The molecular, temporal and region-specific requirements of the beta isoform of Calcium/Calmodulin-dependent protein kinase type 2 (CAMK2B) in mouse locomotion.

Genetic approaches using temporal and brain region-specific restricted gene deletions have provided a wealth of insight in the brain regions and temporal aspects underlying spatial and associative learning. However, for locomotion such extensive studies are still scarce. Previous studies demonstrated that Camk2b(-/-) mice, which lack the β isoform of Calcium/Calmodulin-dependent protein kinase 2 (CAMK2B), show very severe locomotion deficits.

Quantification of topically delivered 5-aminolevulinic acid by lontophoresis across ex vivo human stratum corneum.

Iontophoretic transport of the prodrug 5-aminolevulinic acid (ALA), which is used for photodynamic therapy (PDT), across human stratum corneum (SC) was studied quantitatively in vitro. The experiments were carried out in a three-compartment iontophoresis cell consisting of two electrode chambers equipped with Ag-AgCl electrodes, each separated from a central acceptor chamber by a sheet of SC, supported by a dialysis membrane, to mimic the side-by-side configuration normally used in vivo. Acceptor fluid samples were collected every hour for a period of 30 h in a fraction collector and analyzed by high-performance liquid chromatography-fluorometry after derivatization of the ALA.

X-ray microanalysis of cryopreserved human skin to study the effect of iontophoresis on percutaneous ion transport.

Purpose: To study at the ultrastructural level which part of the skin is associated with percutaneous iodide transport by passive diffusion and iontophoresis.

Methods: Following passive diffusion or iontophoresis of iodide, the morphology and the ion distribution of the skin was preserved by rapid freezing. The skin was kept frozen until and during examination by transmission electron microscopy (TEM) and X-ray microanalysis (XRMA).

Visualization of percutaneous 3H-estradiol and 3H-norethindrone acetate transport across human epidermis as a function of time.

Developing transdermal therapeutic systems for estradiol and norethindrone acetate raised questions about the steroids penetration pathway across and retention in the skin. This paper describes the distribution of 3H-estradiol and 3H-norethindrone acetate in human stratum corneum after topical application to dermatomed skin in vitro. The study involved (a) permeation experiments to determine the steroid flux, (b) autoradiographical visualization of the steroid distribution in the same skin samples, and (c) a correlation between flux and skin distribution in time.

An integrated pharmacokinetic-pharmacodynamic approach to optimization of R-apomorphine delivery in Parkinson's disease.

R-apomorphine is a mixed dopamine D(1)/D(2) receptor agonist which is potentially useful in the management of Parkinson's disease. The delivery of R-apomorphine is complicated however by a number of pharmacokinetic and pharmacodynamic factors. This review describes the development of a transdermal iontophoretic delivery system for R-apomorphine on the basis of integrated pharmacokinetic-pharmacodynamic (PK/PD) investigations in patients with idiopathic Parkinson's disease.

Reflection contrast microscopy for high resolution detection of (3)H-estradiol in ultrathin sections of human stratum corneum.

A single autoradiographical method for light and electron microscopy (LM and EM) is presented. Human skin, containing (3)H-estradiol ((3)H-E2) after an in vitro permeation experiment, was processed via a non-extractive tissue preparation protocol, comprising cryo-fixation, freeze-drying, osmium tetroxide vapor fixation, and Spurr resin embedding. Semithin sections were processed for LM autoradiography, while ultrathin sections were processed both for high-resolution LM and EM autoradiography.

In vivo human skin permeability enhancement by oleic acid: a laser Doppler velocimetry study.

Topical application of a skin permeation enhancer such as oleic acid (OA) can result in: (i) higher skin permeability for many exogenous substances (ii) an irritation reaction. Laser Doppler velocimetry (LDV) is one of few techniques which can assess both effects using appropriate protocols. Direct LDV measurement, measuring cutaneous blood flow, has been preferred as a tool to evaluate skin irritation.

Mechanistic and quantitative prediction of aminopeptidase activity in stripped human skin based on the HaCaT cell sheet model.

