Formulation,optimization and evaluation of transferosomal gel for transdermal insulin delivery

The present study deals with the development of transferosomal gel containing insulin by reverse phase evaporation method for painless insulin delivery for use in the treatment of insulin dependent diabetes mellitus. The effect of independent process variables like ratio of lipids (soya lecithin:cholesterol), ratio of lipids and surfactants, and ratio of surfactants (Tween 80:sodium deoxycholate) on the in vitro permeation flux (μg/cm²/h) of formulated transferosomal gels containing insulin through porcine ear skin was optimized using 2³ factorial design. The optimal permeation flux was achieved as 13.50 ± 0.22 μg/cm²/h with drug entrapment efficiency of 56.55 ± 0.37% and average vesicle diameter range, 625–815 nm. The in vitro insulin permeation through porcine ear skin from these transferosomal gel followed zero-order kinetics (R² = 0.9232–0.9989) over a period of 24 h with case-II transport mechanism. The in vitro skin permeation of insulin from optimized transferosomal gel by iontophoretic influence (with 0.5 mA/cm² current supply) also provided further enhancement of permeation flux to 17.60 ± 0.03 μg/cm²/h. The in vivo study of optimized transferosomal gel in alloxan-induced diabetic rat has demonstrated prolonged hypoglycemic effect in diabetic rats over 24 h after transdermal administration.     

Efficient Ibuprofen Delivery from Anhydrous Semisolid Formulation Based on a Novel Cross-linked Silicone Polymer Network: An In Vitro and In Vivo Study

The use of silicone as a primary polymer in topical semisolid pharmaceutical formulations is infrequent. Recent development of novel silicone materials provides an opportunity to investigate their drug delivery efficiencies. In this study, an anhydrous semisolid formulation was prepared using a novel cross-linked silicone polymer network swollen in isododecane. Similar formulations were prepared using petrolatum, an acrylic, or a cellulose polymer. All formulations contained 5% ibuprofen (IBP). In vitro permeability was evaluated for all formulations and a commercial product using human cadaver epidermis. The silicone formulation delivered IBP more efficiently than all other formulations in terms of flux, cumulative amount, and percent drug release. The silicone formulation showed the maximum flux of 85.9 μg.cm⁻².h⁻¹ and a cumulative IBP release of 261.6 μg in 8 h, whereas the benchmark showed 20.1 μg.cm⁻².h⁻¹ and 30.9 μg, respectively. An in vivo study conducted on rats showed calculated blood AUCs of 59.2 and 17.6 μg.h/g (p < 0.003) for the silicone formulation and the benchmark, respectively. The IBP in excised rat skin was 264 ± 59 μg/g for the silicone formulation and 102 ± 5 μg/g for the benchmark. The results obtained from the in vitro and in vivo studies demonstrate efficient topical IBP delivery by the silicone formulation.     

Iontophoretic transport kinetics of ketorolac in vitro and in vivo: Demonstrating local enhanced topical drug delivery to muscle

