorescence quenching method were 7.286 × 10⁴ M^? 1 at pH 4.0 and 1.45 7 × 10⁵ M^? 1 at pH 6.0. The thermodynamic parameters such as enthalpy change, entropy change, and free energy change were calculated, and the results indicated hydrogen bonds and van der Waals interactions played a major role in the binding reaction. The RLS experiment was performed to study the influence of porphyrin on the rPrP^C aggregation at different pH values. The CD experiments were conducted to investigate the effects of porphyrin on the secondary structure and thermal stability of rPrP^C. Finally, the comparison of SPR measurement and fluorescence quenching measurement was discussed.     

Chemical composition,antiproliferative and antioxidant properties of lipid classes in ordinary and dark muscles from chub mackerel (Scomber japonicus)

This study investigated to compare lipid profiles in ordinary and dark muscles from chub mackerel and to examine antiproliferative and antioxidative properties of lipid classes. The average levels of neutral lipids (NL), glycolipids (GL), and phospholipids (PL) in ordinary muscle were 92.32 ± 0.19%, 5.10 ± 0.48%, and 2.58 ± 0.46%; in dark muscle were 96.88 ± 0.15%, 2.59 ± 0.36%, and 0.54 ± 0.29%, respectively. The fatty acid composition indicated that PL had a higher percentage of PUFA (especially 22:6n?3) with lower percentages of SFA and MUFA compared to NL and GL (p < 0.05). The main ion peaks of GL in ordinary and dark muscles showed that monocharged and bischarged molecular ion were presented at m/z 876.9 and 438.8, respectively. In MTT assay, inhibition of AGS and HT-29 cell proliferation was greatest with the 0.5 and 1.0 mg mL^?1 GL treatments. The GL of ordinary muscle with 0.05 mg mL^?1 concentrations markedly decreased the levels of reactive oxygen species (ROS) induced by H₂O₂ compared to the control (p < 0.05). From our results, GL might have antiproliferative and antioxidant properties based on protective effect against the production of intracellular ROS.     

On the Mechanism and Dynamics of Uptake and Permeation of Polyether-Copolyester Dendrimers Across an In Vitro Blood–Brain Barrier Model

Dendrimers have emerged as a promising drug delivery system due to their well defined size, tailorability, and multifunctional nature. However, their application in brain delivery is relatively a new area of research. The present study was aimed at evaluating the uptake and permeation of polyether-copolyester (PEPE) dendrimers across the blood–brain barrier model and exploring the underlying mechanisms. Saturation was observed in the uptake of rhodamine B labeled PEPE dendrimers by brain vascular endothelial (bEnd.3) cells at high concentrations. Clathrin and caveolin inhibitors produced partial inhibition of the dendrimer uptake, signifying contribution of both pathways in the uptake process. PEPE dendrimers were able to cross in vitro BBB model in high amounts with P_app of 19.7 ± 1.9 × 10^?6 cm/s and 38.6 ± 4.1 × 10^?6 cm/s for den-1-(G2)-400 and den-2-(G2)-400, respectively; and only 11–14% reduction in transendothelial electrical resistance during initial 4 h. The results of this study suggest that architecture of dendrimers plays a major role not only in influencing the extent and mechanism of uptake by bEnd.3 cells but also permeation across the BBB model. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3748–3760, 2009     

Binding of new aminopropan-2-ol compounds to bovine serum albumin, α1 acid glycoprotein and rat serum using equilibrium dialysis and LC/MS/MS

