Discovery of 4-((2S,4S)-4-Ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic Acid (LNP023), a Factor B Inhibitor Specifically Designed To Be Applicable to Treating a Diverse Array of Complement Mediated Diseases.

The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.

Percutaneous absorption between frog species: Variability in skin may influence delivery of therapeutics.

Frogs have permeable skin, so transdermal delivery provides a practical alternative to traditional dosing routes. However, little is known about how frog skin permeability differs interspecifically, and there are different reported clinical outcomes following topical application of the same chemical in different frog species. This study collated in vitro absorption kinetic data previously reported for two frog species: the green tree frog (Litoria caerulea) and the cane toad (Rhinella marina), and used linear mixed-effects modelling to produce a model of absorption. Histology of skin samples from each species was performed to observe morphological differences that may affect absorption. Absorption kinetics differed significantly between species, with the logP of the applied chemical a better predictor of permeability than molecular weight. Application site also influenced permeability, with dorsal permeability consistently higher in cane toads. Ventral permeability was more consistent between species. Skin thickness differed between species and skin regions, and this may explain the differences in absorption kinetics. Guidelines for selecting chemicals and dosing site when treating frogs are presented. The permeability differences identified may explain the poor reproducibility reported in the treatment of disease across frog species, and reinforces the importance of considering interspecies differences when designing therapeutic treatments for frogs.

Effects of skin region and relative lipophilicity on percutaneous absorption in the toad Rhinella marina.

Owing to the dynamic interaction between frog skin and the environment, xenobiotics in frog habitats are of particular concern, and knowledge of percutaneous absorption in frog skin is necessary for risk-mitigation purposes. Baseline transdermal kinetics in adult aquatic and arboreal frog species have recently been reported; however, there is little information regarding absorption kinetics in adult terrestrial species. The present study investigated the in vitro absorption kinetics of 3 model chemicals-caffeine, benzoic acid, and ibuprofen-through different skin regions in the terrestrial toad Rhinella marina. Caffeine flux was consistently higher than that of the other 2 chemicals (p < 0.001), whereas the fluxes of the moderately and highly lipophilic chemicals (benzoic acid and ibuprofen) were similar, regardless of skin region. When considering individual chemicals, caffeine demonstrated increased flux through the ventral pelvic skin compared with the ventral thoracic or dorsal skin regions. Flux did not differ between skin regions for either benzoic acid or ibuprofen. These findings have implications for management of environmental contamination in frog habitats, as many environmental xenobiotics are of moderate to high lipophilicity and would be expected to be equally absorbed from all skin surfaces in terrestrial toads. Environ Toxicol Chem 2019;38:361-367. © 2018 SETAC.

Prolonging food shelf-life by dual actives release from multi-layered polymer particles.

Biodegradable polymer based 'controlled release packaging' technology has ability to release packaging actives in controlled manner to prolong the food shelf-life. Currently available systems are not sufficiently capable of releasing multiple actives in sustainable fashion. Hence, the purpose of this study was to develop dual actives (antioxidant and antibacterial) loaded multilayered microparticles in one step and to release them at rates suitable for long-term inhibition of bacterial growth as well as lipid oxidation in food. In order to achieve this goal, 2 kinds of multilayered polymer particles made up of PLLA (Poly(l-lactic acid)) and PLGA (Poly(dl-lactic-co-glycolic acid) with varying viscosity were developed using emulsion solvent evaporation method. Surprisingly, low viscous PLGA resulted tri-layered particles (PLGA/PLLA/PLGA: shell/middle/core) instead of bi-layered (PLGA/PLLA: shell/core) particles as observed for high viscous PLGA. The mechanism of formation of tri-layered particles was investigated in detail. The outermost layer consisted of relatively more hydrophilic polymer PLGA along with benzoic acid (antibacterial) and the inner core comprised of hydrophobic polymer PLLA and tocopherol (antioxidant). Release study demonstrated that release rate of dual actives were significantly accelerated from tri-layered particles in comparison to bi-layered one and their release profiles can be well explained with the help of Ridger-Peppas model. Both sets of particles exhibited long-term antibacterial (against both Escherichia coli and Staphylococcus aureus) as well as antioxidant effect over a period of 60 days. The results show for the first time the feasibility of using multilayered microparticles to prolong the food shelf-life by simultaneous release of multiple actives.

Regional variation in percutaneous absorption in the tree frog Litoria caerulea.

