In-vitro Permeability of the Human Nail and of a Keratin Membrane from Bovine Hooves: Prediction of the Penetration Rate of Antimycotics through the Nail Plate and their Efficacy

In contrast to the partition coefficient octanol/water the molecular size of penetrating drugs has a noticeable influence on the permeability of the human nail plate and a keratin membrane from bovine hooves. The relationship between permeability and molecular weight is founded on well-established theories. The correlation between the permeability of the nail plate and that of the hoof membrane allows a prediction of the nail permeability after determination of the drug penetration through the hoof membrane. The maximum flux of ten antimycotics (amorolfine, bifonazole, ciclopirox, clotrimazole, econazole, griseofulvin, ketoconazole, naftifine, nystatin and tolnaftate) through the nail plate was predicted on the basis of their penetration rates through the hoof membrane and their water solubilities. An efficacy coefficient against onychomycoses was calculated from the maximum flux and the minimum inhibitory concentration. Accordingly, amorolfine, ciclopirox, econazole and naftifine are expected to be especially effective against dermatophytes, whereas in the case of an infection with yeasts only, amorolfine and ciclopirox are promising.     

In-vitro Permeability of the Human Nail and of a Keratin Membrane from Bovine Hooves: Penetration of Chloramphenicol from Lipophilic Vehicles and a Nail Lacquer

Lipophilic vehicles and especially nail lacquers are more appropriate for topical application on the nail than aqueous systems because of their better adhesion. This work has, therefore, studied the penetration through the human nail plate of the model compound chloramphenicol from the lipophilic vehicles medium chain triglycerides and n-octanol and from a lacquer based on quaternary poly(methyl methacrylates) (Eudragit RL). The results were compared with data obtained with a keratin membrane from bovine hooves. If the swelling of the nail plate or the hoof membrane is not altered by use of lipophilic vehicles, the maximum flux of the drug is independent of its solubility in the vehicle and is the same as that from a saturated aqueous solution. These vehicles are not able to enter the hydrophilic keratin membrane because of their non-polar character and so cannot change the solubility of the penetrating substance in the barrier. If the concentration of the drug in the nail lacquer is sufficiently high, the maximum flux through both barriers equals that from aqueous vehicles or even exceeds it because of the formation of a supersaturated system. Penetration through the nail plate follows first order kinetics after a lag-time of 400 h. The course of penetration through the hoof membrane is initially membrane-controlled and later becomes a matrix-controlled process because of the membrane's greater permeability. Chloramphenicol is dissolved in the lacquer up to a concentration of 31%. The relative release rates from these solution matrices are independent of the drug concentration but they decrease on changing to a suspension matrix. These results show that drug flux is independent of the character of the vehicle and that penetration of the drug is initially membrane-controlled and changes to being matrix-controlled as the drug content of the lacquer decreases.     

In-vitro Permeability of the Human Nail and of a Keratin Membrane from Bovine Hooves: Influence of the Partition Coefficient Octanol/Water and the Water Solubility of Drugs on their Permeability and Maximum Flux

Penetration of homologous nicotinic acid esters through the human nail and a keratin membrane from bovine hooves was investigated by modified Franz diffusion cells in-vitro to study the transport mechanism. The partition coefficient octanol/water PC_Oct/w of the esters was over the range 7 to > 51000. The permeability coefficient P of the nail plate as well as the hoof membrane did not increase with increasing partition coefficient or lipophilicity of the penetrating substance. This indicates that both barriers behave like hydrophilic gel membranes rather than lipophilic partition membranes as in the case of the stratum corneum. Penetration studies with the model compounds paracetamol and phenacetin showed that the maximum flux was first a function of the drug solubility in water or in the swollen keratin matrix. Dissociation hindered the diffusion of benzoic acid and pyridine through the hoof membrane. Since keratin, a protein with an isoelectric point of about 5, is also charged, this reduction can be attributed to an exclusion of the dissociating substance due to the Donnan equilibrium. Nevertheless, the simultaneous enhancement of the water solubility makes a distinct increase of the maximum flux possible. In order to screen drugs for potential topical application to the nail plate, attention has to be paid mainly to the water solubility of the compound. The bovine hoof membrane may serve as an appropriate model for the nail.     