The objective of the study was to investigate the iontophoretic delivery kinetics of ketorolac (KT), a highly potent NSAID and peripherally-acting analgesic that is currently indicated to treat moderate to severe acute pain. It was envisaged that, depending on the amounts delivered, transdermal iontophoretic administration might have two distinct therapeutic applications: (i) more effective and faster local therapy with shorter onset times (e.g. to treat trauma-related pain/inflammation in muscle) or (ii) a non-parenteral, gastrointestinal tract sparing approach for systemic pain relief. The first part of the study investigated the effect of experimental conditions on KT iontophoresis using porcine and human skin in vitro. These results demonstrated that KT electrotransport was linearly dependent on current density – from 0.1875 to 0.5 mA/cm² – (r² > 0.99) and drug concentration – from 5 to 20 mg/ml (r² > 0.99). Iontophoretic permeation of KT from a 2% hydroxymethyl cellulose gel was comparable to that from an aqueous solution with equivalent drug loading (584.59 ± 114.67 and 462.05 ± 66.56 μg/cm², respectively). Cumulative permeation (462.05 ± 66.56 and 416.28 ± 95.71 μg/cm²) and steady state flux (106.72 ± 11.70 and 94.28 ± 15.47 μg/cm² h), across porcine and human skin, were statistically equivalent confirming the validity of the model. Based on the results in vitro, it was decided to focus on topical rather than systemic applications of KT iontophoresis in vivo. Subsequent experiments, in male Wistar rats, investigated the local enhancement of KT delivery to muscle by iontophoresis. Drug biodistribution was assessed in skin, in the biceps femoris muscle beneath the site of iontophoresis (‘treated muscle’; TM), in the contralateral muscle (‘non-treated muscle’; NTM) and in plasma (P). Passive topical delivery and oral administration served as negative and positive controls, respectively. Iontophoretic administration for 30 min was superior to passive topical delivery for 1 h and resulted in statistically significant increases in KT levels in the skin (91.04 ± 15.48 vs. 20.16 ± 8.58 μg/cm²), in the biceps femoris at the treatment site (TM; 6.74 ± 3.80 vs. <LOQ), in the contralateral site (NTM; 1.26 ± 0.54 vs. <LOQ) and in plasma (P; 8.58 ± 2.37 μg/ml vs. <LOD). In addition to increasing bioavailability, iontophoretic administration of KT showed clear selectivity for local delivery to the biceps femoris at the treatment site – the TM:NTM ratio was 5.26 ± 1.45, and the TM:P and NTM:P ratios were 0.75 ± 0.32 and 0.14 ± 0.04, respectively. Furthermore, the post-iontophoretic concentration of KT in the ‘treated’ biceps femoris muscle and the muscle:plasma ratio were also superior to those following oral administration of a 4 mg/kg dose (6.74 ± 3.80 vs. 0.62 ± 0.14 μg/g and 0.75 ± 0.32 vs. 0.14 ± 0.03, respectively). In conclusion, the results demonstrate that iontophoresis of ketorolac enables local enhanced topical delivery to subjacent muscle; this may have clinical application in the treatment of localised inflammation and pain.     

Human skin penetration of cobalt nanoparticles through intact and damaged skin

Cobalt nanoparticles (CoNPs) are produced for several industrial and biomedical applications but there is a lack of data on human cutaneous absorption. Cobalt is also a skin sensitizer that can cause allergic contact dermatitis. Co applied as NPs, due to their small size and high surface, can penetrate into the skin in higher amount that bulk material. The aim of this study was to evaluate the absorption of Co applied as NPs in both intact and damaged skin. Experiments were performed using Franz cells and 1.0 mg cm^?2 of CoNPs was applied as donor phase for 24 h. Mean Co content of 8.5 ± 1.2 ng cm^?2 and 1.87 ± 0.86 μg cm^?2 were found in the receiving solutions of Franz cells when the CoNPs suspension was applied on intact skin and on damaged skin, respectively. Twenty-four hours Co flux permeation was 76 ± 49 ng cm^?2 h^?1 in damaged skin with a lag time of 2.8 ± 2.1 h. This study suggests that Co applied as NPs is able to penetrate the human skin in an in vitro diffusion cell system.     

Bilayered Nail Lacquer of Terbinafine Hydrochloride for Treatment of Onychomycosis

The present study aimed to develop bilayered nail lacquer of terbinafine hydrochloride (TH) for treatment of onychomycosis. The composite nail lacquer formed an underlying drug-loaded hydrophilic layer and overlying hydrophobic vinyl layer. The hydrophilic lacquer made of hydroxylpropyl methylcellulose E-15 contained polyethylene glycol 400 (PEG 400) as a drug permeation enhancer. The vinyl lacquer was composed of poly (4-vinyl phenol) as a water- resistant film former. In vitro permeation studies in Franz diffusion cells indicated that the amount of TH permeated across the human cadaver nail in 6 days was 0.32 ± 0.14, 1.12 ± 0.42, and 1.42 ± 0.53 μg/cm² from control (hydrophilic lacquer devoid of PEG 400), monolayer (hydrophilic lacquer alone), and bilayered nail lacquers, respectively. A higher nail drug load was seen in vitro with the bilayered lacquer (0.59 ± 0.13 μg/mg) as compared to monolayer (0.36 ± 0.09 μg/ mg) and control (0.28 ± 0.07 μg/mg) lacquers. The drug loss despite multiple washing was significantly low (p < 0.001) for the bilayered lacquer owing to the protective vinyl coating. Clinical studies demonstrated the efficacy of bilayered lacquer to achieve better drug load in the nail plate (1.27 ± 0.184 μg/mg) compared to monolayer (0.67 ± 0.18 μg/mg) and control (0.21 ± 0.04 μg/mg) lacquers.     

