Influence of Array Interspacing on the Force Required for Successful Microneedle Skin Penetration: Theoretical and Practical Approaches

Insertion behaviour of microneedle (MN) arrays depends upon the mechanical properties of the skin and, MN geometry and distribution in an array. In addressing this issue, this paper studies MN array insertion mechanism into skin and provides a simple quantitative basis to relate the insertion force with distance between two MNs. The presented framework is based on drawing an analogy between a beam on an elastic foundation and mechanism of needle insertion, where insertion force is separated into different components.A theoretical analysis indicates that insertion force decreases as interspacing increases. For a specified skin type, insertion force decreased from 0.029 to 0.028 N/MN when interspacing at MN tip was increased from 50 μm (350 μm at MN base) to 150 μm (450 μm at MN base).However, dependence of insertion force seems to decrease as the interspacing is increased beyond 150 μm. To assess the validity of the proposed model, a series of experiments was carried out to determine the force required for skin insertion of MN. Experiments performed at insertion speed of 0.5 and 1.0 mm/s yielded insertion force values of 0.030 and 0.0216 N, respectively, for 30 μm interspacing at MN base (330 μm interspacing at tip) and 0.028 and 0.0214 N, respectively, for 600 μm interspacing at MN base (900 μm interspacing at tip). Results from theoretical analysis and finite element modelling agree well with experimental results, which show MN interspacing only begins to affect insertion force at low interspacing (< 150 μm interspacing at MN base). This model provides a framework for optimising MN devices, and should aid development of suitable application method and determination of force for reliable insertion into skin.     

Hydrogel-Forming Microneedles Increase in Volume During Swelling in Skin,but Skin Barrier Function Recovery is Unaffected

We describe, for the first time, quantification of in-skin swelling and fluid uptake by hydrogel-forming microneedle (MN) arrays and skin barrier recovery in human volunteers. Such MN arrays, prepared from aqueous blends of hydrolyzed poly(methylvinylether/maleic anhydride) (15%, w/w) and the cross-linker poly(ethyleneglycol) 10,000 Da (7.5%, w/w), were inserted into the skin of human volunteers (n = 15) to depths of approximately 300 μm by gentle hand pressure. The MN arrays swelled in skin, taking up skin interstitial fluid, such that their mass had increased by approximately 30% after 6 h in skin. Importantly, however, skin barrier function recovered within 24 h after MN removal, regardless of how long the MN had been in skin or how much their volume had increased with swelling. Further research on closure of MN-induced micropores is required because transepidermal water loss measurements suggested micropore closure, whereas optical coherence tomography indicated that MN-induced micropores had not closed over, even 24 h after MN had been removed. There were no complaints of skin reactions, adverse events, or strong views against MN use by any of the volunteers. Only some minor erythema was noted after patch removal, although this always resolved within 48 h, and no adverse events were present on follow-up. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.     

Assessment of DNA damage,cytotoxicity, and apoptosis in human hepatoma (HepG2) cells after flurochloridone herbicide exposure

In vitro effects of flurochloridone (FLC) and its formulations Twin Pack Gold® [25% active ingredient (a.i.)] and Rainbow® (25% a.i.) were evaluated in HepG2 cells. Whereas cytokinesis-blocked micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were employed for genotoxicity, MTT, neutral red, and apoptosis detections were used for cytotoxicity evaluation. Activities were tested within the concentration range of 0.25–15 μg/ml FLC. Results demonstrated that neither FLC nor Rainbow® was able to induce MNs. On the other hand, 5 μg/ml Twin Pack Gold® only increased MN frequency. Furthermore, 10 and 15 μg/ml of both formulations resulted in cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. A marked increase in the genetic damage index was observed after treatment with all compounds. SCGE assay appeared to be more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of FLC than did MN. Our results reveal that FLC and its two formulations trigger apoptosis on HepG2 cells. The results represent the first experimental evidence of the in vitro apoptogenic role exerted on mammalian cells by FLC and the FLC-based formulations Rainbow® and Twin Pack Gold®, at least on HepG2 cells.     