Facts and myths about columns packed with sub-3 μm and sub-2 μm particles

Increasing the separating efficiency enhances the separation power. The most popular solution for improving chromatographic performance is to employ columns packed with small particle diameters (i.e., sub-2 μm particles) to induce a simultaneous improvement in efficiency, optimal velocity and mass transfer, albeit the cost of pressure. In this study a systematic evaluation of the possibilities and limitations of the separations obtained with 5 cm long narrow bore columns packed with 1.5–3.0 μm particles is presented. Several commercially available different sub-3 μm and sub-2 μm packed columns were evaluated by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on different polar neutral real life analytes, to show that the separation time is not obviously shorter if the particle size is reduced. Comparison of low-molecular weight compounds (one steroid and one non-steroid hormone, with molecular weights lower than 500) and a high-molecular weight one (MW ∼ 1000) was conducted. Same efficiency can be achieved with columns packed with 1.9–2.1 μm particles as with smaller particles. The column packed with 3 μm particles had the lowest reduced plate height minimum (h = 2.2) while the column with the smallest particles (1.5 μm) gave the highest reduced plate height minimum (h ∼ 3.0). According to this study, the theoretically expected efficiency of very fine particles (diameter <2 μm) used in practice today is compromised. Investigation of this phenomenon is presented.     

Assessment of Iontophoretic and Passive Ungual Penetration by Laser Scanning Confocal Microscopy

Purpose

To estimate the in vitro ungual penetration depth of sodium fluorescein and nile blue chloride by laser scanning confocal microscopy.

Methods

The depth, uniformity and pathways of penetration of both markers into human nail during passive and iontophoretic experiments were investigated. The penetration of sodium fluorescein into the dorsal, ventral and intermediate layers of the nail was also studied. Transversal images were used to estimate directly the relative penetration of the markers with respect to the complete thickness of the nail. “Exposed layer” images allowed estimating the depth of penetration by taking xy-plans, starting by the exposed layer, and following the z axis into the nail.

Results

The fluorescent markers penetrated 7–12% of the nail thickness. Iontophoresis increased penetration of both markers compared to passive diffusion. However, ungual penetration was not modified by the intensity of current applied. Penetration into the dorsal, ventral, and intermediate nail layers was similar. The method developed allowed inter- and intra- nail variability to be accounted for.

Conclusions

Iontophoresis enhanced moderately the penetration of the two markers into the nail plate as compared to passive diffusion. The confocal images suggested the transcellular pathway to be predominant during both passive and iontophoretic experiments.

     

Drug delivery into the skin by degradable particles

Recently, it was demonstrated that particles could be utilized as carrier systems for drugs into the hair follicles. In the present study, a two-component drug delivery system is presented consisting of degradable particles loaded with fluorescein isothiocyanate and a separate protease formulation for degradation. The particles were applied alone, 30 min previous to the protease application and simultaneously with the protease onto porcine skin. Subsequently, biopsies were removed, and the penetration depths of the particles were analyzed using laser scanning microscopy.The obtained results demonstrate that the particles alone achieved a penetration depth of around 900 μm. Similar results were obtained for the successive application of particles and protease, whereas a release of the fluorescent dye was only observed in the upper 250 μm corresponding to the penetration depth of the protease. In the case of the simultaneous application, the particles were partly dissolved before application, leading to a reduced particle size and diminished penetration depth.The results revealed that degradable particles are a promising tool for drug delivery into the skin.     