Toxicological implications of the delivery of fentanyl from gel extracted from a commercial transdermal reservoir patch

Fentanyl in a rate controlling membrane (RCM) transdermal patch form has been available since the early 1990s for outpatient management of chronic pain. Fatalities associated with misuse or overuse of fentanyl patches have been reported. Concerns have also been raised about the possibility that defects in such patches may result in leaking of the reservoir of the patch onto patients’ skin and consequent overdose. In order to investigate the possibility of fentanyl toxicity arising from leaking of patches, the permeation of fentanyl from the reservoir gel of a commercially available fentanyl transdermal patch was examined in vitro. Finite doses of the formulation were applied to human skin and permeation was monitored, at 32 °C under non-occluded conditions, for 48 h. Similar levels of skin permeation of fentanyl from the 1% gel formulation were obtained for the two skin donor samples tested. After 48 h, the dose of fentanyl that had permeated was 7.4 (±3.6)% and 7.7 (±1.9)% of the respective total amounts applied. At the end of the experiment, most of the drug was found in the residual formulation at the skin surface (i.e. 63–66%). For both the skin samples, a relatively small amount of the fentanyl applied (2–3%) was present in the skin at 48 h after application. The maximum flux from the data generated was between 6 and 24 h over which time frame it was 0.3 μg/cm²/h. Assuming spreading of leaked gel over an area of 100 cm², this would result in a plasma level of 0.6 ng/mL. The anticipated plasma levels from a 100 μg/h patch are known to be approximately 2.5 ng/ml. Thus, the maximum increase in the plasma levels from a patch which leaks gel is calculated to be, at most, about 25%.     

Nicotine withdrawal and craving in adolescents: Effects of sex and hormonal contraceptive use

While sex differences in the nicotine withdrawal (NW) symptoms and craving (NC) have been extensively described in adult cigarette smokers, few studies have investigated these phenomena in adolescents. We investigated the effect of gender and hormonal contraception (HC) on NW and NC during the first 14 days of cessation in adolescent smokers using data from a randomized, placebo-controlled, double-blind trial of the transdermal nicotine replacement therapy for smoking cessation.Analyses showed similar levels of NW severity in males and females, regardless of HC use. However, significantly higher NC was observed in females compared to males, (2.22 ± 0.12 vs. 1.65 ± 1.14; p = 0.003). Further, females not using HC reported the highest level of NC (2.38 ± 0.16) followed by females using HC (2.08 ± 0.25) and males (1.71 ± 0.16; p = 0.007).The current findings suggest that adolescent females experience similar NW severity to males, but have stronger NC. Further, the use of hormonal contraceptives may impact the severity of craving. Addressing these different symptoms in adolescents may be useful in increasing smoking cessation rates in this special population of smokers.     

The chemotherapeutic potential of doxorubicin-loaded PEG-b-PLGA nanopolymersomes in mouse breast cancer model

Vesicles of mPEG-PLGA block copolymer were developed to deliver a therapeutic quantity of doxorubicin (DOX) for breast cancer treatment. The DOX-loaded nanoparticles (NPs) were prepared by the pH-gradient method and then evaluated in terms of morphology, size, DOX encapsulation efficiency and in vitro drug release mechanism.The PEG-PLGA nanopolymersomes were 134 ± 1.2 nm spherical NPs with a narrow size distribution (PDI = 0.121). DOX was entrapped in mPEG-PLGA nanopolymersomes with an encapsulation efficiency and a loading content of 91.25 ± 4.27% and 7.3 ± 0.34%, respectively. The DOX-loaded nanopolymersomes were found to be stable, demonstrating no significant change in particle size and encapsulation efficiency (EE%) during the 6-month storage period of the lyophilized powder at 4 °C. The nanopolymersomes sustained the release of DOX. In cytotoxicity studies of 4T1 cell line samples, free DOX showed a higher cytotoxicity (IC50 = 1.76 μg/mL) than did DOX-loaded nanopolymersomes (15.82 μg/mL) in vitro. In order to evaluate the antitumor efficacy and biodistribution of DOX-loaded nanopolymersomes, murine breast tumors were established on the BALB/c mice, and in vivo studies were performed. The obtained results demonstrated that the prepared drug delivery system was highly effective against a murine breast cancer tumor model and successfully accumulated in the tumor site through an enhanced permeation and retention mechanism.In vivo studies also proved that DOX-loaded nanopolymersomes are stable in blood circulation and could be considered a promising and effective DOX delivery system for breast cancer treatment.     