Characterization of chromatin instabilities induced by glyphosate,terbuthylazine and carbofuran using cytome FISH assay

Possible clastogenic and aneugenic effects of pesticides on human lymphocytes at concentrations likely to be encountered in residential and occupational exposure were evaluated with (and without) the use of metabolic activation (S9). To get a better insight into the content of micronuclei (MN) and other chromatin instabilities, lymphocyte preparations were hybridized using pancentromeric DNA probes. Frequency of the MN, nuclear buds (NB) and nucleoplasmic bridges (NPB) in cultures treated with glyphosate slightly increased from 3.5 μg/ml onward. Presence of S9 significantly elevated cytome assay parameters only at 580 μg/ml. No concentration-related increase of centromere (C+) and DAPI signals (DAPI+) was observed for glyphosate treatment. Terbuthylazine treatment showed a dose dependent increase in the number of MN without S9 significant at 0.0008 μg/ml and higher. At concentration lower than 1/16 LD50 occurrence of C+MN was significantly elevated regardless of S9, but not dose related, and in the presence of S9 only NBs containing centromere signals were observed. Carbofuran treatment showed concentration-dependent increase in the number of MN. The frequency of C + MN was significant from 0.008 μg/ml onward regardless of S9. Results suggest that lower concentrations of glyphosate have no hazardous effects on DNA, while terbuthylazine and carbofuran revealed a predominant aneugenic potential.     

Genotoxicity of quinocetone,cyadox and olaquindox in vitro and in vivo

Quinocetone (QCT) and Cyadox (CYA) are important derivative of heterocyclic N-oxide quinoxaline (QdNO), used actively as antimicrobial feed additives in China. Here, we tested and compared the genotoxic potential of QCT and CYA with olaquindox (OLA) in Ames test, HGPRT gene mutation (HGM) test in V79 cells, unscheduled DNA synthesis (UDS) assay in human peripheral lymphocytes, chromosome aberration (CA) test, and micronucleus (MN) test in mice bone marrow. OLA was found genotoxic in all 5 assays. In Ames test, QCT produced His⁺ mutants at 6.9 μg/plate in Salmonella typhimurium TA 97, at 18.2 μg/plate in TA 100, TA 1535, TA 1537, and at 50 μg/plate in TA 98. CYA produced His⁺ mutants at 18.2 μg/plate in TA 97, TA 1535, and at 50 μg/plate in TA 98, TA 100 and TA 1537. QCT was found positive in HGM and UDS assay at concentrations ⩾10 μg/ml while negative results were reported in CA test and MN test. Collectively, we found that OLA was more genotoxic than QCT and CYA. Genotoxicity of QCT was found at higher concentration levels in Ames test, HGM and UDS assays while CYA showed weak mutagenic potential to bacterial cells in Ames test.     

Does potassium sorbate induce genotoxic or mutagenic effects in lymphocytes?

The present study evaluates the genotoxic potential of potassium sorbate (PS) in cultured and isolated human lymphocytes. To assess the damage caused by PS in humans, we designed in vitro experiments by measuring chromosomal aberrations (CAs), sister-chromatid exchanges (SCEs), micronucleus (MN) and comet assays. Lymphocytes were treated with negative control (sterile distilled water), positive control (MMC for cultured lymphocytes, and H₂O₂ for isolated lymphocytes) and four concentrations (125, 250, 500, and 1000 μg/ml) of PS. According to the results, PS treatment significantly increases the CAs (with or without gaps at 500 and 1000 μg/ml concentrations) and SCEs (at 250, 500, 1000 μg/ml for 24 h and 125, 250, 500, 1000 μg/ml for 48 h) compared with vehicle control. Following treatment of the isolated lymphocytes for 1 h, significant PS-induced DNA strand breaks were observed, at all concentrations. However, PS failed to significantly affect the MN assay. On the contrary, PS does not cause cell cycle delay as noted by the non-significant decrease in the cytokinesis-block proliferation index (CBPI) and replicative index (RI). Only a slight decrease was observed in the mitotic index (MI) at the highest concentration for both treatment times. From the results, PS is clearly seen to be genotoxic to the human peripheral blood lymphocytes in vitro.     

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.     