Transungual delivery of ketoconazole using novel lacquer formulation

Onychomycosis, a common fungal infection of the nail, can have a substantial impact on quality of life. The success of topical therapy for onychomycosis depends on effective penetration, which can be enhanced using an appropriate delivery method. This study evaluated the effectiveness of a novel topical lacquer on enhancing [¹⁴C]-ketoconazole penetration by comparing nail absorption, nail distribution, and nail penetration of [¹⁴C]-ketoconazole dissolved in the novel lacquer versus a commercial ketoconazole cream. Using the in vitro finite dose model, the formulations were applied daily to human nail plates for 7 days. Drug absorption was measured by monitoring rate of appearance in each nail layer and the supporting bed. After the multiple day treatment, cumulative concentrations of ketoconazole formulated in novel lacquer in the deep nail layer and the nail bed were significantly greater than cumulative concentrations of commercial ketoconazole (p < 0.05), as well as several orders of magnitude greater than the minimal inhibitory concentration (MIC) deemed necessary to inhibit the growth of causative dermatophytic and yeast species. These results suggest that this novel ketoconazole lacquer has the potential to be an effective topical treatment for onychomycosis.     

Shell and small particles; Evaluation of new column technology

The performance of 5 cm long columns packed with shell particles was compared to totally porous sub-2 μm particles in gradient and isocratic elution separations of hormones (dienogest, finasteride, gestodene, levonorgestrel, estradiol, ethinylestradiol, noretistherone acetate, bicalutamide and tibolone). Peak capacities around 140–150 could be achieved in 25 min with the 5 cm long columns. The Ascentis Express column (packed with 2.7 μm shell particles) showed similar efficiency to sub-2 μm particles under gradient conditions. Applying isocratic separation, the column of 2.7 μm shell particles had a reduced plate height minimum of approximately h = 1.6. It was much smaller than obtained with totally porous particles (h ≈ 2.8). The impedance time also proved more favorable with 2.7 μm shell particles than with totally porous particles. The influence of extra-column volume on column efficiency was investigated. The extra-column dispersion of the chromatographic system may cause a shift of the HETP curves.     

A direct LC/MS/MS method for the determination of ciclopirox penetration across human nail plate in in vitro penetration studies

Due to severe chelating effect caused by N-hydroxylpyridone group of ciclopirox, there is no published direct HPLC or LC/MS/MS method for the determination of ciclopirox in any in vitro or in vivo matrix. Instead, the time-consuming pre-column derivatization methods have been adapted for indirect analysis of ciclopirox. After overcoming the chelating problem by using K₂EDTA coated tubes, a direct, sensitive and high-throughput LC/MS/MS method was successfully developed and validated to determine the amount of ciclopirox that penetrated across the nail plate during in vitro nail penetration studies. The method involved adding a chemical analog, chloridazon as internal standard (IS) in K₂EDTA coated tubes, mixing IS with ciclopirox in a 96-well plate and then proceeding to LC/MS/MS analysis. The MS/MS was selected to monitor m/z 208.0 → 135.8 and 221.8 → 77.0 for ciclopirox and IS, respectively, using positive electrospray ionization. The method was validated over a concentration range of 8–256 ng/mL, yielding calibration curves with correlation coefficients greater than 0.9991 with a lower limit of quantitation (LLOQ) of 8 ng/mL. The assay precision and accuracy were evaluated using quality control (QC) samples at three concentration levels. Analyzed concentrations ranged from 101% to 113% of their respective nominal concentration levels with coefficients of variation (CV) below 10.6%. The average recovery of ciclopirox from nail matrix was 101%.     

