Dermatokinetics: Advances and Experimental Models, Focus on Skin Metabolism.

Numerous dermal contact products, such as drugs or cosmetics, are applied on the skin, the first protective barrier to their entrance into the organism. These products contain various xenobiotic molecules that can penetrate the viable epidermis. Many studies have shown that keratinocyte metabolism could affect their behavior by biotransformation. While aiming for detoxification, toxic metabolites can be produced. These metabolites may react with biological macromolecules often leading to sensitization reactions. After passing through the epidermis, xenobiotics can reach the vascularized dermis and therefore, be bioavailable and distributed into the entire organism. To highlight these mechanisms, dermatokinetics, based on the concept of pharmacokinetics, has been developed recently. It provides information on the action of xenobiotics that penetrate the organism through the dermal route. The purpose of this review is first to describe and synthesize the dermatokinetics mechanisms to consider when assessing the absorption of a xenobiotic through the skin. We focus on skin absorption and specifically on skin metabolism, the two main processes involved in dermatokinetics. In addition, experimental models and methods to assess dermatokinetics are described and discussed to select the most relevant method when evaluating, in a specific context, dermatokinetics parameters of a xenobiotic. We also discuss the limits of this approach as it is notably used for risk assessment in the industry where scenario studies generally focus only on one xenobiotic and do not consider interactions with the rest of the exposome. The hypothesis of adverse effects due to the combination of chemical substances in contact with individuals and not to a single molecule, is being increasingly studied and embraced in the scientific community.

Preclinical study of the novel vascular occluding agent, WST11, for photodynamic therapy of the canine prostate.

PURPOSE: Vascular targeted photodynamic therapy with WST09 shows promise for recurrent prostate cancer after radiation but hydrophobicity in aqueous solutions limited application. We tested the safety and efficacy of WST11, a novel water soluble vascular occluding agent, for vascular targeted photodynamic therapy of the dog prostate and compared it to WST09 vascular targeted photodynamic therapy. MATERIALS AND METHODS: Optical fibers were inserted in the prostate and connected to diode lasers. WST11 (Steba Biotech, Cedex, France) at varying doses, including a drug control with no light in 34 dogs, and WST09 (Steba Biotech) (2 mg/kg) in 3 dogs were infused during 10 minutes. Illumination was initiated at 5 or 10 minutes, and lasted up to 33.2 minutes based on laser fluence and delivered energy. Blood was collected for analysis and pharmacokinetics. The end point was at 1 week. RESULTS: No vascular targeted photodynamic therapy associated change was observed in blood pressure or blood test values. Circulating WST11 increased with drug infusion and decreased rapidly during 1 hour to reach undetectable levels by 24 hours. All except 1 dog with bowel intussusception did well after vascular targeted photodynamic therapy with only mild urinary symptoms that resolved within 24 to 48 hours. Lung and liver were normal. Hemorrhage was present in all prostates except controls. This translated into necrosis at a WST11 threshold and within a window of doses at fixed illumination. Necrosis was associated with loss of the vessel endothelial layer. Fluence highly impacted necrosis. WST11 vascular targeted photodynamic therapy was advantageously comparable to WST09 vascular targeted photodynamic therapy, and optimally ablated about 5.0 cm(3) of tissue per lobe and about 10 cm(3) of the whole prostate. CONCLUSIONS: The safety and efficacy of WST11 vascular targeted photodynamic therapy in the dog prostate support clinical applications for prostate cancer and benign prostatic hyperplasia.

Temporal transcriptomic analysis of human primary keratinocytes exposed to ß-naphthoflavone highlights the protective efficacy of skin to environmental pollutants.