Interactions between dietary flavonoids apigenin or luteolin and chemotherapeutic drugs to potentiate anti-proliferative effect on human pancreatic cancer cells,in vitro

The objectives were to assess the potential of dietary flavonoids apigenin (Api) and luteolin (Lut) to enhance the anti-proliferative effects of chemotherapeutic drugs on BxPC-3 human pancreatic cancer cells and to investigate the potential molecular mechanism of action. Simultaneous treatment or pretreatment (0, 6, 24 and 42 h) of flavonoids and chemotherapeutic drugs at various concentrations (0–50 μM) were assessed using the MTS cell proliferation assay. Simultaneous treatment with either flavonoid (13, 25 or 50 μM) and chemotherapeutic drugs 5-fluorouracil (5-FU, 50 μM) or gemcitabine (Gem, 10 μM) for 60 h resulted in mostly less-than-additive effects (p < 0.05). Pretreatment for 24 h with 13 μM of either Api or Lut, followed by Gem for 36 h was optimal to inhibit cell proliferation. Pretreatment of cells with 11–19 μM of either flavonoid for 24 h resulted in 59–73% growth inhibition when followed by Gem (10 μM, 36 h). Lut (15 μM, 24 h) pretreatment followed by Gem (10 μM, 36 h), significantly decreased protein expression of nuclear GSK-3β and NF-κB p65 and increased pro-apoptotic cytosolic cytochrome c. Pretreatment of BxPC-3 human pancreatic cancer cells with low concentrations of Api or Lut effectively aid in the anti-proliferative activity of chemotherapeutic drugs.     

Studies on percutaneous penetration of chemicals – Impact of storage conditions for excised human skin

According to international guidelines skin penetration experiments can be carried out using freshly excised or frozen stored skin. However, this recommendation refers to data obtained in experiments with human cadaver skin. In our study, the percutaneous penetration of the occupationally relevant chemicals anisole, cyclohexanone and 1,4-dioxane was investigated for freshly excised as well as for 4 and 30 days at ?20 °C stored human skin using the diffusion cell technique. As indicator for the impairment of skin barrier by freezing cholesterol dissolution was determined in the solvents in exposure chambers of diffusion cells. Considering the percutaneously penetrated amounts, the following ranking was determined: 1,4-dioxane > anisole > cyclohexanone (decline to a factor of 5.9). The differences of fluxes between freshly excised and frozen stored skin (4 and 30 days) were not significant (p > 0.05). Cholesterol dissolved from the skin indicates no significant differences between freshly excised and frozen stored skin. This study shows that freezing of human skin for up to 30 days does not alter the skin barrier function and the permeability of chemicals.     

Evaluation of 8-hydroxy-2-deoxyguanosine and NFkB activation,oxidative stress response,acetylcholinesterase activity,and histopathological changes in rainbow trout brain exposed to linuron

Linuron is a widely used herbicide to control grasses and annual broad leaf weeds. It is known that linuron has toxic effects on different organisms. However, the toxic effects of linuron on aquatic organisms, especially fish, is completely unknown. Thus, we aimed to investigate changes in 8-hydroxy-2-deoxyguanosine (8-OHdG) and nuclear factor kappa B (NFkB) activity, histopathological changes, antioxidant responses and acetylcholinesterase (AChE) activity in rainbow trout brain after exposure to linuron. Fish were exposed to 30 μg/L, 120 μg/L and 240 μg/L concentrations of linuron for twenty-one days. Brain tissues were taken from fish for 8-OHdG and NFkB activity, histopathological examination and determination of superoxide dismutase (SOD), catalase (CAT) enzyme activity, lipid peroxidation (LPO), and reduced glutathione (GSH) levels. Our data indicated that high linuron concentrations caused a decrease in GSH levels, SOD and CAT activities in brain tissues (p < 0.05). LPO levels were significantly increased by 240 μg/L linuron. All concentrations caused a significant inhibition in brain AChE enzyme activity (p < 0.05). Immunopositivity was detected for 8-OHdG and NFkB, and linuron exposure caused histopathological damage to the brain tissues. The results of this study can provide useful information for understanding of linuron-induced toxicity.     

