Mechanistic studies of the transdermal iontophoretic delivery of 5-OH-DPAT in vitro

A characterization and optimization of the in vitro transdermal iontophoretic transport of 5-hydroxy-2-(N,N,-di-n-propylamino)tetralin (5-OH-DPAT) is presented. The utility of acetaminophen as a marker of electroosmotic flow was studied as well. The following parameters of iontophoretic transport of 5-OH-DPAT were examined: drug donor concentration, electroosmotic contribution, influence of co-ions, current density, and composition of the acceptor phase. The steady-state flux (Flux_ss) of acetaminophen was linearly correlated with the donor concentration and co-iontophoresis of acetaminophen did not influence the iontophoretic flux of 5-OH-DPAT, indicating that acetaminophen is an excellent marker of electroosmotic flow. Lowering the Na⁺ concentration from 78 to 10 mM in the donor phase, resulted in a 2.5-fold enhancement of the Flux_ss. The Flux_ss showed a nonlinear relation with the drug donor concentration and an excellent linear correlation with the current density. Reducing the pH of the acceptor phase from 7.4 to 6.2 resulted in a dramatic decrease of the Flux_ss of 5-OH-DPAT, explained by a reduced electroosmotic flow and an increased counter-ion flow. Optimization of the conditions resulted in a maximum Flux_ss of 5-OH-DPAT of 1.0 µmol · cm^?2 h^?1 demonstrating the potential of the iontophoretic delivery of this dopamine agonist for the symptomatic treatment of Parkinson's disease. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:275–285, 2010     

Highly pathogenic avian influenza subtype H5Nx clade 2.3.4.4 outbreaks in Dutch poultry farms, 2014–2018: Clinical signs and mortality

In recent years, different subtypes of highly pathogenic avian influenza (HPAI) viruses caused outbreaks in several poultry types worldwide. Early detection of HPAI virus infection is crucial to reduce virus spread. Previously, the use of a mortality ratio threshold to expedite notification of suspicion in layer farms was proposed. The purpose of this study was to describe the clinical signs reported in the early stages of HPAI H5N8 and H5N6 outbreaks on chicken and Pekin duck farms between 2014 and 2018 in the Netherlands and compare them with the onset of an increased mortality ratio (MR). Data on daily mortality and clinical signs from nine egg‐producing chicken farms and seven Pekin duck farms infected with HPAI H5N8 (2014 and 2016) and H5N6 (2017–2018) in the Netherlands were analysed. In 12 out of 15 outbreaks for which a MR was available, MR increase preceded or coincided with the first observation of clinical signs by the farmer. In one chicken and two Pekin duck outbreaks, clinical signs were observed prior to MR increase. On all farms, veterinarians observed clinical signs of general disease. Nervous or locomotor signs were reported in all Pekin duck outbreaks, but only in two chicken outbreaks. Other clinical signs were observed less frequently in both chickens and Pekin ducks. Compared to veterinarians, farmers observed and reported clinical signs, especially respiratory and gastrointestinal signs, less frequently. This case series suggests that a MR with a set threshold could be an objective parameter to detect HPAI infection on chicken and Pekin duck farms at an early stage. Observation of clinical signs may provide additional indication for farmers and veterinarians for notifying a clinical suspicion of HPAI infection. Further assessment and validation of a MR threshold in Pekin ducks are important as it could serve as an important tool in HPAI surveillance programs.     

Transdermal iontophoresis of rotigotine: influence of concentration, temperature and current density in human skin in vitro.