Determining the effect of ocular chemical injuries on topical drug delivery

Ocular chemical injuries (OCIs) commonly cause ocular damage and visual loss and treatment uses topical therapies to facilitate healing and limit complications. However, the impact of chemical injury on corneal barrier function and treatment penetration is unknown. Therefore, the aim of this study was to determine the effect of OCI on drug penetration and absorption. Porcine corneal explants were used to assess histological damage, electrical resistance, and the trans-corneal penetration/corneal adsorption of reference compounds (sodium fluorescein and rhodamine B) and dexamethasone. Corneal explants were injured with either 1 M sulfuric acid, or 1 M sodium hydroxide. Dexamethasone penetration was measured using high-performance liquid chromatography (HPLC) and that of fluorescein and rhodamine using fluorescence. Dexamethasone corneal adsorption was measured using enzyme-linked immunoabsorbant assay (ELISA). Both acid and alkaline injuries reduced trans-corneal electrical resistance. NaOH injury increased hydrophilic fluorescein penetration (NaOH 8.59 ± 1.50E–05 cm.min⁻¹ vs. Hanks'' Balanced Salt Solution (HBSS) 1.64 ± 1.01E–06 cm.min⁻¹) with little impact on hydrophobic rhodamine B (1 M NaOH 6.55 ± 2.45E–04 cm.min⁻¹ vs. HBSS 4.60 ± 0.972E–04 cm.min⁻¹) and dexamethasone penetration (1 M NaOH 3.00 ± 0.853E–04 cm.min⁻¹ vs. HBSS 2.69 ± 0.439E–04 cm.min⁻¹). By contrast, H₂SO₄ decreased trans-corneal penetration of hydrophilic fluorescein (H₂SO₄ 1.16 ± 14.2E–07 cm.min⁻¹) and of hydrophobic dexamethasone (H₂SO₄ 1.88 ± 0.646E–04 cm.min⁻¹) and rhodamine B (H₂SO₄ 4.60 ± 1.42E–05 cm.min⁻¹). Acid and alkaline OCI differentially disrupted the corneal epithelial barrier function. Acid injury reduced penetration of hydrophobic dexamethasone and rhodamine B as well as hydrophilic fluorescein, which may translate clinically into reduced drug penetration after OCI, while alkaline injury increased fluorescein penetration, with minimal effect on dexamethasone and rhodamine B penetration.     

Quantitative analysis of colistin A and colistin B in plasma and culture medium using a simple precipitation step followed by LC/MS/MS

An analytical method for quantitation of colistin A and colistin B in plasma and culture medium is described. After protein precipitation with acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), the supernatants were diluted with 0.03% TFA. The compounds were separated on an Ultrasphere C18 column, 4.6 mm × 250 mm, 5 μm particle size with a mobile phase consisting of 25% ACN in 0.03% TFA and detected with tandem mass spectrometry. The instrument was operating in ESI negative ion mode and the precursor–product ion pairs were m/z 1167.7 → 1079.6 for colistin A and m/z 1153.7 → 1065.6 for colistin B. The lower limit of quantification (LLOQ) for 100 μL plasma was 19.4 and 10.5 ng/mL for colistin A and B, respectively, with CV <6.2% and accuracy < ±12.6%. For culture medium (50 μL + 50 μL plasma), LLOQ was 24.2 and 13.2 ng/mL for colistin A and B, respectively, with CV <11.4% and accuracy < ±8.1%. The quick sample work-up method allows for determination of colistin A and B in clinical samples without causing hydrolysis of the prodrug colistin methanesulfonate (CMS).     

Determination of rhodamine B in soft drink, waste water and lipstick samples after solid phase extraction

A new solid phase extraction method is described for sensitive and selective determination of trace levels of rhodamine B in soft drink, food and industrial waste water samples. The method is based on the adsorption of rhodamine B on the Sepabeads SP 70 resin and its elution with 5 mL of acetonitrile in a mini chromatographic column. Rhodamine B was determined by using UV visible spectrophotometry at 556 nm. The effects of different parameters such as pH, amount of rhodamine B, flow rates of sample and eluent solutions, resin amount, and sample volume were investigated. The influences of some alkali, alkali earth and transition metals on the recoveries of rhodamine B were investigated. The preconcentration factor was found 40. The detection limit based on three times the standard deviation of the reagent blank for rhodamine B was 3.14 μg L⁻¹. The relative standard deviations of the procedure were found as 5% in 1 × 10⁻⁵ mol L⁻¹ rhodamine B. The presented procedure was successfully applied to real samples including soft drink, food and industrial waste water and lipstick samples.     