Development of In Vivo Impedance Spectroscopy Techniques for Measurement of Micropore Formation Following Microneedle Insertion

Microneedles (MNs) provide a minimally invasive means to enhance skin permeability by creating micron-scale channels (micropores) that provide a drug delivery pathway. Adequate formation of the micropores is critical to the success of this unique drug delivery technique. The objective of the current work was to develop sensitive and reproducible impedance spectroscopy techniques to monitor micropore formation in animal models and human subjects. Hairless guinea pigs, a Yucatan miniature pig, and human volunteers were treated with 100 MN insertions per site following an overnight prehydration period. Repeated measurements were made pre- and post-MN treatment using dry and gel Ag/AgCl electrodes applied with light verses direct pressure to hold the electrode to the skin surface. Impedance measurements dropped significantly post-MN application at all sites (p < 0.05, irrespective of electrode type or gel application), confirming micropore formation. In the Yucatan pig and human subjects, gel electrodes with direct pressure yielded the lowest variability (demonstrated by lower %relative standard deviation), whereas dry electrodes with direct pressure were superior in the guinea pigs. These studies confirm that impedance measurements are suitable for use in both clinical and animal research environments to monitor the formation of new micropores that will allow for drug delivery through the impermeable skin layers.     

The mycotoxin patulin,modulates tight junctions in caco-2 cells

The mycotoxin patulin is a common contaminant of fruit. Here, we demonstrate that patulin reduces the barrier properties of the intestinal cell line, caco-2 by specific effects on tight junction components. Within 5 h of exposure to 100 μM toxin, the transepithelial electrical resistance of caco-2 monolayers was reduced by approximately 95% and the monolayer became more permeable to FITC-labelled dextrans of 4–40 kDa. Immunoblotting revealed occludin proteolysis and a significant reduction in ZO-1 levels. Patulin had no influence on claudin levels but marked changes in their distribution were observed. These data indicate that patulin decreases the barrier properties of caco-2 monolayers by modulation of the tight junction.     

Cell damage induced by copper: An explant model to study anemone cells

Sea anemones are benthic organisms, of low mobility and can be directly affected by water pollution. This work studied the defense mechanisms and DNA damage caused by copper toxicity in cells from the anemone Bunodosoma cangicum. For this, exposure of anemones cells were held, kept in primary culture through explant of podal disk to copper (7.8 and 15.6 μg/L), and the control group, for 6 and 24 h. Cytotoxicity was seen through the viability and cell number, MXR phenotype through the accumulation of rhodamine-B, ROS generation by H₂DCF-DA and DNA damage by comet assay. The results obtained show that there is a drop in viability and number of cells, especially after exposure of 24 h in 15.6 μg/L. There is an induction of the MXR activity only at 7.8 μg/L for 24 h. As for ROS, there is an increase in the generation of reactive species in greatest concentration of copper for 6 h, and in both for 24 h, which leads to oxidative stress, which culminates with a DNA damage. What was evidenced by the increase of the tail size, % DNA presented and moment of tail. Therefore, the copper represents an adversity to the anemones cells, being cytotoxic and genotoxic.     

Poly (ethylene)-glycol conjugated solid lipid nanoparticles of noscapine improve biological half-life,brain delivery and efficacy in glioblastoma cells

Noscapine crosses blood–brain-barrier and inhibits proliferation of glioblastoma cells. However, short plasma half-life and rapid elimination necessitate the administration of multiple injections for successive chemotherapy. Noscapine bearing solid lipid nanoparticles, Nos-SLN and poly (ethylene)-glycol conjugated solid lipid nanoparticles of noscapine, Nos-PEG-SLN of 61.3 ± 9.3-nm and 80.5 ± 8.9-nm containing 80.4 ± 3.2% and 83.6 ± 1.2% of Nos, were constructed. First order kinetic and Higuchi equation were followed to release the Nos at intracellular pH ~ 4.5. Further, a decrease in IC₅₀ (Nos; 40.5 μM > Nos-SLN; 27.2 μM > 20.8 μM) and enhanced subG1 population were observed in U87cells. Plasma half-life was enhanced up to ~ 11-fold and ~ 5-fold by Nos-PEG-SLN and Nos-SLN which significantly (P < 0.05) deposits 400.7 μg/g and 313.1 μg/g of Nos in comparison to 233.2 μg/g by drug solution. This is first report demonstrating a workable approach to regulate the administration of multiple injections of Nos, warranting further in vivo tumor regression study for superior management of brain cancer.From the Clinical EditorThis report describes a possible approach to regulate the administration of multiple injections of Noscapine using solid lipid nanoparticles. The data warrant further in vivo tumor regression studies for optimal management of glioblastoma, a generally very poorly treatable brain cancer.     