HaCaT cell culture sheets were recently demonstrated to be a useful tool to study epidermal metabolism. Here we report on a mechanistic and quantitative correlation between the kinetics of aminopeptidase-based cleavage of L-Ala-4-methoxy-2-naphthylamide (Ala-MNA) in HaCaT sheets versus stripped human skin. Fresh human skin (breast or abdominal) was obtained from cosmetic surgery, tape-stripped, and dermatomed.

Electroperturbation of human stratum corneum fine structure by high voltage pulses: a freeze-fracture electron microscopy and differential thermal analysis study.

Application of high voltage pulses (HVP) to the skin has been shown to promote the transdermal drug delivery by a mechanism involving skin electroporation. The aim of this study was to detect potential changes in lipid phase and ultrastructure induced in human stratum corneum by various HVP protocols, using differential thermal analysis and freeze-fracture electron microscopy. Due to the time involved between the moment the electric field is switched off and the analysis, only "secondary" phenomena rather than primary events could be observed.

Validation and testing of a new iontophoretic continuous flow through transport cell.

A new continuous flow through transport cell is presented. The design is based on a minimisation of contributions of the cell conformation and experimental protocol to the overall transport kinetics. The system is validated by measuring the washout period of methylene blue and apomorphine.

Pharmacokinetics, enantiomer interconversion, and metabolism of R-apomorphine in patients with idiopathic Parkinson's disease.

The pharmacokinetics and metabolism of R-apomorphine were determined in 10 patients with idiopathic Parkinson's disease after intravenous infusion of 30 micrograms.kg-1 in 15 min. Specifically, emphasis was on enantiomeric interconversion into S-apomorphine and on the formation of apocodeine and isoapocodeine, since these metabolites may interfere with the pharmacodynamics of R-apomorphine.

Stepwise intravenous infusion of apomorphine to determine the therapeutic window in patients with Parkinson's disease.

A new experimental strategy was applied to determine the concentration-effect relation and the therapeutic window of apomorphine in individual patients with Parkinson's disease. Apomorphine was administered by a stepwise intravenous infusion. The infusion rate was increased by 10 micrograms/kg/h every 20 minutes, up to 100 micrograms/kg/h or less when adverse effects occurred.

Validation of freeze-drying to visualize percutaneous 3H-estradiol transport: the influence of skin hydration on the efficacy of the method.

A study was made of the validity of freeze-drying to visualize the distribution of 3H-estradiol in human stratum corneum after topical application of a dry dose, a patch or a buffer solution. Each of these donor formulations was applied to human dermatomed skin for 24 h using Franz permeation cells. Subsequently, small pieces of skin were subjected to cryofixation, freeze-drying, osmium tetroxide vapor fixation, Spurr resin embedding and electron microscopic autoradiography.

In vivo human skin barrier modulation by topical application of fatty acids.

The in vivo effects of fatty acids on skin barrier function were assessed by measuring: (i) transepidermal water loss (TEWL), (ii) diffusion lag times for hexyl nicotinate (HN), and (iii) irritant skin response using laser Doppler velocimetry (LDV) in combination with visual scoring. Two classes of fatty acids have been investigated: straight-chain saturated fatty acids (SFA), having 6-12 carbon atoms, and unsaturated fatty acids (UFA): oleic, linoleic, alpha-linolenic and arachidonic acids. It has been reported that these acids can enhance the permeation of various compounds across the skin.

Iontophoretic delivery of apomorphine. II: An in vivo study in patients with Parkinson's disease.

Purpose: Transdermal transport rates of the dopamine agonist R-apomorphine were determined in patients with idiopathic Parkinson's disease (IPD). Apomorphine was applied by iontophoresis at two current densities.

Methods: In ten patients apomorphine was applied passively for one hour.

Iontophoretic delivery of apomorphine. I: In vitro optimization and validation.

Purpose: To investigate the feasibility of transdermal iontophoretic delivery of apomorphine in patients with Parkinson's disease, transdermal transport rates were optimized and validated across human stratum corneum and freshly dermatomed human skin in vitro.

Methods: In all experiments R-apomorphine hydrochloride was applied in the anodal compartment. The effect on the flux of the following parameters was studied, using a flow through transport cell current density, pH, concentration, ionic strength, osmolarity, buffer strength, temperature and skin type.

Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease.

Analytical methods are described for the selective, rapid and sensitive determination of R- and S-apomorphine, apocodeine and isoapocodeine and the glucuronic acid and sulfate conjugates in plasma and urine. The methods involve liquid-liquid extraction followed by high-performance liquid chromatography with electrochemical detection. The glucuronide and sulfate conjugates are determined after enzymatic hydrolysis.

An in vivo-In vitro study of the use of a human skin equivalent for irritancy screening of fatty acids.

A human skin equivalent (HSE) consisting of reconstructed epidermis on a fibroblast-populated collagen gel was evaluated as a model for irritancy screening. The irritancy potential of a series of saturated and unsaturated fatty acids was investigated in vivo under short-term exposure conditions using transepidermal water loss (TEWL), laser Doppler velocimetry (LDV) and the penetration of hexyl nicotinate as parameters. The effects of the fatty acids in vitro were studied after topical application on HSE using changes in epidermal morphology, changes in interleukin (IL)-1alpha and interleukin-8 mRNA expression and protein levels, and alterations in activity of plasminogen activators as endpoints.

Development of an optimal protocol for the ultrastructural examination of skin by transmission electron microscopy.

The intercellular lipid bilayers of the stratum corneum provide the permeability barrier of the skin. To perform an electron microscopical examination of the ultrastructure of these bilayers, ruthenium tetroxide fixation is required. In this study an optimal fixation protocol was developed and selected upon comparing seven different fixation procedures, using glutaraldehyde (GA) and the postfixatives ruthenium red, osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) in combination with potassium ferrocyanide (K4Fe(CN)6) and potassium ferricyanide (K3Fe(CN)6).

Electroperturbation of the human skin barrier in vitro: II. Effects on stratum corneum lipid ordering and ultrastructure.

In transdermal iontophoresis, drugs can be driven across the skin by electrorepulsion, but their transport can also be enhanced by electrical perturbation of the skin barrier. Our objective was to study perturbing effects of electrical current on human stratum corneum lipid fine structure combining techniques including freeze-fracture electron microscopy. Human stratum corneum was subjected to pulsed constant currents, varying from 0.

In vitro human skin barrier modulation by fatty acids: skin permeation and thermal analysis studies.

Purpose: This study aims to elucidate the skin permeation enhancement and the skin perturbation effects of a number of fatty acids, i.e. straight-chain saturated (SFA), monounsaturated (MUFA) and polyunsaturated acids (PUFA).

Localization of aminopeptidase activity in freshly excised human skin: direct visualization by confocal laser scanning microscopy.

The aim of this study was to localize and visualize aminopeptidase activity within freshly excised, dermatomed human skin without perturbation of its histologic integrity. The use of confocal laser scanning microscopy (CLSM) is introduced as a novel approach by which to monitor the degradation of suitable substrates in the skin. The fluorescence of the metabolites originating from the cleavage of the aminopeptidase probe bis-Leu-rhodamine 110 (Leu2-R11O) was interpreted to reflect the local aminopeptidase activity in the tissue.

Assessment of the potential irritancy of oleic acid on human skin: Evaluation in vitro and in vivo.

As skin barrier modulating compounds, fatty acids are frequently used in formulations for transdermal or topical delivery. In this study the effects of oleic acid on keratinocytes in vitro was compared with its in vivo skin irritancy in humans. Dose- and time-dependent effects of oleic acid were examined in submerged human keratinocyte cultures, in reconstructed human epidermis (RE-DED), and in excised human skin, using alterations in morphology and changes in interleukin-1alpha mRNA levels as endpoints.

In vivo buccal delivery of the peptide drug buserelin with glycodeoxycholate as an absorption enhancer in pigs.

Purpose: To study the potential of buccal delivery of the peptide drug in pigs.

Methods: Intravenous administration and buccal delivery without and with 10 mM sodium glycodeoxycholate (GDC) as absorption enhancer were investigated as a randomised cross-over study in six pigs. The buccal delivery device consisted of an application chamber with a solution of buserelin and was attached to the buccal mucosa for 4 hours using an adhesive patch.