Influence of the flavonoids apigenin, kaempferol, and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1

OATP1A2 and OATP2B1 are uptake transporters of the human organic anion transporting polypeptide (OATP) family with a broad substrate spectrum including several endogenous compounds as well as drugs such as the antihistaminic drug fexofenadine and HMG-CoA reductase inhibitors. Both transporters are localized in the apical membrane of human enterocytes. Flavonoids, abundantly occurring in plants, have previously been shown to interact with drug metabolizing enzymes and transporters. However, the impact of flavonoids on OATP1A2 and OATP2B1 transport function has not been analyzed in detail. Therefore, HEK293 cell lines stably expressing OATP1A2 and OATP2B1 were used to investigate the influence of the Ginkgo flavonoids apigenin, kaempferol, and quercetin on the transport activity of OATP1A2 and OATP2B1. K_i values of all three flavonoids determined from Dixon plot analyses using BSP as substrate indicated a competitive inhibition with quercetin as the most potent inhibitor of OATP1A2 (22.0 μM) and OATP2B1 (8.7 μM) followed by kaempferol (OATP1A2: 25.2 μM, OATP2B1: 15.1 μM) and apigenin (OATP1A2: 32.4 μM OATP2B1: 20.8 μM). Apigenin, kaempferol, and quercetin led to a concentration-dependent decrease of the OATP1A2-mediated fexofenadine transport with IC₅₀ values of 4.3 μM, 12.0 μM, and 12.6 μM, respectively. The OATP1A2- and OATP2B1-mediated transport of atorvastatin was also efficiently inhibited by apigenin (IC₅₀ for OATP1A2: 9.3 μM, OATP2B1: 13.9 μM), kaempferol (IC₅₀ for OATP1A2: 37.3 μM, OATP2B1: 20.7 μM) and quercetin (IC₅₀ for OATP1A2: 13.5 μM, OATP2B1: 14.1 μM). These data indicate that modification of OATP1A2 and OATP2B1 transport activity by apigenin, kaempferol, and quercetin may be a mechanism for food-drug or drug-drug interactions in humans.     

Investigation of nail permeation enhancement by chemical modification using water as a probe

Our objective was to screen molecules that could interact with keratin in the human nail and thereby improve the topical penetration of actives into and through the nail plate. We used specialized Franz-type diffusion cells for our permeation experiments and water as a marker molecule. Aqueous/hydroalcoholic gels containing the enhancers were spiked with tritiated water and compared with a control (without enhancer). We computed the normalized water flux (defined as a product of flux and nail thickness) for each gel. We defined an enhancement factor for water as the ratio of the normalized water flux from a gel containing enhancer to that of the control. Our results indicate that the chemical structure of the modifier is most important in determining its ability to enhance penetration. The best enhancement effect was obtained using N-(2-mercaptopropionyl) glycine, a mercaptan derivative of an amino acid, in combination with urea. The concentration of each chemical modifier was linearly related to normalized water flux and mercaptan levels were more important that urea levels in penetration enhancement. Barrier integrity of nails was compromised after treatment with effective chemical modifiers. Thus, we have developed a suitable technique to screen nail penetration enhancers using water as a probe.     