Silver nanoparticle induced toxicity to human sperm by increasing ROS(reactive oxygen species) production and DNA damage

Silver nanoparticles (AgNPs) have been used in medical products and industrial coatings, due to their antimicrobial properties. Excessive use of AgNPs can have adverse effects on the human body, however, their toxicity characteristics to human sperm and the potential mechanisms are not entirely clear. In this study, we exposed human sperm to different doses of AgNPs (0, 50 μg ml⁻¹, 100 μg ml⁻¹, 200 μg ml⁻¹ or 400 μg ml⁻¹) for various times (15 min, 30 min, or 60 min), followed by analyses of the sperm viability, motility and the ratio of abnormal to normal sperm.Then, transmission electron microscopy(TEM) was used to explore the sperm ultrastructural characteristics. Reactive oxygen species production and DNA fragmentation were tested using standard kits and the sperm chromatin dispersion method, respectively. The results showed a dose- and time-dependent decline in sperm viability and motility and an increased ratio of abnormal to normal sperm after 30 min and 60 min of exposure to AgNPs at 200 μg ml⁻¹ and 400 μg ml⁻¹. The most common abnormalities were sperm heads with disrupted chromatin or absent acrosomes, bent tails, and curved mid-pieces. The ultrastructural characteristics of AgNP-treated sperm included disrupted, swollen, granular and vacuolar defects of the chromatin. In addition, ROS(reactive oxygen species)production and DNA fragmentation were markedly increased after 60 min of exposure to AgNPs at 200 μg ml⁻¹ and 400 μg ml⁻¹. Our results indicated that AgNPs caused detrimental changes in human sperm characteristics, and the excessive use of AgNPs should be carried out with caution.     

A study of genotoxicity and oxidative stress induced by mercuric chloride in the marine polychaete Perinereis aibuhitensis

The marine polychaete worm Perinereis aibuhitensis was used to study the genotoxic effects of mercuric chloride by means of the comet assay and micronucleus (MN) test. P. aibuhitensis was subjected in vivo to two different concentrations of mercuric chloride (0.05 mg L⁻¹ and 0.5 mg L⁻¹) for 96 h. The comet assay of coelomocytes demonstrated that TailDNA% values increased with extended exposure to or increased concentrations of HgCl₂ (p < 0.01). The frequency of MNs was the highest in the treatment with 96 h of exposure at all concentrations (p < 0.01). The genotoxic effect of HgCl₂ was both dose- and time-dependent in exposed P. aibuhitensis. The activities of the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidases (GPx) were also estimated. Significant variations in antioxidant enzyme activities depended on the sampling time and the concentrations of mercuric chloride. Compared with the control, the activities of the antioxidant enzymes (SOD and GPx) were elevated at the lower concentration of mercuric chloride (0.05 mg L⁻¹) (p < 0.05) for shorter exposure periods (24 h and 72 h). At the higher concentration of mercury (0.5 mg L⁻¹), the activities of GPx and SOD were inhibited; no variation was observed. These results proved that the use of the comet assay and MN test in coelomocytes of P. aibuhitensis is appropriate for determining the levels of DNA damage and that P. aibuhitensis is a species that is sensitive to mercury pollutants. This species may be considered a suitable candidate for monitoring marine heavy metal pollution.     

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.     