Iontophoretic transport of rotigotine across human stratum corneum (HSC) was studied in vitro in side by side diffusion cells according to the following protocol: 6 h of passive diffusion, 9 h of iontophoresis followed by 5 h of passive diffusion. A current density of 0.5 mA cm(-2) was applied. The parameters studied were the influence of the rotigotine concentration in donor phase and the influence of the molecular weight of the co-ions. To this end, Na(+) was replaced by tetra ethyl ammonium (TEA(+)) or tetra butyl ammonium (TBA(+)) (both at pH 5 and 6). In addition, the influence of the acceptor phase temperature (32 degrees C versus room temperature), the replacement of HSC by dermatomed human skin (DHS), and the relation between drug transport and current density were examined. The estimated steady-state flux (Flux(ss)) gradually increased with the drug concentration in the donor phase in a linear manner. The flux was also linearly correlated with the applied current density providing a convenient approach to individual dose titration. The use of TEA(+) as co-ion increased the rotigotine iontophoretic flux significantly, while TBA(+) did not. Replacing HSC by DHS reduced the iontophoretic rotigotine transport, while an increase in temperature to 32 degrees C increased the rotigotine flux. The maximum Flux(ss) achieved was around 80 nmol cm(-2) h(-1) indicating that by means of iontophoresis, a therapeutic level of rotigotine might be achieved with a reasonable patch size.     

Niosomes as a novel peroral vaccine delivery system.

The feasibility to develop a peroral vaccine delivery system based on non-ionic surfactant vesicles (niosomes) was evaluated using BALB/c mice. Ovalbumin was encapsulated in various lyophilized niosome preparations consisting of sucrose esters, cholesterol and dicetyl phosphate. Two different formulations were compared in this study. The specific antibody titres within serum, saliva and intestinal washings were monitored by ELISA on days 7, 14, 21 and 28 after intragastric administration. Only encapsulation of ovalbumin into Wasag7 (70% stearate sucrose ester, 30% palmitate sucrose ester (40% mono-, 60% di/tri-ester)) niosomes resulted in a significant increase in antibody titres. Administration of ovalbumin and empty niosomes did not exert a similar effect, neither did administration of any control formulation. In contrast to ovalbumin loaded Wasag7 niosomes, application of the more hydrophilic Wasag15 (30% stearate sucrose ester, 70% palmitate sucrose ester (70% mono-, 30% di/tri-ester)) niosome preparations did not result in an increase in antibody titres.     

The Effect of Two Azones on the Lateral Lipid Organization of Human Stratum Corneum and Its Permeability

Purpose. Investigation of the relationship between changes in humanSC lipid organization induced by N-alkyl-azocycloheptane-2-one andSC permeability to the model compound HgCl₂. Methods. Human dermatomed skin was treated with propylene glycol(PG), oleyl-Azone (OAz) or dodecyl-Azone (DAz) in 0.15 M PG.Untreated skin served as control. The lateral lipid organization wasstudied by electron diffraction. Hg was measured on tape-strips by X-raymicroanalysis and in the acceptor phase by atom absorptionspectrometry. Results. In control and PG treated samples, the lipid packing wasmainly orthorhombic, while a small fraction was hexagonal. In OAz andDAz treated samples, the orthorhombic lipid organization remained,however, the hexagonal packing was recorded less frequently. Theamount of Hg decreased as a function of depth in all SC samples,however, the penetration profile increased significantly upon OAztreatment. The cumulative amount of Hg in the acceptor phase of OAztreated samples also increased significantly compared to control andPG treated samples. Conclusions. The increased penetration of Hg into OAz treated skincould not be related to an orthorhombic-hexagonal phase transition.Alternatively, phase separation of OAz and/or formation of grainboundaries might affect SC permeability, hereby increasing Hgpenetration. A similar mechanism is proposed for DAz.     

