Delta opioid receptor expression correlates to skin ageing and melanin expression in Asian women.

While the evidence for the implication of opioid receptors (OPr) in ageing is growing, there is, to our knowledge, no study focusing directly on changes in vivo cutaneous OPr expression with increasing age. We thus investigated OPr expression in 30 healthy female Asian volunteers in Southern China whose ages range from the early 20s to the early 60s. Excisional biopsies were taken from the sun-exposed extensor area of the lower arm and the photo-protected area of the upper inner arm. The thickness of the epidermal layers, melanin content, as well as expression of mu-opioid receptors (MOPr) and delta-opioid receptors (DOPr) were compared between different age ranges and photo-exposure status. Significant increased epidermal hypertrophy on the extensor surface was observed. There was significant reduction of DOPr in the epidermis with increasing age, independent of photo-ageing. The increase of melanin was significantly correlated with epidermal DOPr expression, not with MOPr expression. DOPr expression could thus serve as a marker for real biological ageing unaffected by chronic photo-exposure. Additionally, DOPr expression was inversely correlated with the deposition of melanin. Based on these results, we hypothesise that regulation of DOPr expression could be used to improve aged skin, including hyperpigmentation.

Strat-M® positioning for skin permeation studies: A comparative study including EpiSkin® RHE, and human skin.

In the development and optimization of dermatological products, In Vitro Permeation Testing (IVPT) is pivotal for controlled study of skin penetration. To enhance standardization and replicate human skin properties reconstructed human skin and synthetic membranes are explored as alternatives. Strat-M® is a membrane designed to mimic the multi-layered structure of human skin for IVPT. For instance, in Strat-M®, the steady-state fluxes (JSS) of resorcinol in formulations free of permeation enhancers were found to be 41 ± 5 µg/cm2·h for the aqueous solution, 42 ± 6 µg/cm2·h for the hydrogel, and 40 ± 6 µg/cm2·h for the oil-in-water emulsion. These results were closer to excised human skin (5 ± 3, 9 ± 2, 13 ± 6 µg/cm2·h) and surpassed the performance of EpiSkin® RHE (138 ± 5, 142 ± 6, and 162 ± 11 µg/cm2·h). While mass spectrometry and Raman microscopy demonstrated the qualitative molecular similarity of EpiSkin® RHE to human skin, it was the porous and hydrophobic polymer nature of Strat-M® that more faithfully reproduced the skin's diffusion-limiting barrier. Further validation through similarity factor analysis (∼80-85%) underscored Strat-M®'s significance as a reliable substitute for human skin, offering a promising approach to enhance realism and reproducibility in dermatological product development.

A novel physiologically based pharmacokinetic model of rectal absorption, evaluated and verified using clinical data on 10 rectally administered drugs.

We present a physiologically based pharmacokinetic (PBPK) model simulating systemic drug concentrations following administration to the human rectum. Rectum physiology is parameterized based on literature data. The model utilizes in vitro release (IVRT) profiles from which drug mass transfer through the rectal fluid and tissue and into the systemic circulation are predicted. Due to a lack of data, rectal fluid and tissue absorption parameters are predicted either from colon absorption, with modifications relevant to rectal physiology, or optimized. The PBPK model is evaluated by simulating 29 clinical studies for 10 drugs. For 8 drugs (diazepam, diclofenac, indomethacin, naproxen, paracetamol, pentobarbital, phenobarbital and theophylline) the bias (average fold error, AFE) and precision (absolute average fold error, AAFE) of Cmax, AUC0-t and AUC0-inf simulations range from 0.87 to 2.22, indicating good agreement with observed values. For prochlorperazine and promethazine, the AFEs and AAFEs of Cmax predictions are 1.30 and 2.52, respectively. TheAUC0-t and AUC0-inf are overpredicted for both compounds(AFEs and AAFEs from 2.66 to 4.90). This results from a lack of reliable elimination data for prochlorperazine and the relevance of the IVRT profiles used in the promethazine model. The model paves the way for more mechanistic rectal drug absorption studies and virtual bioequivalence methods for rectal drug products.

Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum.

Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR-partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400-1500 cm-1) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R2 = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R2 (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies.

Highlighting the efficiency of ultrasound-based emulsifier-free emulsions to penetrate reconstructed human skin.

OBJECTIVE: The cosmetic industry endeavours to strengthen the greener and safer claims of processes to respond to the high demand from customers for natural and environmentally friendly products. High-frequency ultrasonication technology (HFUT) is a physical process enabling the stabilization of emulsions without requiring additional ingredients, such as emulsifying surfactants (ES) to be introduced into the formulations. In this study, key formulation characteristics of an emulsion synthesized by HFUT and a reference emulsion (RE) were compared, as well as the permeation kinetics of caffeine, used as a model active cosmetic ingredient, from both types of emulsions. METHODS: The pH, droplet size and viscosity of emulsions prepared by the HFUT and the RE were determined and compared. The permeation of caffeine from the HFUT emulsion and the RE applied to the surface of reconstructed human epidermis (RHE) models was compared. RESULTS: The ES-free formulations prepared by HFUT displayed a nearly 2-fold lower average droplet size and over 3-fold greater viscosity, compared to the RE. Despite these differences, the absence of ES in the HFUT emulsion did not significantly alter the permeation kinetics of caffeine through RHE. The caffeine steady-state flux, lag time and permeability coefficients differed by 20%-30% only. CONCLUSION: This study demonstrates the potential of the HFUT to yield topical cosmetic products with lower requirements ingredients-wise, without losing efficacy, supporting the possible implementation of the technology in the cosmetic industry.

OBJECTIF: l'industrie cosmétique œuvre à renforcer les revendications plus écologiques et plus sûres des processus pour répondre à la forte demande des clients de produits naturels et plus respectueux de l'environnement. La technologie d'ultrasons à haute fréquence (High-Frequency Ultrasonication Technology, HFUT) est un processus physique permettant de stabiliser les émulsions sans qu'il soit nécessaire d'ajouter des ingrédients supplémentaires, tels que des surfactants émulsifiants, aux formulations. Dans cette étude, les principales caractéristiques de formulation d'une émulsion synthétisée par HFUT et d'une émulsion de référence ont été comparées, ainsi que la cinétique de perméation de la caféine, utilisée comme ingrédient cosmétique actif modèle, dans les deux types d'émulsion. MÉTHODES: le pH, la taille des gouttelettes, et la viscosité de l'émulsion préparée par HFUT et de l'émulsion de référence ont été déterminés et comparés. La perméation de la caféine de l'émulsion HFUT et de l'émulsion de référence appliquées à la surface de modèles d'épiderme humain reconstruit a été comparée. RÉSULTATS: la formulation sans surfactants émulsifiants préparée par HFUT présentait une taille moyenne de gouttelettes presque 2 fois plus faible et une viscosité plus de 3 fois supérieure comparée à l'émulsion de référence. Malgré ces différences, l'absence de surfactants émulsifiants dans l'émulsion HFUT n'a pas significativement modifié la cinétique de perméation de la caféine dans l'épiderme humain reconstruit. Le flux à l'état d'équilibre de la caféine, le temps de latence et les coefficients de perméabilité différaient de 20 à 30 % uniquement. CONCLUSION: cette étude démontre le potentiel de la technologie HFUT à générer des produits cosmétiques topiques possédant des exigences plus faibles en termes d'ingrédients, sans perte d'efficacité, soutenant la mise en œuvre éventuelle de la technologie dans l'industrie cosmétique.

Investigations on detoxification mechanisms of novel para-phenylenediamine analogues through N-acetyltransferase 1 (NAT-1).