Folate-mediated poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) nanoparticles for targeting drug delivery

A novel targeting drug delivery system (TDDS) has been developed. Such a TDDS was prepared by W₁/O/W₂ solvent extraction/evaporation method, adopting poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] as the drug carrier, folic acid (FA) as the targeting ligand, and doxorubicin (DOX) as the model anticancer drug. The average size, drug loading capacity and encapsulation efficiency of the prepared DOX-loaded, folate-mediated P(HB-HO) nanoparticles (DOX/FA–PEG–P(HB-HO) NPs) were found to be around 240 nm, 29.6% and 83.5%. The in vitro release profile displayed that nearly 50% DOX was released in the first 5 days. The intracellular uptake tests of the nanoparticles (NPs) in vitro showed that the DOX/FA–PEG–P(HB-HO) NPs were more efficiently taken up by HeLa cells compared to non-folate-mediated P(HB-HO) NPs. In addition, DOX/FA–PEG–P(HB-HO) NPs (IC₅₀ = 0.87 μM) showed greater cytotoxicity to HeLa cells than other treated groups. In vivo anti-tumor activity of the DOX/FA–PEG–P(HB-HO) NPs showed a much better therapeutic efficacy in inhibiting tumor growth, and the final mean tumor volume was 178.91 ± 17.43 mm³, significantly smaller than normal saline control group (542.58 ± 45.19 mm³). All these results have illustrated that our techniques for the preparing of DOX/FA–PEG–P(HB-HO) NPs developed in present work are feasible and these NPs are effective in selective delivery of anticancer drug to the folate receptor-overexpressed cancer cells. The new TDDS may be a competent candidate in application in targeting treatment of cancers.     

Clinical Pharmacokinetics of Paclitaxel Liposome with a New Route of Administration in Human Based on the Analysis with Ultra Performance Liquid Chromatography

We investigated the clinical pharmacokinetics of paclitaxel liposome with a new route of administration, which was intrapleural infusion, in nine advanced nonsmall-cell lung cancer (NSCLC) patients with malignant pleural effusions after a single administration. Paclitaxel concentrations were measured in pleural fluid and plasma using a simple and rapid ultra performance liquid chromatography (UPLC) method following intra-and inter-day validations. In subjects, AUC_0–96h values in pleural fluid and plasma were 17831 ± 6439 μgh/mL and 778 ± 328 μgh/mL, respectively, and T_max values were initial time and 6.67 h after administration and the corresponding C_max values were 558 ± 44 μg/mL and 12.89 ± 6.86 μg/mL, respectively. The T_{1/2 IP}, CL_IP and Vd_IP values in pleural fluid were 76 ± 48 h, 0.005 ± 0.002 L/hm² and 0.53 ± 0.23 L/m², respectively. The T_1/2,_pla, CL_pla, and Vd_pla values in plasma were 68.34 ± 56.74 h, 0.184 ± 0.080 L/hm², and 17.53 ± 16.57 L/m², respectively. However, some paclitaxel concentrations from several patients in plasma could not be detected at some designed time-points. Our results might offer new opportunities to design and determine individually appropriate therapeutic dosage regimens based on a pharmacokinetic profile.     