The skin covers almost the entire body and plays an important role in detoxification and elimination of xenobiotics. These processes are initiated following the binding of xenobiotics to the aryl hydrocarbon receptor (AhR), which leads to the expression of several detoxification enzymes. To gain some insights on their impacts on skin cells over time, a temporal transcriptional analysis using gene expression arrays was performed in human primary epidermal keratinocyte (HEK) cells exposed for 6, 24 and 48 h to ß-naphthoflavone (ßNF), a potent agonist of AhR. Our results demonstrated that expression of genes related to xenobiotic, inflammation, and extracellular matrix remodeling was increased upon ßNF treatment from 6 h onwards. In contrast, the anti-oxidative response was seen mainly starting at 24 h. While some of the genes controlled by the epidermal differentiation complex was induced as soon as 6 h, expression of most of the S100 related genes located within the same chromosomal locus and keratin genes was increased at later times (24 and 48 h). Altogether our transcriptomic data highlight that following ßNF exposure, HEK cells elicited a protective xenobiotic response together with the activation of inflammation and keratinocyte regeneration. Later on these processes were followed by the stimulation of anti-oxidant activity and terminal differentiation.

Aloe-emodin-induced DNA fragmentation in the mouse in vivo comet assay.

Aloe-emodin (AE) and derivatives may be present as undesired components co-extracted during extraction of plants containing anthraquinonic derivatives for preparation of diacetylrhein. AE is a well-known in vitro mutagen, but up to now it failed to induce any clear in vivo genotoxic activity in the chromosome aberration assay in rat bone marrow or the in vivo/in vitro UDS test in liver. However, the two target organs noted during rodent carcinogenicity studies with danthron and emodin, two other well-known anthraquinone derivatives, are the colon and the kidney. Therefore, the choice of the organs for testing the genotoxicity of AE, i.e. bone marrow and liver, may be considered inadequate to demonstrate a possible in vivo genotoxic activity. In this context, the in vivo mouse comet assay was performed on both isolated kidney and colon cells in order to demonstrate a possible organospecific genotoxicity after oral administration of AE. Concurrently, the Ames test and the in vitro micronucleus assay with TK6 human lymphoblastoid cells were performed in their microscale version both with S9 from Aroclor 1254-induced liver or kidney, and without S9. AE induced primary DNA damage in the liver and in the kidney as observed between 3 and 6h after two oral administrations at 500, 1000 and 2000mg/kg bw, underlining an in vivo genotoxic mechanism of action. Furthermore, AE induced a clear genotoxic activity both in the Salmonella typhimurium strains TA1537 and TA98 and in the in vitro micronucleus assay in the absence as well as in the presence of metabolic activation. As no significant variation in the genotoxic activity of AE was noted when using either liver or kidney S9-mix, it seems that no quantitatively and/or qualitatively specific renal metabolism occurs. The kidney may be a target organ of AE as it is the major route of excretion. Under such conditions the separation of AE components should take place and the residual content of undesired AE derivatives should be made as low as reasonably achievable. AE present in plant extracts should be considered as an in vivo genotoxin and this property should be taken into account in the risk assessment for human exposure.

Evaluation of the new photosensitizer Stakel (WST-11) for photodynamic choroidal vessel occlusion in rabbit and rat eyes.

PURPOSE: To evaluate the photodynamic potential of a new hydrosoluble photosensitizer (WST-11, Stakel; Steba Biotech, Toussus-Le-Noble, France), for use in occlusion of normal choroidal vessels in the rabbit eye and CNV (choroidal neovascularization) in the rat eye. METHODS: Occlusive and nonocclusive parameters of Stakel and verteporfin photodynamic therapy (PDT) were investigated in pigmented rabbits. Eyes were followed by fluorescein angiography (FA) and histology at various intervals after PDT. RESULTS: When occlusive parameters (fluence of 50 J/cm(2), 5 mg/kg drug dose and DLI [distance to light illumination] of 1 minute) were used, Stakel PDT was efficient immediately after treatment without associated structural damage of the RPE and retina overlying the treated choroid in the rabbit eye. Two days later, total occlusion of the choriocapillaries was seen in 100% of the treated eyes, along with accompanying histologic structural changes in the overlying retina. When the occlusive parameters (fluence, 100 J/cm2; drug dose, 12 mg/m2; and DLI, 5 minutes) of verteporfin PDT were used, occlusion of the choriocapillaries was observed in 89% of the treated eyes. Histology performed immediately after treatment demonstrated structural damage of the overlying retina and RPE layer. Weaker, nonocclusive Stakel PDT parameters (25 J/cm2, 5 mg/kg, and DLI of 10 minutes) did not induce choriocapillary occlusion or retinal lesions on FA or histology. Weaker, nonocclusive verteporfin PDT parameters (10 J/cm2, 0.2 mg/kg, and DLI of 5 minutes) did not induce choriocapillary occlusion. However, histology of these eyes showed the presence of damage in the retinal and choroidal tissues. Moreover, preliminary results indicate that selective CNV occlusion can be achieved with Stakel PDT in the rat eye. CONCLUSIONS: Unlike verteporfin PDT, Stakel PDT does not cause direct damage to the RPE cell layer or retina. These observations indicate that Stakel PDT may have a high potential for beneficial therapeutic outcomes in treatment of AMD.