Frog skin structure and physiology differs between skin regions, however little is known about how these differences affect transdermal absorption of chemicals. Further, no information is available regarding how the relative lipophilicity of a chemical influences its transdermal pharmacokinetics in frog skin. This study investigated the in vitro percutaneous absorption of three model chemicals - benzoic acid, caffeine, and ibuprofen - through dorsal and ventral skin of the tree frog Litoria caerulea. Flux was significantly higher through the ventral skin for all chemicals. Relative lipophilicity affected flux differently in different skin regions. These differences are likely due to significantly thicker dorsal skin increasing absorption path length, and also possibly owing to lipoid secretions on the dorsum providing an additional diffusional barrier. This knowledge can advise risk mitigation of xenobiotics in agricultural and industrial settings, and also guide selection of chemicals and doses when considering transdermal drug therapy in captive frogs.

Comparison of the Skin Penetration of 3 Metabolically Stable Chemicals Using Fresh and Frozen Human Skin.

BACKGROUND: The Cosmetics Europe ADME Task Force is developing in vitro and in silico tools for predicting skin and systemic concentrations after topical application of cosmetic ingredients. There are conflicting reports as to whether the freezing process affects the penetration of chemicals; therefore, we evaluated whether the storage of human skin used in our studies (8-12 weeks at -20°C) affected the penetration of model chemicals. METHODS: Finite doses of trans-cinnamic acid (TCA), benzoic acid (BA), and 6-methylcoumarin (6MC) (non-volatile, non-protein reactive and metabolically stable in skin) were applied to fresh and thawed frozen skin from the same donors. The amounts of chemicals in different skin compartments were analysed after 24 h. RESULTS: Although there were some statistical differences in some parameters for 1 or 2 donors, the penetration of TCA, BA, and 6MC was essentially the same in fresh and frozen skin, i.e., there were no biologically relevant differences in penetration values. Statistical differences that were evident indicated that penetration was marginally lower in frozen than in fresh skin, indicating that the barrier function of the skin was not lost. CONCLUSION: The penetration of the 3 chemicals was essentially unaffected by freezing the skin at -20°C for up to 12 weeks.

Metabolism and antioxidant effect of malaxinic acid and its corresponding aglycone in rat blood plasma.

Malaxinic acid (MA) is a phenolic acid compound, found mainly in pear fruits (Pyrus pyrifolia N.), that is isoprenylated on the C-3 position of benzoic acid. Recently, the effects of prenylated phenolics on health have received much interest owing to their reported potent beneficial biological effects. We conducted a comparative study in rats to determine the metabolism, pharmacokinetics, and antioxidative activities of MA and its corresponding aglycone (MAA). MA and MAA were orally administered to rats (Sprague-Dawley, male, 6 weeks old) and their metabolites in plasma were analyzed. In addition, the MA metabolites in plasma were separated and the structures were confirmed via NMR and HR-MS analyses. The antioxidative activities of MA and MAA were evaluated by measuring their inhibitory effects on the 2,2'-azobis(2-amidinopropane)dihydrochloride- or copper ion-induced lipid peroxidation of rat plasma. MA was not absorbed in the intact form (the glucoside); both MA and MAA were absorbed as MAA and its metabolite form (glucuronide or sulfate). Moreover, the observed metabolite was the glucuronate of MAA rather than the glucuronide or sulfate. Concentrations of the free form of aglycone (MA administration, 4.6 ± 2.2µM; MAA administration, 7.2 ± 2.3µM) and total MAA (MA administration, 19.6 ± 4.4µM; MAA administration, 21.7 ± 3.3µM) in plasma reached a maximum at 15min after the oral administration of MA and MAA, respectively. The relative inhibitory effects on the formation of cholesteryl ester hydroperoxides in plasma collected at 15min after the oral administration of MA, MAA, and p-hydroxybenzoic acid (p-HBA) were as follows: MAA > MA ≥ p-HBA > control. Although the majority of MA and MAA is metabolized to conjugates, the compounds may contribute to the antioxidant defenses in the blood circulation owing to the presence of a phenolic hydroxyl group in the free form.

Pharmacokinetic data reduce uncertainty in the acceptable daily intake for benzoic acid and its salts.