BackgroundThe binding of three new aminopropan-2-ol compounds briefly called 2F109, ANBL and TWo8 with potential cardiovascular activity to bovine serum albumin (BSA), α₁-acid glycoprotein (AGP) and to rat serum was studied. The chemical structures of these compounds are related to carvedilol. They possess an antiarrhythmic and hypotensive activity, and β- and α-adrenolytic mechanism of action. All analogues are weak bases with pKa values 8.65,8.85 and 8.26 for 2F109, ANBL and TWo8, respectively, and they possess lipophilic character (log P > 1.9584).MethodsThe extent of protein binding was determined using equilibrium dialysis in the range 2.5 – 900 μM, and 2.5 – 300 μM for binding of investigated compounds to BSA and AGP, respectively, and the quantitative measurement was done by LC/ESI-MS/MS assay.ResultsThe studied compounds bound to a single class of binding sites on BSA which was characterized by low affinity (K_d for 2F109 = 8.49 × 10^–5 M, for ANBL = 1.92 × 10^–5 M, and for TWo8 = 1.71 × 10^–5 M) and low capacity(n = 0.53 for 2F109,0.132 for ANBL and 0.13 for TWo8). The binding of 2F109, ANBL and TWo8 to AGP revealed one class of binding sites, with moderate affinity (K_d for 2F109 = 4.67 × 10^–6 M, for ANBL = 3.48 × 10^–5 M, and for TWo8 = 1.13 × 10^–5 M) and higher capacity (n = 2.21 for 2F109, 2.76 for ANBL and 2.28 for TWo8).ConclusionThe obtained data indicate that 2F109, ANBL and TWo8 moderately bind to BSA (34.2 – 71.2%) with low capacity (K_a = 6.21 × 10³–7.61 × 10³ M^–1)and strongly bind to AGP(71.5–85.5%)with moderate affinity (K_a = 7.94 × 104⁴.73 ×10⁵ M^–1).     

Using transdermal iontophoresis to increase granisetron delivery across skin in vitro and in vivo: Effect of experimental conditions and a comparison with other enhancement strategies

The objectives of the study were (i) to investigate the effect of experimental parameters on the iontophoretic transport of granisetron, (ii) to identify the relative contributions of electromigration (EM) and electroosmosis (EO), (iii) to determine the feasibility of delivering therapeutic amounts of drug for the treatment of chemotherapy-induced nausea and vomiting and (iv) to test the in vitro results in a simple animal model in vivo. Preliminary in vitro studies using aqueous granisetron formulations investigating the effect of drug concentration (5, 10, 20 and 40 mM) and current density (0.1, 0.2, 0.3 mA cm^?2) were performed using porcine ear skin. As expected, cumulative delivery in vitro at the 20 and 40 mM concentrations was significantly greater than that at 5 and 10 mM, which were not statistically different (p < 0.05). Increasing the applied current density from 0.1 to 0.3 mA cm^?2 resulted in a ～4.2-fold increase in iontophoretic flux. Furthermore, in the absence of Na⁺ in the formulation, no dependence of iontophoretic flux on drug concentration was reported (at a granisetron concentration of 40 mM, the transport rate was 2.93 ± 0.62 μg cm^?2 min^?1). Co-iontophoresis of acetaminophen was used to show that EM was the predominant transport mechanism accounting for 71–86% of total granisetron delivery. In vivo studies in Wistar rats (40 mM granisetron; application of 0.3 mA cm^?2 for 5 h with Ag/AgCl electrodes and salt bridges) showed an average iontophoretic input rate (k_input) of 0.83 ± 0.26 μg min^?1 and a maximum plasma concentration (C_max) of 0.092 ± 0.004 μg ml^?1. Based on these results and given the known pharmacokinetics, transdermal iontophoresis could achieve therapeutic drug levels for the management of chemotherapy-induced emesis using a reasonably sized (4–6 cm²) patch.     

Mitochondrial toxicity of selective COX-2 inhibitors via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria

Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC₅₀: 6.48 ± 2.74 μM) > celecoxib (IC₅₀: 14.92 ± 6.40 μM) > valdecoxib (IC₅₀: 161.4 ± 28.6 μM) > rofecoxib (IC₅₀: 238.4 ± 79.2 μM) > etoricoxib (IC₅₀: 405.1 ± 116.3 μM). Mechanism based inhibition of ATP synthesis (K_inact 0.078 min^− 1 and K_I 21.46 μM and K_inact/K_I ratio 0.0036 min^− 1 μM^− 1) was shown by lumiracoxib and data suggest that the opening of the MPT pore may not be the mechanism of toxicity. A positive correlation (with r² = 0.921) was observed between the potency of inhibition of ATP synthesis and the log P values. The in vitro metabolism of coxibs in rat liver mitochondria yielded for each drug substance a major single metabolite and identified a hydroxy metabolite with each of the coxibs and these metabolites did not alter the inhibition profile of ATP synthesis of the parent compound. The results suggest that coxibs themselves could be involved in the hepatotoxic action through inhibition of ATP synthesis.     

Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor

IntroductionP-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood–brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1–40) HiLyte Fluor? TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine.MethodsMale Wistar rats' everted intestines were divided into sacs, each sac was filled with 10 μM Ab-HiLyte with or without 50 μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594 nm at each appropriate time.ResultsThe Ab-HiLyte P-gp mediated efflux had a K = 1.00 × 10^? 2 min^? 1 and t_1/2 = 68.74 min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K = 1.65 × 10^? 3 min^? 1) and the half life is extremely increased (t_1/2 = 419 min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K = 1.81 × 10^? 2 min^? 1 and t_1/2 = 38.28 min).DiscussionThe results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound.     

In vivo and in vitro evaluation of the estrogenic properties of the 17β-(butylamino)-1,3,5(10)-estratrien-3-ol (buame) related to 17β-estradiol

BackgroundBuame [17β-(butylamino)-1,3,5(10)-estratrien-3-ol] possesses anticoagulant and antiplatelet activities that are potentially antithrombotic. Since its estrogenicity is unknown, it was evaluated by established methods.MethodsBuame (10, 100, 500, and 1,000 μg/kg), 17β-estradiol (E₂) (100 μg/kg), or propylene glycol (10 ml/kg) were subcutaneously (sc) administered for three days to immature Wistar female rats (21 days old). The relative uterotrophic effect to E₂ was 78 (E₂ = 100) with a relative uterotrophic potency of 1.48 (E₂ = 100). Adult ovariectomized Wistar rats received an sc injection at 8:00 h (reversed cycle) of: 7.5 μg of E₂ (≈ 30 μg/kg), buame (≈ 750, 1,500, 3,000 μg/kg), or corn oil (≈ 1.2 ml/kg). After 24 h, progesterone (4–5 mg/kg) was administered. Sexual receptivity was assessed 5 to 7 h later, and the lordosis quotient (LQ; number lordosis/number mounts × 100) was evaluated.ResultsBuame induced lordosis (LQmax 85 ± 9; ED50 952 ± 19 μg/kg) and E₂ LQmax 56 ± 8; ED50 10 ± 2 μg/kg; the relative LQpotency was 0.51 (E₂ = 100). Buame competed with [³H]E₂ for the estrogen receptor (Buame RBA = 0.15 and Ki = 5.9 × 10^?7 M; E₂ RBA = 100; Ki = 6.6 × 10^?9 M). Buame increased MCF-7 cells proliferation, from 10^?11 to 10^?9 M, its proliferative effect was 1.73–1.79 (E₂ = 3.0–3.9); its relative proliferative effect to E₂ was 33–40% (E₂ = 100%) and relative potency 10.4–10.7 (E₂ = 100). Tamoxifen and fulvestrant (ICI 182,780) inhibited buame's proliferation indicating mediation through estrogen receptors in this response.ConclusionBuame is therefore an estrogen partial agonist with a weak estrogenic activity.     

Behavioral sequelae following acute diisopropylfluorophosphate intoxication in rats: Comparative effects of atropine and cannabinomimetics