Layer-by-Layer Assembly of Nanosized Membrane Fractions for the Assessment of Cytochrome P450 Xenobiotic Metabolism.

Herein, we report the use of sequential layer-by-layer (LbL) assembly to design nanostructured films made of recombinant bacterial membrane fractions (MF), which overexpress cytochrome P450 (CYP) and cytochrome P450 reductase. The ability to incorporate MF in LbL multilayered films is demonstrated by an in situ quartz crystal microbalance with dissipation monitoring using poly-l-lysine or poly-l-ornithine as a polycation. Results show that MF preserve a remarkable CYP1A2 catalytic property in the adsorbed phase. Moreover, atomic force microscopy images reveal that MF mostly adopt a flattened conformation in the adsorbed phase with an extensive tendency to aggregate within the multilayered films, which is more pronounced when increasing the number of bilayers. Interestingly, this behavior seems to enhance the ability of embedded MF to remain active after repeated uses. The proposed strategy constitutes a practical alternative for the immobilization of active CYP enzymes. Besides their fundamental interest, MF-based multilayers are useful nano-objects for the creation of new biomimetic reactors for the assessment of xenobiotic metabolism.

Selection of dosing regimen with WST11 by Monte Carlo simulations, using PK data collected after single IV administration in healthy subjects and population PK modeling.

WST11, a novel generation of photo sensitizers to be used for vascular-targeted phototherapy (VTP), is effective at short interval between injection and illumination and it is expected to enable selective destruction of neovasculature with minimal side effects or skin photo toxicity. This study was conducted to evaluate the clinical and laboratory safety, tolerability and pharmacokinetic profile of WST11 given as a single intravenous administration (1.25, 2.5, 5, 7.5, 10, or 15 mg/kg) during an escalating dose study in healthy male subjects. This article describes WST11 population pharmacokinetic modeling and simulations performed to optimize the IV infusion-dosing regimen in combination with illumination, the target PK profile being plateau concentrations during approximately 30 min. The study included 42 healthy male subjects, administered 1.25, 2.5, 5, 7.5, 10, or 15 mg/kg as a 10-min IV infusion. A population pharmacokinetic model was developed using nonlinear mixed effects modeling (NONMEM). Monte Carlo simulations of the population PK dataset (NONMEM) were performed to select series of dosing regimen which would result in a plateau of concentration lasting at least 30 min and allow laser illumination. A two-compartment model with nonlinear elimination best described the data. No demographic factor was shown to affect the WST11 pharmacokinetics. The clearance was shown to decreases with the dose administered, ranging from 6 L/h (dose of 79 mg) to 2 L/h (dose of 1110 mg). The duration of the infusion was estimated at 12 min. The volume of distribution of the central compartment was 3 L and the volume of the peripheral compartment was 1.15 L. The apparent inter-compartmental clearance was 0.137 L/h. The between subjects variability on clearance and on volume was low. Residual variability was moderate with a CV of 21%. Due to the dose effect on clearance and the rapid elimination, simulations showed that different dosing inputs are necessary: for 5 and 10 mg/kg BW, a sufficiently good dosing scenario is to administer 80% of the dose over 5 min, 15% over 10 min and the remaining 5% over 10 min. For lower doses, the sequence 70% in 5 min/20% in 10 min/10% in 10 min is preferable. The pharmacokinetic profile of WST11 by IV administration would allow a treatment by laser illumination in good clinical conditions using controlled infusions. The study results do not indicate that the dose should be adjusted for body size. The only factor that determines the drug input profile is the dose level, since the elimination half-life decreases when the dose administered increases. The use of the population PK model for simulations has shown that, at dose level of 5 mg/kg or more, a loading dose of 80% dose given over 5 min followed by 15% of dose during 10 min and remained dose to give over 10 min would result in a favorable PK profile.