Transdermal Macromolecular Delivery: Real-Time Visualisation of Iontophoretic and Chemically Enhanced Transport Using Two-Photon Excitation Microscopy

Purpose. To investigate the transdermal delivery of a modelmacromolecule by passive and iontophoretic means following pretreatment withC₁₂-penetration enhancers and to visualise transport across humanstratum corneum (SC) in real time. Methods. Transport studies of dextran, labelled with fluorescentCascade Blue® (D-CB; M_R = 3 kDa) across human stratum corneum,were conducted during passive and iontophoretic modes of deliveryfollowing pretreatment with either dodecyltrimethylammoniumbromide (DTAB), sodium dodecyl sulphate (SDS) or Azone®.Size-exclusion chromatography was used to assess maintenance of dextranstructural integrity throughout experimental lifetime. Two-photonexcitation microscopy was employed to visualise real-time dextran transportduring current application. Results. The positively charged C₁₂-enhancer DTAB elevated passiveD-CB steady-state flux (J_ss) and was the only enhancer to do soabove control during iontophoresis. The negatively charged SDS had theleast effect during both stages. On-line macromolecular transport wasvisualised, indicating both inter- and intra-cellular pathways across SCduring current application. No transport was visible across untreatedSC during passive transport. Conclusions. Use of a positively charged enhancer may improve J_ssof anionic macromolecular penetrants during passive and iontophoreticdelivery. On-line visualisation of iontophoresis across SC was possibleand can provide mechanistic insight into SC transport pathways.     

Skin barrier disruption by acetone: observations in a hairless mouse skin model

To disrupt the barrier function of the skin, different in vivo methods have been established, e.g., by acetone wiping or tape-stripping. In this study, the acetone-induced barrier disruption of hairless mice was investigated in order to establish a reliable model to study beneficial, long-term effects on barrier recovery after topical application. For both treatments (i.e., acetone treatment and tape-stripping) the transepidermal water loss directly after disruption and the subsequent barrier recovery profile were similar. Histological assessment showed significant lower number of corneocyte layers in acetone-treated and tape-stripped skin compared to untreated skin, while there was no statistical difference between the two treatments. Lipid analysis of acetone-treated skin revealed that only small fraction of lipids were extracted consisting of predominantly nonpolar lipids. Importantly, the ratio of the barrier lipids, i.e., cholesterol, free fatty acids and ceramides, remained similar between control and acetone-treated skin. This reflects the undisrupted lipid organization, as determined by small-angle X-ray diffraction measurements: the long-periodicity lamellar phase was still present after acetone treatment. Our results contradict earlier studies which reported no mechanical stratum corneum removal, a substantial extraction of lipids and disruption in lipid organization. In conclusion, our studies demonstrate that barrier disruption due to acetone treatment is mainly due to removal of corneocytes.     

Development of a murine model to evaluate the effect of vernix caseosa on skin barrier recovery

Abstract:  The aim of this study was twofold, that is the generation of a reliable model for skin barrier disruption and repair and to evaluate recovery of damaged skin after application of vernix caseosa (VC). VC was selected as its wound healing properties were suggested previously, but never clearly demonstrated. Five different levels of barrier disruption in mice, accomplished by tape-stripping, were evaluated. Disruption models such as moderate, severe #1 and #2 (transepidermal water loss (TEWL) of 31 ± 2, 59 ± 4 and 66 ± 3 g/m²/h, respectively) showed complete recovery within 72 h. However, not all corneocytes were removed after tape-stripping. Additionally, models such as severe #3 and #4 (TEWL of 73 ± 5 and 79 ± 6 g/m²/h, respectively) with a more severe disruption were evaluated. After tape-stripping, all corneocytes were removed and the remaining epidermis was intact. However, model #3 still showed complete recovery within 72 h. With model #4, a crust was formed and almost complete recovery (approximately 90%) was obtained within only 8 days. The effect of VC application on recovery of disrupted skin was evaluated with model #3 and #4. Model #3 showed that application of VC predominantly influenced initial recovery and is therefore merely appropriate to study the effect of formulations in the initial recovery period. Topical application of VC on model #4 considerably increased initial and long-term recovery. Moreover, VC application promoted rapid formation of stratum corneum and prevented epidermal thickening. These observations not only confirm the ability of VC to enhance barrier recovery, but also suggest potential use of this treatment clinically.