Human neuroblastoma cells transfected with tyrosine hydroxylase gain increased resistance to methylmercury-induced cell death

In a previous study we demonstrated that human neuroblastoma SH-SY5Y cells transfected with human tyrosine hydroxylase isoform 1 (SH + TH cells) were substantially more resistant to cell death induced by pro-oxidants than wild type SH-SY5Y cells (SH cells). In the present communication we used methylmercury as a model of cell stress in order to test whether SH + TH cells would behave in a similar manner in response to this stressor. Incubation with methylmercury (0.1–3 μM) for 24 h caused a significant reduction in cell viability and increased apoptotic markers in both cell types. However, the effects were significantly reduced in the SH + TH cells when compared to the SH cells. Activation of p38^MAPK was also reduced in the SH + TH compared to the SH cells after methylmercury exposure. Since p38^MAPK is known to participate in signal transduction pathways during cell stress, our data suggest that SH + TH cells develop an increased resistance to environmental stress caused by neurotoxins such as methylmercury. In conclusion our results show that insertion of the human TH gene in cells that originally do not express this protein leads to alterations in cell homeostasis and triggers defense mechanisms against pro-oxidative insults.     

Evaluation of EpiDerm full thickness-300 (EFT-300) as an in vitro model for skin irritation: Studies on aliphatic hydrocarbons

The aim of this study was to understand the skin irritation effects of saturated aliphatic hydrocarbons (HCs), C9–C16, found jet fuels using in vitro 3-dimensional EpiDerm full thickness-300 (EFT-300) skin cultures. The EFT-300 cultures were treated with 2.5 μl of HCs and the culture medium and skin samples were collected at 24 and 48 h to measure the release of various inflammatory biomarkers (IL-1α, IL-6 and IL-8). To validate the in vitro results, in vivo skin irritation studies were carried out in hairless rats by measuring trans epidermal water loss (TEWL) and erythema following un-occlusive dermal exposure of HCs for 72 h. The MTT tissue viability assay results with the EFT-300 tissue show that 2.5 μl/tissue (≈4.1 μl/cm²) of the HCs did not induce any significant changes in the tissue viability for exposure times up to 48 h of exposure. Microscopic observation of the EFT-300 cross-sections indicated that there were no obvious changes in the tissue morphology of the samples at 24 h, but after 48 h of exposure, tridecane, tetradecane and hexadecane produced a slight thickening and disruption of stratum corneum. Dermal exposures of C12–C16 HCs for 24 h significantly increased the expression of IL-1α in the skin as well as in the culture medium. Similarly, dermal exposure of all HCs for 24 h significantly increased the expression of interleukin-6 (IL-6) and IL-8 in the skin as well as in the culture medium in proportion to the HC chain length. As the exposure time increased to 48 h, IL-6 concentrations increased 2-fold compared to the IL-6 values at 24 h. The in vivo skin irritation data also showed that both TEWL and erythema scores increased with increased HCs chain length (C9–C16). In conclusion, the EFT-300 showed that the skin irritation profile of HCs was in the order of C9 ⩽ C10 ⩽ C11 ⩽ C12 < C13 ≈ C14 ≈ C16 and that the tissue was an excellent in vitro model to predict in vivo irritation and to understand the structural activity relationship of HCs.     

Significance of individual adjustment of initial loading dosage of teicoplanin based on population pharmacokinetics

An initial loading dose of teicoplanin is required to reach the optimal trough concentration (≥10 μg/mL) rapidly. To attain the optimal teicoplanin concentration efficiently, an individual loading dose regimen based on population pharmacokinetics, in which the target trough concentration was set to 15 μg/mL, was defined. Among 70 patients, 33 patients received the individual loading dose regimen, 33 patients received the conventional loading dose regimen (200 mg or 400 mg every 12 h on Day 1 followed by 200 mg once daily) and 4 patients received no loading dose. The proportion of patients showing an optimal plasma concentration was 88% in the individual loading dose regimen but only 33% in the conventional loading dose regimen. No patient without a loading dose showed the optimal concentration. Both total loading dose and plasma concentration were significantly (P < 0.001) higher in the individual loading dose group than in the conventional loading dose group. Notably, the trough concentration was almost constant in patients with individual loading doses ranging from 800 mg to 1800 mg. These findings suggest that individual adjustment of the initial loading dose of teicoplanin is potentially useful to attain the optimal concentration rapidly.