Para-phenylenediamine (PPD) is one of the most used chemicals in oxidative hair dyes. However, its use has been associated with adverse effects on health, including contact dermatitis and other systemic toxicities. Novel PPD derivatives have been proposed as a safer replacement for PPD. This can be achieved if these molecules minimally permeate the skin and/or are easily metabolised by enzymes in the skin (e.g., N-acetyltransferase-1 (NAT-1)) into innocuous compounds before gaining systemic entry. This study investigated the detoxification pathway mediated by NAT-1 enzymes on 6 synthesized PPD analogues (namely, P1-P6) with different chemical properties, to study the role of functional groups on detoxification mechanisms in HaCaT skin cells. These compounds were carefully designed with different chemical properties (whereby the ortho position of PPD was substituted by nucleophile and electrophile groups to promote N-acetylation reactions, metabolism and clearance). Compounds P2-P4 N-acetylated at 54-49 nmol/mg/min, which is 1.6 times higher than N-acetylation of PPD, upregulated NAT-1 activity from 8-7% at 50 µM to 22-11% at 100 µM and showed 4 times higher rate of elimination (k equal to 0.141 ± 0.016-0.124 ± 0.01 h-1) and 3 times faster rate of clearance (0.172 ± 0.007-0.158 ± 0.005 h-1mgprotein-1) than PPD (0.0316 ± 0.0019 h-1, 0.0576 ± 0.003 h-1mg protein-1, respectively). The data suggest that nucleophile substituted compounds detoxify at a faster rate than PPD. Our metabolic and detoxification mechanistic studies revealed significantly higher rates of N-acetylation, NAT-1 activity and higher detoxification of P2-P4 in keratinocytes, suggesting the importance of nucleophilic groups at the ortho position in PPD to reduce toxicity of aniline-based dyes on human skin cells.

Development of novel alternative hair dyes to hazardous para-phenylenediamine.

Most of the permanent hair dye products contain p-phenylenediamine (PPD), a well-known skin sensitizer. PPD may cause cutaneous reactions and leads to allergic contact dermatitis (ACD), a condition with major medical and financial repercussions. Hair dye-induced ACD represents a growing concern both for consumers and the cosmetics industry. In this study we introduced novel side chains on the PPD molecule with the goal of overcoming the hazard potential of PPD. Our strategy relies on the replacement of the colorless PPD with new, larger and intrinsically colorled PPD derivatives to reduce dermal penetration and thus the skin sensitization potential. We synthesized two oligomers with bulky side-chains, which displayed 7-8 times lower cytotoxicity than PPD, a significantly weaker sensitization potential (22.0 % and 23.8 % versus 55.5 % for PPD) in the Direct Peptide Reactivity Assay, minimal cumulative penetration through excised skin and an intrinsic ability to colour and preserve the nuance when applied on bleached hair. The lower skin permeation and sensitizing potential are absolutely crucial and give a clear advantage of our products over other standards. These novel PPD hair dyes show significantly less hazard potential than PPD and may, upon further risk assessment studies, replace PPD in consumer care products.

Freezing Weakens the Barrier Function of Reconstructed Human Epidermis as Evidenced by Raman Spectroscopy and Percutaneous Permeation.

The development and characterization of reconstructed human epidermis (RHE) is an active area of R&D. RHE can replace animal tissues in pharmaceutical, toxicological and cosmetic sciences, yielding scientific and ethical advantages. RHEs remain costly, however, due to consumables and time required for their culture and a short shelf-life. Storing, i.e., freezing RHE could help reduce costs but to date, little is known on the effects of freezing on the barrier function of RHE. We studied such effects using commercial EpiSkin™ RHE stored at -20, -80 and -150 °C for 1 and 10 weeks. We acquired intrinsic Raman spectra in the stratum corneum (SC) of the RHEs as well as spectra obtained following topical application of resorcinol in an aqueous solution. In parallel, we quantified the effects of freezing on the permeation kinetics of resorcinol from time-dependent permeation experiments. Principal component analyses discriminated the intrinsic SC spectra and the spectra of resorcinol-containing RHEs, in each case on the basis of the freezing conditions. Permeation of resorcinol through the frozen RHE increased 3- to 6-fold compared to fresh RHE, with the strongest effect obtained from freezing at -20 °C for 10 weeks. Due to the extensive optimization and standardization of EpiSkin™ RHE, the effects observed in our work may be expected to be more pronounced with other RHEs.

Opioids and skin homeostasis, regeneration and ageing - What's the evidence?