Genotoxicity of nanomaterials: DNA damage and micronuclei induced by carbon nanotubes and graphite nanofibres in human bronchial epithelial cells in vitro

Despite the increasing industrial use of different nanomaterials, data on their genotoxicity are scant. In the present study, we examined the potential genotoxic effects of carbon nanotubes (CNTs; >50% single-walled, ∼40% other CNTs; 1.1 nm × 0.5–100 μm; Sigma–Aldrich) and graphite nanofibres (GNFs; 95%; outer diameter 80–200 nm, inner diameter 30–50 nm, length 5–20 μm; Sigma–Aldrich) in vitro. Genotoxicity was assessed by the single cell gel electrophoresis (comet) assay and the micronucleus assay (cytokinesis-block method) in human bronchial epithelial BEAS 2B cells cultured for 24 h, 48 h, or 72 h with various doses (1–100 μg/cm², corresponding to 3.8–380 μg/ml) of the carbon nanomaterials. In the comet assay, CNTs induced a dose-dependent increase in DNA damage at all treatment times, with a statistically significant effect starting at the lowest dose tested. GNFs increased DNA damage at all doses in the 24-h treatment, at two doses (40 and 100 μg/cm²) in the 48-h treatment (dose-dependent effect) and at four doses (lowest 10 μg/cm²) in the 72-h treatment. In the micronucleus assay, no increase in micronucleated cells was observed with either of the nanomaterials after the 24-h treatment or with CNTs after the 72-h treatment. The 48-h treatment caused a significant increase in micronucleated cells at three doses (lowest 10 μg/cm²) of CNTs and at two doses (5 and 10 μg/cm²) of GNFs. The 72-h treatment with GNFs increased micronucleated cells at four doses (lowest 10 μg/cm²). No dose-dependent effects were seen in the micronucleus assay. The presence of carbon nanomaterial on the microscopic slides disturbed the micronucleus analysis and made it impossible at levels higher than 20 μg/cm² of GNFs in the 24-h and 48-h treatments. In conclusion, our results suggest that both CNTs and GNFs are genotoxic in human bronchial epithelial BEAS 2B cells in vitro. This activity may be due to the fibrous nature of these carbon nanomaterials with a possible contribution by catalyst metals present in the materials—Co and Mo in CNTs (<5 wt.%) and Fe (<3 wt.%) in GNFs.     

Interaction between arsenic exposure from drinking water and genetic susceptibility in carotid intima–media thickness in Bangladesh

Epidemiologic studies that evaluated genetic susceptibility for the effects of arsenic exposure from drinking water on subclinical atherosclerosis are limited. We conducted a cross-sectional study of 1078 participants randomly selected from the Health Effects of Arsenic Longitudinal Study in Bangladesh to evaluate whether the association between arsenic exposure and carotid artery intima–media thickness (cIMT) differs by 207 single-nucleotide polymorphisms (SNPs) in 18 genes related to arsenic metabolism, oxidative stress, inflammation, and endothelial dysfunction. Although not statistically significant after correcting for multiple testing, nine SNPs in APOE, AS3MT, PNP, and TNF genes had a nominally statistically significant interaction with well-water arsenic in cIMT. For instance, the joint presence of a higher level of well-water arsenic (≥ 40.4 μg/L) and the GG genotype of AS3MT rs3740392 was associated with a difference of 40.9 μm (95% CI = 14.4, 67.5) in cIMT, much greater than the difference of cIMT associated with the genotype alone (β = − 5.1 μm, 95% CI = − 31.6, 21.3) or arsenic exposure alone (β = 7.2 μm, 95% CI = − 3.1, 17.5). The pattern and magnitude of the interactions were similar when urinary arsenic was used as the exposure variable. Additionally, the at-risk genotypes of the AS3MT SNPs were positively related to the proportion of monomethylarsonic acid (MMA) in urine, which is indicative of arsenic methylation capacity. The findings provide novel evidence that genetic variants related to arsenic metabolism may play an important role in arsenic-induced subclinical atherosclerosis. Future replication studies in diverse populations are needed to confirm the findings.     