Comparative assessment of local tolerance of alcohols commonly used in alcohol-based hand rubs for hand hygiene.

Hand hygiene plays a key role in nosocomial infection prevention. To achieve users' adherence, products' dermal tolerance is essential. We aimed at making a comparative assessment of skin irritation and phototoxicity of the 3 alcohols commonly used in alcohol-based hand rubs (Ethanol, Propan-2-ol, Propan-1-ol) at 60, 70, 80 or 85% w/w in water or with co-formulates (hydrating, emollient and skin protective agents). In vitro validated OECD methods 439 and 432 were used. For irritation, EpiSkin™ Small Model was the chosen Reconstructed Human Epidermis (RhE). For phototoxicity, co-formulates alone or in mixture with and without alcohol were tested using BALB/c 3T3 cell cultures. Whilst Ethanol and Propan-2-ol could not be differentiated and displayed good skin tolerance profiles, Propan-1-ol based products lead to significant viability impairments of RhE at 60, 70 or 80% and at 60% in the presence of co-formulates. However, these results could not be reproduced in another RhE model. Taking also into account bibliographic data on Propan-1-ol, this suggests that our results are probably related to a lack of specificity of the used RhE. Therefore, it can be relevant in case of significant results to use two different RhE models before performing any classification and/or performing any complementary tests.

Characterization of the inhibitory activity of tenatoprazole on the gastric H+,K+ -ATPase in vitro and in vivo.

Tenatoprazole is a prodrug of the proton pump inhibitor (PPI) class, which is converted to the active sulfenamide or sulfenic acid by acid in the secretory canaliculus of the stimulated parietal cell of the stomach. This active species binds to luminally accessible cysteines of the gastric H+,K+ -ATPase resulting in disulfide formation and acid secretion inhibition. Tenatoprazole binds at the catalytic subunit of the gastric acid pump with a stoichiometry of 2.6 nmol mg(-1) of the enzyme in vitro. In vivo, maximum binding of tenatoprazole was 2.9 nmol mg(-1) of the enzyme at 2 h after IV administration. The binding sites of tenatoprazole were in the TM5/6 region at Cys813 and Cys822 as shown by tryptic and thermolysin digestion of the ATPase labeled by tenatoprazole. Decay of tenatoprazole binding on the gastric H+,K+ -ATPase consisted of two components. One was relatively fast, with a half-life 3.9 h due to reversal of binding at cysteine 813, and the other was a plateau phase corresponding to ATPase turnover reflecting binding at cysteine 822 that also results in sustained inhibition in the presence of reducing agents in vitro. The stability of inhibition and the long plasma half-life of tenatoprazole should result in prolonged inhibition of acid secretion as compared to omeprazole. Further, the bioavailability of tenatoprazole was two-fold greater in the (S)-tenatoprazole sodium salt hydrate form as compared to the free form in dogs which is due to differences in the crystal structure and hydrophobic nature of the two forms.

Effects of rhein on human articular chondrocytes in alginate beads.