The current acceptable daily intake (ADI) for benzoic acid and its salts as food additives is 0-5 mg/kg body weight. This accounts for a total uncertainty factor (UF) of 100, which includes a default factor of 10 for interspecies differences. Based on pharmacokinetic data in rodents and humans, we derived a chemical-specific adjustment factor (CSAF) of 2 for the pharmacokinetic component of the interspecies UF. Additional analyses indicate that this CSAF is conservative and interspecies differences between rats and humans are likely closer to unity. Human clinical studies indicate that the pharmacokinetics of benzoic acid and its salts are similar in children and adults, and that there is a lack of adverse events in humans at doses comparable to the no observed adverse effect level (NOAEL) in rodents; this suggests that the pharmacokinetic UF for intraspecies variability, as well as the pharmacodynamic components of the UFs, may also be reduced, although we did not calculate to what degree. In conclusion, the total UF can be reduced to 50 (2 for interspecies differences in pharmacokinetics, 2.5 for interspecies differences in pharmacodynamics, and 10 for intraspecies variability), which would increase the ADI to 0-10 mg/kg body weight.

Further development of an in vitro model for studying the penetration of chemicals through compromised skin.

A new in vitro model based on the electrical resistance properties of the skin barrier has been established in this laboratory. The model utilises a tape stripping procedure in dermatomed pig skin that removes a specific proportion of the stratum corneum, mimicking impaired barrier function observed in humans with damaged skin. The skin penetration and distribution of chemicals with differing physicochemical properties, namely; Benzoic acid, 3-Aminophenol, Caffeine and Sucrose has been assessed in this model. Although, skin penetration over 24h differed for each chemical, compromising the skin did not alter the shape of the time course profile, although absorption into receptor fluid was higher for each chemical. Systemic exposure (receptor fluid, epidermis and dermis), was marginally higher in compromised skin following exposure to the fast penetrant, Benzoic acid, and the slow penetrant Sucrose. The systemically available dose of 3-Aminophenol increased to a greater extent and the absorption of Caffeine was more than double in compromised skin, suggesting that Molecular Weight and Log Pow, are not the only determinants for assessing systemic exposure under these conditions. Although further investigations are required, this in vitro model may be useful for prediction of dermal route exposure under conditions where skin barrier is impaired.

Vehicle effects on human stratum corneum absorption and skin penetration.

This study evaluated the effects of three vehicles-ethanol (EtOH), isopropyl alcohol (IPA), and isopropyl myristate (IPM)-on stratum corneum (SC) absorption and diffusion of the [14C]-model compounds benzoic acid and butenafine hydrochloride to better understand the transport pathways of chemicals passing through and resident in SC. Following application of topical formulations to human dermatomed skin for 30 min, penetration flux was observed for 24 h post dosing, using an in vitro flow-through skin diffusion system. Skin absorption and penetration was compared to the chemical-SC (intact, delipidized, or SC lipid film) binding levels. A significant vehicle effect was observed for chemical skin penetration and SC absorption. IPA resulted in the greatest levels of intact SC/SC lipid absorption, skin penetration, and total skin absorption/penetration of benzoic acid, followed by IPM and EtOH, respectively. For intact SC absorption and total skin absorption/penetration of butenafine, the vehicle that demonstrated the highest level of sorption/penetration was EtOH, followed by IPA and IPM, respectively. The percent doses of butenafine that were absorbed in SC lipid film and penetrated through skin in 24 h were greatest for IPA, followed by EtOH and IPM, respectively. The vehicle effect was consistent between intact SC absorption and total chemical skin absorption and penetration, as well as SC lipid absorption and chemical penetration through skin, suggesting intercellular transport as a main pathway of skin penetration for model chemicals. These results suggest the potential to predict vehicle effects on skin permeability with simple SC absorption assays. As decontamination was applied 30 min after chemical exposure, significant vehicle effects on chemical SC partitioning and percutaneous penetration also suggest that skin decontamination efficiency is vehicle dependent, and an effective decontamination method should act on chemical solutes in the lipid domain.

Depth-dependent stratum corneum permeability in human skin in vitro.