Genotoxicity and antileishmanial activity evaluation of Physalis angulata concentrated ethanolic extract

Antileishmanial in vitro tests, as well as Ames and micronucleus assays were performed with a concentrated ethanolic extract of Physalis angulata (EEPA)ResultsEEPA did not present mutagenic effect in Salmonella typhimurium strains at concentration reaching 3000 μg/plate and did not induce mutagenic effects after two oral administrations with a 24 h interval at a dose level of 2000 mg/kg. EEPA presented antileishmanial activity and presented an IC₅₀ value of 5.35 ± 2.50 μg/mL and 4.50 ± 1.17 μg/mL against Leishmania amazonensis and Leishmania braziliensis promastigotes, respectively. In the cytotoxicity test against macrophages, the EEPA had a LC₅₀ of 6.14 ± 0.59 μg/mL. Importantly, the IC₅₀ against L. amazonensis intracellular amastigotes was 1.23 ± 0.11 μg/mL.ConclusionEEPA extract is non-mutagenic and presented a promising pharmacological effect against Leishmania parasites.     

Penetration of doripenem into prostatic tissue following intravenous administration in prostatectomy patients

This study examined the prostatic penetration of doripenem in prostatectomy patients. Doripenem 500 mg was administered by a 0.5-h infusion and venous blood and prostatic tissue samples were obtained up to 5 h afterwards. Drug concentrations in plasma and prostatic tissue were measured chromatographically. The observed maximum concentration (C_max) (mean ± standard deviation; n = 9) was 27.5 ± 5.1 μg/mL in plasma and 5.09 ± 1.94 μg/g in prostate tissue and the prostate/plasma C_max ratio was 0.189 ± 0.078. The area under the drug concentration–time curve (AUC) was 49.7 ± 6.9 μg h/mL in plasma and 3.93 ± 1.89 μg h/g in prostate tissue and the prostate/plasma AUC ratio was 0.081 ± 0.047. Based on a three-compartment pharmacokinetic analysis, average drug exposure times above 0.25 μg/mL (the minimum inhibitory concentration for isolates of common pathogens) in the prostate were 23.2% for 500 mg once daily, 46.2% for 500 mg twice daily and 69.9% for 500 mg three times daily. The 500-mg regimens all achieved the drug exposure time target (bacteriostatic 20% or bactericidal 40%) in the prostate, despite the relatively low penetrability of doripenem.     

Impact of iron oxide nanoparticles on brain,heart, lung,liver and kidneys mitochondrial respiratory chain complexes activities and coupling

The present study evaluates the effects of iron oxide nanoparticles (ION) on mitochondrial respiratory chain complexes activities in five organs characterized by different oxidative capacities and strongly involved in body detoxification. Isolated mitochondria were extracted from brain, heart, lung, liver and kidneys in twelve Wistar rats (8 weeks) using differential centrifugations. Maximal oxidative capacities (Vmax), mitochondrial respiratory chain complexes activity using succinate (Vsucc, complexes II, III, and IV activities) or N, N, N′, N′-tetramethyl-p-phenylenediaminedihydrochloride (tmpd)/ascorbate (Vtmpd, complex IV activity) and, mitochondrial coupling (Vmax/Vo) were determined in controls and after exposure to 100, 200, 300 and 500 μg/ml Fe₃O₄. Data showed that baseline maximal oxidative capacities were 26.3 ± 4.7, 48.9 ± 4.6, 11.3 ± 1.3, 27.0 ± 2.5 and 13.4 ± 1.7 μmol O₂/min/g protein in brain, heart, lung, liver, and kidneys mitochondria, respectively. Complexes II, III, and IV activities also significantly differed between the five organs. Interestingly, as compared to baseline values and in all tissues examined, exposure to ION did not alter mitochondrial respiratory chain complexes activities whatever the nanoparticles (NPs) concentration used. Thus, ION did not show any toxicity on mitochondrial coupling and respiratory chain complexes I, II, III, and IV activities in these five major organs.     