Assessment of a dry extract from milk thistle (Silybum marianum) for interference with human liver cytochrome-P450 activities

The effect of a standardised dry extract from Silybum marianum (HEPAR-PASC®) on the enzyme kinetics of cytochrome-P450 isoenzymes (CYP) was investigated with primary human hepatocytes and human liver microsomes in order to assess the potential for drug-drug interactions. A cytotoxic effect on hepatocytes was observed at concentrations at and above 50 μg/ml. The EC₅₀ value was calculated to be 72.0 μg/ml. Therefore, the chosen test concentrations for CYP induction on human hepatocytes were 50, 10, and 1.5 μg/ml, which allowed for interpretation of the clinical significance of the data with a range of 50-1-fold c_max at maximal recommended doses. No induction was observed at the lowest concentration of 1.5 μg/ml, which is close to c_max. The extract did not induce CYP 3A4 at any of the tested concentrations. A low or marginal induction of 1A2, 2B6, and 2E1 at the maximum concentration of 50 μg/ml was observed. CYP inhibition on human microsomes was tested at concentrations of 150, 15, and 1.5 μg/ml. No or minor CYP inhibition was observed for all CYPs tested at the lowest concentration of 1.5 μg/ml, i.e. CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. At concentrations of 15 and 150 μg/ml the extract significantly inhibited CYP 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4. In these cases, K_i values were determined. All K_i values exceeded c_max by at least a factor of 10-fold. According to FDA regulations 1 > c_max/K_i > 0.1 indicates, that drug-drug interactions are possible for CYPs 2C8, and 2C9, but not likely, and are remote for CYPs 2C19, 2D6, and 3A4.     

Venom of Collett's snake (Pseudechis colletti) blocks the binding of α-bungarotoxin to acetylcholine receptors at chick but not human neuromuscular junctions: A histochemical study

Transverse cryosections, 6-8 μm thick, were cut from unfixed biventer cervicis muscles of chicks and quadriceps muscles of humans, mounted on glass slides and incubated for 1 h in either isotonic phosphate buffered saline, pH 7.3 (PBS), or crude venom of venom of Pseudechis colletti at concentrations between 2.1 and 210 μg ml⁻¹ in PBS. They were then exposed to a fluoresceine-conjugated α-bungarotoxin to label ACh receptor sites. Exposure to the crude venom of P. colletti prevented the labelling of acetylcholine (ACh) receptors in chick muscle in a dose-dependant manner; at a concentration of 2.1 μg ml⁻¹ labelling fell by 20% and at a concentration of 21 μg ml⁻¹ by more than 90%. In contrast, exposure to the venom at concentrations as high as 210 μg ml⁻¹ had no effect on receptor labelling in human skeletal muscle. The results suggest that ACh receptors in human skeletal muscle are relatively resistant to the postsynaptically active neurotoxins in the venom of P. colletti. The data explain the apparent anomaly that the venom blocks neuromuscular transmission in isolated nerve-muscle preparations of the chick whilst human subjects of envenoming bites by P. colletti exhibit no overt signs of neurotoxicity.     

Quantitative analysis of six polyynes and one polyene in Oplopanax horridus and Oplopanax elatus by pressurized liquid extraction and on-line SPE–HPLC

A pressurized liquid extraction and on-line SPE–HPLC method was developed for simultaneous determination of six polyynes, including falcarindiol, oplopandiol, (11S,16S,9Z)-9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate, oplopandiol acetate, oplopantriol A, oplopantriol B, and one polyene, (S,E)-nerolidol, in Oplopanax horridus and Oplpanax elatus. The analysis was conducted on a Grace Prevail C₁₈ column (3 μm, 7 mm × 33 mm) with gradient elution of acetonitrile and water after the sample loaded and washed with 42%ACN in 0.3 min on a phenomenex Strata-X on-line Extraction Cartridge SPE column (2.5 μm, 2.0 × 20 mm). All calibration curves of seven analytes showed good linearity within the test ranges. The validated method was successfully applied to quantify six polyynes and one polyene in two species of Oplopanax, O. horridus and O. elatus.     

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.