The stratum corneum (SC), a permeable membrane, limits percutaneous penetration. As SC chemical and structural properties responsible for skin barrier function appear depth-related, we conducted an in vitro dermatopharmacokinetic study on intact and adhesive tape-stripped skin samples to clarify whether SC is a homogeneous barrier for chemical transport. SC concentration-thickness profiles were determined for four C-14 labeled model chemicals, panthenol, benzoic acid, paraoxon and butenafine, using the tape-stripping approach. Data analysis with the unsteady-state diffusion equation of Fick's second law permitted a chemical diffusion coefficient in SC. To evaluate the consistency of SC permeability from its surface to lower levels, the skin was tape-stripped five to 10 times to remove the upper cell layers before chemical application, such that diffusion coefficients could be determined from three SC depth levels (0, 5 and 10 tape strips). Results suggested the depth-dependency of SC permeability to panthenol, benzoic acid and butenafine; the diffusion coefficient of panthenol decreased significantly after the first five tape strips and subsequently remained consistent. A progressive increase in diffusion coefficients of benzoic acid and butenafine was observed as tape-stripping levels increased. The removal of superficial SC did not result in a significant difference in the paraoxon diffusion coefficient. For individual chemicals, a variation in the diffusion coefficient from SC surface to deeper layers agreed with the change of the diffusion coefficient over time in intact skin. Characterization of the SC properties contributing to the depth-dependent SC permeability will hopefully provide further insight to skin penetration/decontamination. Copyright © 2016 John Wiley & Sons, Ltd.

Discovery of potent aryl-substituted 3-[(3-methylpyridine-2-carbonyl) amino]-2,4-dimethyl-benzoic acid EP4 antagonists with improved pharmacokinetic profile.

Two new series of EP4 antagonists containing a 3-methylaryl-2-carbonyl core have been identified. One series has a 3-substituted-phenyl core, while the other one incorporates a 3-substituted pyridine. Both series led to compounds with potent activity in functional and human whole blood (hWB) assays. In the pyridine series, compound 7a was found to be a highly potent and selective EP4 antagonist, with suitable rat and dog pharmacokinetic profiles.

Effects of soap-water wash on human epidermal penetration.

Skin decontamination is a primary interventional method used to decrease dermal absorption of hazardous contaminants, including chemical warfare agents, pesticides and industrial pollutants. Soap and water wash, the most common and readily available decontamination system, may enhance percutaneous absorption through the "wash-in effect." To understand better the effect of soap-water wash on percutaneous penetration, and provide insight to improving skin decontamination methods, in vitro human epidermal penetration rates of four C(14) -labeled model chemicals (hydroquinone, clonidine, benzoic acid and paraoxon) were assayed using flow-through diffusion cells. Stratum corneum (SC) absorption rates of these chemicals at various hydration levels (0-295% of the dry SC weights) were determined and compared with the results of the epidermal penetration study to clarify the effect of SC hydration on skin permeability. Results showed accelerated penetration curves of benzoic acid and paraoxon after surface wash at 30 min postdosing. Thirty minutes after washing (60 min postdosing), penetration rates of hydroquinone and benzoic acid decreased due to reduced amounts of chemical on the skin surface and in the SC. At the end of the experiment (90 min postdosing), a soap-water wash resulted in lower hydroquinone penetration, greater paraoxon penetration and similar levels of benzoic acid and clonidine penetration compared to penetration levels in the non-wash groups. The observed wash-in effect agrees with the enhancement effect of SC hydration on the SC chemical absorption rate. These results suggest SC hydration derived from surface wash to be one cause of the wash-in effect. Further, the occurrence of a wash-in effect is dependent on chemical identity and elapsed time between exposure and onset of decontamination. By reducing chemical residue quantity on skin surface and in the SC reservoir, the soap-water wash may decrease the total quantity of chemical absorbed in the long term; however, the more immediate accelerated absorption of chemical toxins, particularly chemical warfare agents, may be lethal. Copyright © 2015 John Wiley & Sons, Ltd.

Skin Concentrations of Topically Applied Substances in Reconstructed Human Epidermis (RHE) Compared with Human Skin Using in vivo Confocal Raman Microscopy.

Detailed knowledge about the skin concentration of topically applied substances is important to understand their local pharmacological activity. In particular since in vitro models of reconstructed human epidermis are increasingly used as models for diseased skin. In general, diffusion cell experiments are performed to determine the diffusion flux of test substances through either skin models or excised skin both from humans and animals. Local concentrations of the test substances within the skin are then calculated applying diffusion laws and suitable boundary conditions. In this study we used a direct approach to reveal the local concentrations of test substances within skin using confocal Raman microscopy. This non-invasive method can also be applied in vivo and therefore we directly compared in vivo concentrations with those obtained from commercially available reconstructed human epidermis (RHE). Hydrophilic and lipophilic test substances with log Pow from -0.07 to 5.91 were topically applied on human skin in vivo and RHE from SkinEthic was used as the commercial skin model. Local concentration profiles in the stratum corneum (SC) showed substantial differences between the RHE model and the in vivo situation. Differences between RHE models and human skin in vivo were also observed in their molecular composition, in particular in terms of their water profile, lipid content and the presence of natural moisturizing factor (NMF). Confocal Raman is shown to be a powerful non-invasive method for qualitative and quantitative comparative studies between RHE models and human skin in vivo. This method can also be applied to validate RHE models for future use in clinical studies.