Continuous versus intermittent infusion of cefepime in neurosurgical patients with post-operative intracranial infections

Cefepime is administered as an intermittent infusion (II); however, continuous infusion (CI) may be advantageous because β-lactam antibiotics exhibit time-dependent antibacterial activity. This retrospective, non-randomised, comparative study included 68 neurosurgical patients with post-operative intracranial infections treated with 4 g/day cefepime over 24 h as a CI (n = 34) or 2 g every 12 h as II (n = 34). CI controlled the intracranial infection more rapidly and effectively than II (6.6 ± 1.9 days vs. 7.8 ± 2.6 days; P = 0.036). By considering the minimum inhibitory concentrations (MICs) to be 4 μg/mL and 8 μg/mL, the percentage of time when the cefepime plasma or CSF concentrations were higher than the MIC (%T_>MIC) was calculated for each patient. For plasma cefepime concentrations, the %T_>MIC in the CI group was higher than in the II group (for MICs of 8 μg/mL, 100% vs. 75%, respectively). The mean calculated area under the curve (AUC) in the CI group was similar to the II group (1197.99 ± 72.15 μg h/mL vs. 890.84 ± 140.78 μg h/mL; P = 0.655). For CSF cefepime concentrations, the %T_>MIC in the CI group was higher than in the II group (for MICs of 4 μg/mL and 8 μg/mL, 83.3% and 75% vs. 25% and 0%, respectively). The mean calculated AUC for the CI group was higher than the II group (220.56 ± 13.59 μg h/mL vs. 86.34 ± 5.69 μg h/mL; P = 0.003). Therefore, CI of cefepime significantly enhanced the antibacterial effect and reduced the treatment duration in neurosurgical patients with post-operative intracranial infections.     

Delivery of Nerve Growth Factor to Brain Via Intranasal Administration and Enhancement of Brain Uptake

The main objective of the study was to investigate the efficacy of chitosan to facilitate brain bioavailability of intranasally administered nerve growth factor (NGF). In vitro permeability studies and electrical resistance studies were carried out across the bovine olfactory epithelium using Franz diffusion cells. The bioavailability of intranasally administered NGF in rat hippocampus was determined by carrying out brain microdialysis in Sprague–Dawley rats. The in vitro permeation flux across the olfactory epithelium of NGF solution without chitosan (control) was found to be 0.37 ± 0.06 ng/cm²/h. In presence of increasing concentration of chitosan (0.1%, 0.25%, and 0.5%, w/v) the permeation flux of NGF was found to be 2.01 ± 0.12, 3.88 ± 0.19, and 4.12 ± 0.21 ng/cm²/h respectively. Trans-olfactory epithelial electrical resistance decreased ～34.50 ± 4.06% in presence of 0.25% (w/v) chitosan. The C_max in rats administered with 0.25% (w/v) chitosan and NGF was 1008.62 ± 130.02 pg/mL, which was significantly higher than that for rats administered with NGF only 97.38 ± 10.66 pg/mL. There was ～14-fold increase in the bioavailability of intranasally administered NGF with chitosan than without chitosan. Chitosan can enhance the brain bioavailability of intranasally administered NGF. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3640–3646, 2009     

Nanosized ethosomes bearing ketoprofen for improved transdermal delivery

The potential of ethosomes for delivering ketoprofen via skin was evaluated. The ethosomes were prepared, optimized and characterized. Vesicular shape, size and entrapment efficiency were determined by transmission electron microscopy, dynamic light scattering and minicolumn centrifugation technique, respectively. Vesicle sizes varied from 120.3±6.1 to 410.2±21.8 nm depending on the concentrations of soya phosphatidyl choline (SPC) and ethanol. Entrapment efficiency increased with concentrations of SPC and ethanol. The formulations exhibited entrapment efficiencies of 42–78%. In vitro release through cellophane membrane showed sustained release of drug from ethosomal formulations in contrast to hydroalcoholic drug solution (HA), which released most of the drug within 2–3 h. In vitro drug permeation across human skin revealed improved drug permeation and higher transdermal flux with ethosomal formulations compared to hydroethanolic drug solution. Kinetics of in vitro skin permeation showed zero order drug release from formulations. Based on in vitro transdermal flux, the estimated steady state in vivo plasma concentration from ethosomes attained therapeutic drug levels whereas hydroalcoholic drug solution exhibited sub therapeutic drug concentration with a patch size of 50 cm². Skin permeation of ethosomal formulations assessed by confocal microscopy revealed enhanced permeation of Rhodamine 123 loaded formulation in comparison to the hydroalcoholic solution.     

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.     