This study was designed to investigate the effects of rhein, the active metabolite of diacerhein, on the metabolic functions of human chondrocytes cultured in alginate beads. Enzymatically isolated osteoarthritic (OA) chondrocytes were cultured in alginate beads in a well-defined culture medium for 12 days. Rhein was tested in a range of concentrations comprised between 10(-7) and 4 x 10(-5)M, in the presence or absence of 10(-10)M IL-1beta. Interleukin (IL)-6 and -8, macrophage inflammatory protein (MIP-1beta), stromelysin-1 (MMP-3), aggrecan (AGG), tissue inhibitor of metalloproteinases-1 (TIMP-1), prostaglandin E(2) (PGE(2)) and nitric oxide (NO) productions were assayed. Cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) mRNA steady-state levels were also quantified. In the basal condition, 10(-5)M rhein increased by 46.5% the production of AGG, decreased by 17-30% the production of IL-6, MMP-3, NO and MIP-1beta but enhanced by 50% the production of PGE(2). IL-1beta increased IL-6, IL-8, MIP-1beta, NO, PGE(2) and MMP-3 productions, but inhibited AGG and TIMP-1 synthesis. Rhein partially reversed the effect of IL-1beta on TIMP-1 and NO production, had no effect on AGG, IL-6 and MIP-1beta production, but up-regulated the IL-1beta stimulated PGE(2) production. The COX-2 and iNOS mRNA levels and IL-8 production were not modified by rhein.Overall, these results contribute to explain the clinical efficiency of rhein and give new information on its mechanisms of action.

Rhein inhibits interleukin-1 beta-induced activation of MEK/ERK pathway and DNA binding of NF-kappa B and AP-1 in chondrocytes cultured in hypoxia: a potential mechanism for its disease-modifying effect in osteoarthritis.

In the present report, we show that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL-1beta (10 ng/mL) by an increased DNA binding activity of NF-kappaB and AP-1 transcription factors. Incubation of the cells with 10(-5) M rhein for 24 h was found to reduce this activity, particularly in the case of AP-1. Mitogen activated kinases (ERK-1 and ERK-2) were activated by exposure of the chondrocytes to 1-h treatment with IL-1beta. This effect was greater in hypoxia (3% O2) than in normoxia (21% O2). Rhein was capable of reducing the IL-1beta-stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The level of c-jun protein, an element of AP-1 complex, was increased by exposure of the chondrocytes to IL-1beta after 2, 6, and 24 h. Addition of rhein at 10(-5) M for 24 h did not reduce the c-jun protein amount. The mRNA steady-state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24-h treatment with 10(-5) M rhein. This stimulating effect was also observed in the presence of IL-1beta, suggesting that the drug could prevent or reduce the IL-1beta-induced inhibition of extracellular matrix synthesis. IL-1-induced collagenase (MMPI) expression was significantly decreased by rhein in the same conditions. In conclusion, rhein can effectively inhibit the IL-1-activated MAPK pathway and the binding of NF-kappaB and AP-1 transcription factors, two key factors involved in the expression of several proinflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMPI synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the antiosteoarthritic properties of rhein and its disease-modifying effects on OA cartilage, in spite of absence of inhibition at prostaglandin level.

Pharmacokinetics of tenatoprazole, a newly synthesized proton pump inhibitor, in healthy male Caucasian volunteers.

The pharmacokinetics of tenatoprazole (CAS 113712-98-4), a newly synthesized proton pump inhibitor, and its metabolites TU-501 (sulfide form) and TU-502 (sulfone form) were investigated in an ascending-dose parallel-group study at the dose levels of 10, 20, 40, 80 and 120 mg. A total of 30 healthy Caucasian male volunteers (6 in each dose group) received a single dose at Day 1 (fasted state) and repeated doses from Day 14 to Day 20. CYP2C19 genotype status was determined in all subjects. Concentrations of tenatoprazole, TU-501 and TU-502 in plasma and urine were measured by a validated HPLC/MS/MS method. The single and multiple-dose study provided reliable tolerance. After the single administrations, plasma concentrations reached a maximum between 2.5 and 4.3 h post dose, and thereafter decreased according to a terminal half live (T1/2) ranging from 4.8 to 7.7 h. Similar T1/2 were obtained on first and the last administration, and the steady state was reached after 5 days. Cmax and AUC increased linearly between 10 to 80 mg. However, with the 120 mg dose, the observed Cmax was higher than expected, especially at steady state. For TU-501 and TU-502 metabolites, Cmax and AUC increased linearly after repeated administration between 40 and 120 mg.