Systemic exposure to benzoic acid and hippuric acid following topical application of clindamycin 1%/benzoyl peroxide 3% fixed-dose combination gel in Japanese patients with acne vulgaris.

Clindamycin 1%/benzoyl peroxide 3% fixed-dose combination gel (CLDM/BPO3%) is a topical product for the treatment of acne vulgaris. In this study, plasma and urine concentrations of benzoic acid (BA) and hippuric acid (HA) were analyzed to estimate the pharmacokinetics (PK) of BPO after application of CLDM/BPO3% twice-daily for 7 days in Japanese patients with acne vulgaris. Seven-day repeated application of CLDM/BPO3% appears to be safe in this patient population. Concentrations of plasma and urine BA were below the limit of quantification before and after repeated application in most of the 12 adult male patients. Mean difference in Cmax and AUC0-last for plasma HA indicated increased exposures after repeated application, but with wide 90% confidence intervals. Mean Ae0-12 for urine HA was similar before and after repeated application. Repeated application of CLDM/BPO3% is thus unlikely to result in accumulation of BA and HA. The study suggests negligible systemic exposure to BPO metabolites from CLDM/BPO3% after 7-day repeated application in male patients with acne vulgaris.

Species differences in intestinal glucuronidation activities between humans, rats, dogs and monkeys.

1. Glucuronidation via UDP-glucuronosyltransferase (UGT) in the intestine has been reported to influence the pharmacokinetics (PK) of drugs; however, information concerning the differences in activity between species is limited. Here, we investigated the in vitro and in vivo activities of intestinal glucuronidation for 17 UGT substrates in humans, rats, dogs and monkeys. 2. Although in vitro intrinsic clearance (CLint,u,UGT) in intestinal microsomes showed a good correlation between humans and laboratory animals, values tended to be lower in humans than in laboratory animals. The ratio of CLint,u,UGT in the absence and presence of bovine serum albumin differed between species. In vivo, the fraction of drug absorbed (FaFg) in humans correlated with that in dogs and monkeys, but not in rats. 3. While an inverse correlation between CLint,u,UGT and FaFg was observed in each species, the CLint,u,UGT values in the intestinal microsomes corresponding to FaFg values in dogs were three to four times higher than in other animals. 4. These results indicate the need for a degree of caution when extrapolating PK data from laboratory animals to humans.

Preparation, characterization, and evaluation of dipfluzine-benzoic acid co-crystals with improved physicochemical properties.

PURPOSE: To prepare and characterize the co-crystal of dipfluzine and benzoic acid. To investigate the feasibility of the co-crystal for improving solubility and a faster dissolution rate in vitro and evaluate the bioavailability and tissue distribution of co-crystal in vivo. METHODS: A novel dipfluzine-benzoic acid co-crystal prepared using the solvent-assisted co-grinding and the solvent ultrasonic methods were identified and characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), as well as Raman, solid-state nuclear magnetic resonance (ssNMR), and terahertz (THz) spectroscopy. Pharmacokinetics and tissue distribution were tested in vivo using murine models. Statistics analysis for dissolution data of co-crystal in vitro and animal experiment data in vivo were evaluated using t-test. RESULTS: Results of PXRD and DSC identified the dipfluzine-benzoic acid co-crystals were formed with a molar ratio of 1:2. The IR, Raman, and ssNMR spectra verified the formation of O-H · · · O and O-H · · · F hydrogen bonds. The complex constant, K, was evaluated to be 10(9) orders of magnitude with Δ r G < 0. The co-crystal solubility, the rate of drug dissolution and the relative bioavailability were approximately 500 times, five times and double that of dipfluzine, respectively. Increased solubility of co-crystal did not reduce distribution in the brain; the mean concentrations in the brain increased, but the differences had no statistic significance (p > 0.05). CONCLUSIONS: The co-crystal of dipfluzine-benzoic acid improved the physicochemical properties of dipfluzine, such as solubility and dissolution rate. Furthermore, the increased relative bioavailability of co-crystal indicated the potential use in further clinical study.

Comparison of open-flow microperfusion and microdialysis methodologies when sampling topically applied fentanyl and benzoic acid in human dermis ex vivo.