Plasma protein binding of tetrodotoxin in the marine puffer fish Takifugu rubripes

To elucidate the involvement of plasma protein binding in the disposition of tetrodotoxin (TTX) in puffer fish, we used equilibrium dialysis to measure protein binding of TTX in the plasma of the marine puffer fish Takifugu rubripes and the non-toxic greenling Hexagrammos otakii, and in solutions of bovine serum albumin (BSA) and bovine alpha-1-acid glycoprotein (AGP). TTX (100–1000 μg/mL) bound to protein in T. rubripes plasma with low affinity in a non-saturable manner. The amount of bound TTX increased linearly with the TTX concentration, reaching 3.92 ± 0.42 μg TTX/mg protein at 1000 μg TTX/mL. Approximately 80% of the TTX in the plasma of T. rubripes was unbound in the concentration range of TTX examined, indicating that TTX exists predominantly in the unbound form in the circulating blood of T. rubripes at a wide range of TTX concentrations. TTX also bound non-specifically to H. otakii plasma proteins, BSA, and bovine AGP. The amount of the bound TTX in the plasma of H. otakii and BSA, respectively, was 1.86 ± 0.36 and 4.65 ± 0.70 μg TTX/mg protein at 1000 μg TTX/mL, and that in the bovine AGP was 8.78 ± 0.25 μg TTX/mg protein at 200 μg TTX/mL.     

Influence of MRP1 G1666A and GSTP1 Ile105Val genetic variants on the urinary and blood arsenic levels of Turkish smelter workers

To understand the cellular mechanisms responsible for arsenic metabolism and transport pathways plays a fundamental role in order to prevent the arsenic-induced toxicity. The effect of MRP1 G1666A and GSTP1 Ile105Val polymorphisms on blood and urinary arsenic levels were determined in 95 Turkish smelter workers. Blood and urinary arsenic concentrations were measured by GF-AAS with Zeeman correction and gene polymorphisms were investigated by PCR-RFLP method. The mean blood and urinary arsenic levels were 21.60 ± 12.28 μg/L and 5.58 ± 4.37 μg/L, respectively. A significant association between MRP1 1666A allele and urinary arsenic levels was found (p = 0.001). GSTP1 Ile105Val polymorphism was detected not to be associated with either blood or urinary arsenic levels (p = 0.384, p = 0.440, respectively). Significant association was also detected between MRP1A^-/GSTP1Val⁻ genotypes and urinary arsenic levels (p = 0.001). This study suggested that MRP1 G1666A alone and, also, combined with GSTP1 Ile105Val were associated with inter-individual variations in urinary arsenic levels, but not with blood arsenic levels.     

Comparative in vitro and ex-vivo myelotoxicity of aflatoxins B1 and M1 on haematopoietic progenitors (BFU-E,CFU-E,and CFU-GM): Species-related susceptibility

Haemato- and myelotoxicity are adverse effects caused by mycotoxins. Due to the relevance of aflatoxins to human health, the present study, employing CFU-GM-, BFU-E- and CFU-E-clonogenic assays, aimed at (i) comparing, in vitro, the sensitivity of human vs. murine haematopoietic progenitors to AFB1 and AFM1 (0.001–50 μg/ml), (ii) assessing whether a single AFB1 in vivo treatment (0.3–3 mg/kg b.w.) alters the ability of murine bone marrow cells to form myeloid and erythroid colonies, and (iii) comparing the in vitro with the in vitro ex-vivo data.We demonstrated (i) species-related sensitivity to AFB1, showing higher susceptibility of human myeloid and erythroid progenitors (IC₅₀ values: about 4 times lower in human than in murine cells), (ii) higher sensitivity of CFU-GM and BFU-E colonies, both more markedly affected, particularly by AFB1 (IC₅₀: 2.45 ± 1.08 and 1.82 ± 0.8 μM for humans, and 11.08 ± 2.92 and 1.81 ± 0.20 μM for mice, respectively), than the mature CFU-E (AFB1 IC₅₀: 12.58 ± 5.4 and 40.27 ± 6.05 μM), irrespectively of animal species, (iii) regarding AFM1, a species- and lineage-related susceptibility similar to that observed for AFB1 and (iv) lack of effects after AFB1 in vivo treatment on the proliferation of haematopoietic colonies.