The comparative effects of atropine and the indirect cannabinomimetics URB597 (a fatty acid amide hydrolase inhibitor) and URB602 (a monoacylglycerol lipase inhibitor) on functional and neurobehavioral endpoints following acute diisopropylfluorophosphate intoxication were studied. Male Sprague–Dawley rats were treated with vehicle or DFP (2.5 mg/kg, sc), immediately post-treated with either vehicle, atropine (16 mg/kg), URB597 (3 mg/kg), URB602 (10 mg/kg) or a combination of URB597 and URB602, and functional signs of toxicity as well as nocturnal motor activity were measured daily for seven consecutive days. Performance in the elevated plus maze (for anxiety-like behavior) and the forced swimming test (for depression-like behavior) was measured at days 6–8 and 27–29 after dosing. Twenty-four hours after dosing, DFP markedly reduced cholinesterase activity in selected brain regions and peripheral tissues (diaphragm and plasma). Substantial recovery of cholinesterase activity was noted at both 8 and 29 days after dosing but significant inhibition was still noted in some brain regions at the latest time-point. DFP elicited body weight reductions and typical signs of cholinergic toxicity, and reduced nocturnal ambulation and rearing. Atropine and the cannabinomimetics (alone and in combination) partially attenuated DFP-induced functional signs of toxicity. None of the post-treatments reversed the DFP-induced reduction in ambulation or rearing, however. No significant treatment-related effects on elevated plus maze performance were noted. DFP-treated rats exhibited decreased swimming and increased immobility in the forced swimming test at both time-points. None of the post-treatments had any effect on DFP-induced changes in immobility or swimming at day 8. At day 29, atropine and the combination of URB597/URB602 significantly blocked DFP-induced changes in immobility, while URB597 and the combination reversed DFP-induced changes in swimming. The results suggest that early blockade of muscarinic receptors and enhancement of eCB signaling can attenuate both acute and delayed effects elicited by DFP.     

Preparation of estradiol chitosan nanoparticles for improving nasal absorption and brain targeting

The estradiol(E₂)-loaded chitosan nanoparticles (CS-NPs) were prepared by ionic gelation of chitosan with tripolyphosphate anions (TPP). The CS-NPs had a mean size of (269.3 ± 31.6) nm, a zeta potential of +25.4 mV, and loading capacity of E₂ CS-NPs suspension was 1.9 mg ml⁻¹, entrapment efficiency was 64.7% on average. Subsequently, this paper investigated the levels of E₂ in blood and the cerebrospinal fluid (CSF) in rats following intranasal administration of E₂ CS-NPs. E₂-loaded CS-NPs were administered to male Wister rats either intranasally or intravenously at the dose of 0.48 mg kg⁻¹. The plasma levels achieved following intranasal administration (32.7 ± 10.1 ng ml⁻¹; t_max 28 ± 4.5 min) were significantly lower than those after intravenous administration (151.4 ± 28.2 ng ml⁻¹), while CSF concentrations achieved after intranasal administration (76.4 ± 14.0 ng ml⁻¹; t_max 28 ± 17.9 min) were significantly higher than those after intravenous administration (29.5 ± 7.4 ng ml⁻¹ t_max 60 min). The drug targeting index (DTI) of nasal route was 3.2, percent of drug targeting (DTP%) was 68.4%. These results showed that the E₂ must be directly transported from the nasal cavity into the CSF in rats. Finally, compared with E₂ inclusion complex, CS-NPs improved significantly E₂ being transported into central nervous system (CNS).     

Effect of melamine on potassium currents in rat hippocampal CA1 neurons

As an industrially synthesized chemical, melamine has been applied in a wide range of areas. However, many questions on the adverse effect and toxicity of melamine have been emerged, recently. In this investigation, the cytotoxicity of melamine on PC12 cells was evaluated. Furthermore, the effect of melamine on the transient outward potassium current (I_A) and the delayed rectifier potassium current (I_K) in hippocampal CA1 pyramidal neurons of rat was studied using whole-cell patch-clamp technique. The results showed that melamine-induced cell death in a concentration and time-dependent manner, and produced a concentration-dependent inhibition in amplitudes of I_A and I_K at any concentrations (5 × 10⁻⁴, 5 × 10⁻⁵, and 5 × 10⁻⁶ g/ml). Moreover, at higher concentration (5 × 10⁻⁴ g/ml), melamine had observable effects of the steady-state inactivation of I_A, that is melamine shifted inactivation curve of I_A towards hyperpolarization. The spontaneous firing frequency was increased as well. These results suggest that the regulation of I_A and I_K induced by melamine would make neurons display aberrant firing properties and abnormal neuronal discharge, which could be a possible underlying mechanism for the melamine-induced neurotoxicity.