PURPOSE: The purpose of this study is to compare two sampling methods--dermal Open-Flow Microperfusion (dOFM) and dermal Microdialysis (dMD) in an international joint experiment in a single-laboratory setting. We used human ex-vivo skin and sampled topically administered Fentanyl and Benzoic Acid. The second purpose was to provide guidance to researchers in choosing the most efficient method for a given penetrant and give suggestions concerning critical choices for successful dermal sampling. METHODS: The dOFM and dMD techniques are compared in equal set-ups using three probe-types (one dOFM probe and two dMD probe-types) in donor skin (n = 9)--27 probes of each type sampling each penetrant in solutions applied in penetrationchambers glued to the skin surface over a time range of 20 h. RESULTS: Pharmacokinetic results demonstrated concordance between dOFM and dMD sampling technique under the given experimental conditions. The methods each had advantages and limitations in technical, practical and hands-on comparisons. CONCLUSION: When planning a study of cutaneous penetration the advantages and limitations of each probe-type have to be considered in relation to the scientific question posed, the physico-chemical characteristics of the substance of interest, the choice of experimental setting e.g. ex vivo/in vivo and the analytical skills available.

Glove material, reservoir formation, and dose affect glove permeation and subsequent skin penetration.

Protective gloves are used to reduce dermal exposure when managing chemical exposures at the work place. Different glove materials may offer different degrees of protection. The present study combined the traditional ASTM (American Society for Testing and Materials) model with the Franz diffusion cell to evaluate overall penetration through glove and skin as well as the deposition in the different reservoirs. Benzoic acid was applied on latex or nitrile gloves placed on top of human skin. The amounts of chemical were quantified in the glove material, between glove and skin, within the skin, and in the receptor chamber. Both glove materials reduce total penetration of benzoic acid, but nitrile gloves offer a significantly better protection than latex gloves. This difference was less pronounced at the higher of the two concentrations of benzoic acid applied. Thus, glove types that offer relevant protection at low concentrations does not necessarily give appropriate protection at high concentrations. Significant amounts of benzoic acid could be extracted from the glove materials after exposure. If a chemical is accumulated in the glove material, reuse of single-use gloves should be cautioned. The reuse of gloves is generally not to be recommended without effective decontamination.

Probe depth matters in dermal microdialysis sampling of benzoic acid after topical application: an ex vivo study in human skin.

Microdialysis (MD) in the skin - dermal microdialysis (DMD) - is a unique technique for sampling of topically as well as systemically administered drugs at the site of action, e.g. sampling of dermatological drug concentrations in the dermis. Debate has concerned the existence of a correlation between the depth of the sampling device - the probe - in the dermis and the amount of drug sampled following topical drug administration. This study evaluates the relation between probe depth and drug sampling using dermal DMD sampling ex vivo in human skin. We used superficial (<1 mm), intermediate (1-2 mm) and deep (>2 mm) positioning of the linear MD probe in the dermis of human abdominal skin, followed by topical application of 4 mg/ml of benzoic acid (BA) in skin chambers overlying the probes. Dialysate was sampled every hour for 12 h and analysed for BA content by high-performance liquid chromatography. Probe depth was measured by 20-MHz ultrasound scanning. The area under the time-versus-concentration curve (AUC) describes the drug exposure in the tissue during the experiment and is a relevant parameter to compare for the 3 dermal probe depths investigated. The AUC(0-12) were: superficial probes: 3,335 ± 1,094 µg·h/ml (mean ± SD); intermediate probes: 2,178 ± 1,068 µg·h/ml, and deep probes: 1,159 ± 306 µg·h/ml. AUC(0-12) sampled by the superficial probes was significantly higher than that of samples from the intermediate and deeply positioned probes (p value <0.05). There was a significant inverse correlation between probe depth and AUC(0-12) sampled by the same probe (p value <0.001, r(2) value = 0.5). The mean extrapolated lag-times (±SD) for the superficial probes were 0.8 ± 0.1 h, for the intermediate probes 1.7 ± 0.5 h, and for the deep probes 2.7 ± 0.5 h, which were all significantly different from each other (p value <0.05). In conclusion, this paper demonstrates that there is an inverse relationship between the depth of the probe in the dermis and the amount of drug sampled following topical penetration ex vivo. The result is of relevance to the in vivo situation, and it can be predicted that the differences in sampling at different probe depths will have a more significant impact in the beginning of a study or in studies of short duration. Based on this study it can be recommended that studies of topical drug penetration using DMD sampling should include measurements of probe depth and that efforts should be made to minimize probe depth variability.