What has the opioid receptor system, known for beneficial as well as disastrous effects in the central nervous system, to do with skin? The question is appropriate considering the fact that the nervous system and the skin both derive from the ectoderm. As part of the skin neuroendocrine system, the opioid receptor system exemplifies the closeness between the nervous system and the skin. Overexpression of the δ-opioid receptor in keratinocytes yields dysregulation of involucrin, loricrin, and filaggrin, proteins essential to the integrity of the skin barrier. The µ-opioid receptor ligand ß-endorphin, produced in the pituitary gland and a variety of skin cells, promotes wound healing via regulation of cytokeratin 16 and TGF-ß type II receptor expression in keratinocytes. These and other published results discussed in this viewpoint are evidence for the fundamental role of the skin opioid receptor system in skin homeostasis, regeneration and ageing. While considerable progress in understanding the opioid receptors' function on the cellular level has been made, there is a need to link these results to physiological observations for the development of local skin therapies.

What happens in the skin? Integrating skin permeation kinetics into studies of developmental and reproductive toxicity following topical exposure.

Animal-based developmental and reproductive toxicological studies involving skin exposure rarely incorporate information on skin permeation kinetics. For practical reasons, animal studies cannot investigate the many factors which can affect human skin permeation and systemic uptake kinetics in real-life scenarios. Traditional route-to-route extrapolation is based on the same types of experiments and requires assumptions regarding route similarity. Pharmacokinetic modeling based on skin physiology and structure is the most efficient way to incorporate the variety of intrinsic skin and exposure-dependent parameters occurring in clinical and occupational settings into one framework. Physiologically-based pharmacokinetic models enable the integration of available in vivo, in vitro and in silico data to quantitatively predict the kinetics of uptake at the site of interest, as needed for 21st century toxicology and risk assessment. As demonstrated herein, proper interpretation and integration of these data is a multidisciplinary endeavor requiring toxicological, risk assessment, mathematical, pharmaceutical, biological and dermatological expertise.

Estimation of in vivo dose of dermally applied chemicals leading to estrogen/androgen receptor-mediated toxicity from in vitro data--Illustration with four reproductive toxicants.

We present a quantitative in vitro-in vivo extrapolation framework enabling the estimation of the external dermal exposure dose from in vitro experimental data relevant to a toxicity pathway of interest. The framework adapts elements of the biological pathway altering dose (BPAD) method [Judson et al. Chem Res Toxicol 2011;24:451] to the case of dermal exposure. Dermal doses of four toxicants equivalent to concentrations characterizing their effect on estrogen receptor α or androgen receptor activity in chemical-activated luciferase expression (CALUX) assays are estimated. The analysis shows that dermal BPADs, calculated from one in vitro concentration, can differ by up to a factor of 55, due to the impact applied dose and dermal exposure scenarios can have on skin permeation kinetics. These features should therefore be taken into account in risk assessment of dermally applied chemicals.

Open source software implementation of an integrated testing strategy for skin sensitization potency based on a Bayesian network.

An open-source implementation of a previously published integrated testing strategy (ITS) for skin sensitization using a Bayesian network has been developed using R, a free and open-source statistical computing language. The ITS model provides probabilistic predictions of skin sensitization potency based on in silico and in vitro information as well as skin penetration characteristics from a published bioavailability model (Kasting et al., 2008). The structure of the Bayesian network was designed to be consistent with the adverse outcome pathway published by the OECD (Jaworska et al., 2011, 2013). In this paper, the previously published data set (Jaworska et al., 2013) is improved by two data corrections and a modified application of the Kasting model. The new data set implemented in the original commercial software package and the new R version produced consistent results. The data and a fully documented version of the code are publicly available (http://ntp.niehs.nih.gov/go/its).

Bayesian integrated testing strategy to assess skin sensitization potency: from theory to practice.

Frameworks to predict in vivo effects by integration of in vitro, in silico and in chemico information using mechanistic insight are needed to meet the challenges of 21(st) century toxicology. Expert-based approaches that qualitatively integrate multifaceted data are practiced under the term 'weight of evidence', whereas quantitative approaches remain rare. To address this gap we previously developed a methodology to design an Integrated Testing Strategy (ITS) in the form of a Bayesian Network (BN). This study follows up on our proof of concept work and presents an updated ITS to assess skin sensitization potency expressed as local lymph node assay (LLNA) potency classes. Modifications to the ITS structure were introduced to include better mechanistic information. The parameters of the updated ITS were calculated from an extended data set of 124 chemicals. A detailed validation analysis and a case study were carried out to demonstrate the utility of the ITS for practical application. The improved BN ITS predicted correctly 95% and 86% of chemicals in a test set (n = 21) for hazard and LLNA potency classes, respectively. The practical value of using the BN ITS is far more than a prediction framework when all data are available. The BN ITS can develop a hypothesis using subsets of data as small as one data point and can be queried on the value of adding additional tests before testing is commenced. The ITS represents key steps of the skin sensitization process and a mechanistically interpretable testing strategy can be developed. These features are illustrated in the manuscript via practical examples.

Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo.

There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

Mathematical and pharmacokinetic modelling of epidermal and dermal transport processes.

Topical delivery to the various regions of the skin and underlying tissues, transdermal drug delivery and dermal exposure to environmental chemicals are important areas of research. Mathematical models of epidermal and dermal transport, involving penetration of a solute through various layers of the skin, metabolism in the skin and its subsequent distribution and clearance into systemic circulation from underlying tissues, play an essential role in this research area and are reviewed in this work.

Design and performance of a spreadsheet-based model for estimating bioavailability of chemicals from dermal exposure.

A comprehensive transient model of chemical penetration through the stratum corneum, viable epidermis and dermis formulated in terms of an Excel™ spreadsheet and associated add-in is presented. The model is a one-dimensional homogenization of underlying microscopic transport models for stratum corneum and dermis; viable epidermis is treated as unperfused dermis. The model's salient features are a detailed structural description of the skin layers, a combination of first-principles based transport equations and empirical partition and diffusion coefficients, and the capability of simulating a variety of exposure scenarios. Model predictions are compared with representative in vitro skin permeation data obtained from the literature using as summary parameters total absorption (Q(abs)), maximum flux (J(max)) and skin permeability coefficient (k(p)). The results of this evaluation demonstrate the current state-of-the-art in prediction of transient skin absorption and highlight areas in which further elaborations are needed to obtain satisfactory predictions.

Modeling the human skin barrier--towards a better understanding of dermal absorption.

Many drugs are presently delivered through the skin from products developed for topical and transdermal applications. Underpinning these technologies are the interactions between the drug, product and skin that define drug penetration, distribution, and elimination in and through the skin. Most work has been focused on modeling transport of drugs through the stratum corneum, the outermost skin layer widely recognized as presenting the rate-determining step for the penetration of most compounds. However, a growing body of literature is dedicated to considering the influence of the rest of the skin on drug penetration and distribution. In this article we review how our understanding of skin physiology and the experimentally observed mechanisms of transdermal drug transport inform the current models of drug penetration and distribution in the skin. Our focus is on models that have been developed to describe particular phenomena observed at particular sites of the skin, reflecting the most recent directions of investigation.

A framework incorporating the impact of exposure scenarios and application conditions on risk assessment of chemicals applied to skin.

PURPOSE: 1. To develop a framework for exposure calculation via the dermal route to meet the needs of 21st century toxicity testing and refine current approaches; 2. To demonstrate the impact of exposure scenario and application conditions on the plasma concentration following dermal exposure. METHOD: A workflow connecting a dynamic skin penetration model with a generic whole-body physiologically-based pharmacokinetic (PBPK) model was developed. The impact of modifying exposure scenarios and application conditions on the simulated steady-state plasma concentration and exposure conversion factor was investigated for 9 chemicals tested previously in dermal animal studies which did not consider kinetics in their experimental designs. RESULTS: By simulating the animal study scenarios and exposure conditions, we showed that 7 studies were conducted with finite dose exposures, 1 with both finite and infinite dose exposures (in these 8 studies, an increase in the animal dose resulted in an increase in the simulated steady-state plasma concentrations (C p,ss)), while 1 study was conducted with infinite dose exposures only (an increase in the animal dose resulted in identical C p,ss). Steady-state plasma concentrations were up to 30-fold higher following an infinite dose scenario vs. a finite dose scenario, and up to 40-fold higher with occlusion vs. without. Depending on the chemical, the presence of water as a vehicle increased or decreased the steady-state plasma concentration, the largest difference being a factor of 16. CONCLUSIONS: The workflow linking Kasting's model of skin penetration and whole-body PBPK enables estimation of plasma concentrations for various applied doses, exposure scenarios and application conditions. Consequently, it provides a quantitative, mechanistic tool to refine dermal exposure calculations methodology for further use in risk assessment.

Convective transport of highly plasma protein bound drugs facilitates direct penetration into deep tissues after topical application.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Many products are applied to human skin for local effects in deeper tissues. Animal studies suggest that deep dermal and/or subcutaneous delivery may be facilitated by both dermal diffusion and transport via the cutaneous vasculature. However, the relationship between the extent and pathways of penetration, drug physicochemical properties and deeper tissue physiology is not well understood. WHAT THIS STUDY ADDS: We have used a physiologically based pharmacokinetic model to analyze published human cutaneous microdialysis data, complemented by our own in vitro skin penetration studies. We found that convective blood, lymphatic and interstitial flow led to significant deep tissue concentrations for drugs that are highly plasma protein bound. In such cases, deeper tissue concentrations will occur earlier and may be several orders of magnitude greater than predicted by passive dermal diffusion alone. AIMS: To relate the varying dermal, subcutaneous and muscle microdialysate concentrations found in man after topical application to the nature of the drug applied and to the underlying physiology. METHODS: We developed a physiologically based pharmacokinetic model in which transport to deeper tissues was determined by tissue diffusion, blood, lymphatic and intersitial flow transport and drug properties. The model was applied to interpret published human microdialysis data, estimated in vitro dermal diffusion and protein binding affinity of drugs that have been previously applied topically in vivo and measured in deep cutaneous tissues over time. RESULTS: Deeper tissue microdialysis concentrations for various drugs in vivo vary widely. Here, we show that carriage by the blood to the deeper tissues below topical application sites facilitates the transport of highly plasma protein bound drugs that penetrate the skin, leading to rapid and significant concentrations in those tissues. Hence, the fractional concentration for the highly plasma protein bound diclofenac in deeper tissues is 0.79 times that in a probe 4.5 mm below a superficial probe whereas the corresponding fractional concentration for the poorly protein bound nicotine is 0.02. Their corresponding estimated in vivo lag times for appearance of the drugs in the deeper probes were 1.1 min for diclofenac and 30 min for nicotine. CONCLUSIONS: Poorly plasma protein bound drugs are mainly transported to deeper tissues after topical application by tissue diffusion whereas the transport of highly plasma protein bound drugs is additionally facilitated by convective blood, lymphatic and interstitial transport to deep tissues.

Multiphoton microscopy and fluorescence lifetime imaging provide a novel method in studying drug distribution and metabolism in the rat liver in vivo.

Multiphoton microscopy has been shown to be a useful tool in studying drug distribution in biological tissues. In addition, fluorescence lifetime imaging provides information about the structure and dynamics of fluorophores based on their fluorescence lifetimes. Fluorescein, a commonly used fluorescent probe, is metabolized within liver cells to fluorescein mono-glucuronide, which is also fluorescent. Fluorescein and its glucuronide have similar excitation and emission spectra, but different fluorescence lifetimes. In this study, we employed multiphoton fluorescence lifetime imaging to study the distribution and metabolism of fluorescein and its metabolite in vivo in rat liver. Fluorescence lifetime values in vitro were used to interpret in vivo data. Our results show that the mean fluorescence lifetimes of fluorescein and its metabolite decrease over time after injection of fluorescein in three different regions of the liver. In conclusion, we have demonstrated a novel method to study a fluorescent compound and metabolite in vivo using multiphoton fluorescence lifetime imaging.