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1.  Effect of rubbing on the in vitro skin permeation of diclofenac-diethylamine 1.16% gel 
BMC Research Notes  2012;5:321.
Rubbing a topical NSAID (non steroidal anti-inflammatory drug) on the skin may increase local drug permeation, affecting its distribution to the site of pain and inflammation. The present study evaluates this hypothesis, by assessing in vitro the effect on skin permeation of applying diclofenac-dieythylamine 1.16% gel with or without rubbing.
A single dose of 5 mg/cm2 diclofenac-diethylamine 1.16% gel was applied on excised human skin mounted in Franz-type diffusion cells without or with rubbing for 45 s. Drug penetration into the skin layers was determined after 1 h using the tape stripping technique. In vitro cutaneous permeation into the receptor fluid of the diffusion chamber was measured up to 24 h. Skin electrical resistance was also recorded.
Application of diclofenac-diethylamine 1.16% gel with rubbing resulted to a 5-fold higher flux of diclofenac through the skin than when applied without rubbing at 8 h (P = 0.04). Skin rubbing for 45 s decreased by 2-fold skin electrical resistance when compared to the standard application. Application of diclofenac-diethylamine 1.16% gel with rubbing tended to result in higher accumulation in the stripped skin vs. the superficial skin layers when applied without rubbing (P = 0.2).
These results suggest that rubbing may alter the superficial skin layer resulting in a transient faster initial diffusion of topically applied diclofenac through the stratum corneum into the deeper skin layer of the dermis to the tissue target.
PMCID: PMC3424122  PMID: 22720797
2.  Imaging Drug Delivery to Skin with Stimulated Raman Scattering Microscopy 
Molecular pharmaceutics  2011;8(3):969-975.
Efficient drug delivery to the skin is essential for the treatment of major dermatologic diseases, such as eczema, psoriasis and acne. However, many compounds penetrate the skin barrier poorly and require optimized formulations to ensure their bioavailability. Here, stimulated Raman scattering (SRS) microscopy, a recently-developed, label-free chemical imaging tool, is used to acquire high resolution images of multiple chemical components of a topical formulation as it penetrates into mammalian skin. This technique uniquely provides label-free, non-destructive, three-dimensional images with high spatiotemporal resolution. It reveals novel features of (trans)dermal drug delivery in the tissue environment: different rates of drug penetration via hair follicles as compared to the intercellular pathway across the stratum corneum are directly observed, and the precipitation of drug crystals on the skin surface is visualized after the percutaneous penetration of the co-solvent excipient in the formulation. The high speed three-dimensional imaging capability of SRS thus reveals features that cannot be seen with other techniques, providing both kinetic information and mechanistic insight into the (trans)dermal drug delivery process.
PMCID: PMC3109166  PMID: 21548600
Skin; topical drug delivery; stimulated Raman scattering microscopy; skin penetration pathways; dermatopharmacokinetics
3.  Enhanced skin permeation using polyarginine modified nanostructured lipid carriers 
The objective of the present study was to investigate the effect of polyarginine chain length on topical delivery of surface modified NLCs. Design of experiments (DOE) was used to optimize number of arginines required to deliver active drug into deeper skin layers. The NLCs were prepared by hot-melt technique and the surface of NLCs was modified with six-histidine tagged cell penentrating peptides (CPPs) or YKA. In vivo confocal microscopy and raman confocal spectroscopy studies were performed using fluorescent dye encapsulated NLCs and NLC-CPPs. Spantide II (SP) and ketoprofen (KP) were used as model drugs for combined delivery. In vitro skin permeation and drug release studies were performed using Franz diffusion cells. Inflammatory response corresponding to higher skin permeation was investigated in allergic contact dermatitis (ACD) mouse model. NLCs had a particle size of 140 ± 20 nm with higher encapsulation efficiencies. The negative charge of NLC was reduced from −17.54 to −8.47 mV after surface modification with CPPs. In vivo confocal microscopy and raman confocal spectroscopy studies suggested that a peptide containing 11 arginines (R11) had significant permeation enhancing ability than other polyarginines and TAT peptides. The amount of SP and KP retained in dermis after topical application of NLC-R11 was significantly higher than solution and NLC after 24 h of skin permeation. SP was not found in receiver compartment. However, KP was found in receiver compartment and the amount of KP present in receiver compartment was increased approximately 7.9 and 2.6 times compared to the control solution and NLCs, respectively. In an ACD mouse model, SP+KP-NLC-R11 showed significant reduction (p<0.05) in ear thickness compared to SP+KP solution and SP+KP-NLC. Our results strongly suggest that the surface modification of NLC with R11 improved transport of SP and KP across the deeper skin layers and thus result in reduction of inflammation associated with ACD.
PMCID: PMC3412947  PMID: 22617521
Skin delivery; nanostructured lipid carriers (NLC); cell penetrating peptide (CPP); allergic contact dermatitis (ACD); polyarginine peptide
4.  Skin barrier disruption by acetone: observations in a hairless mouse skin model 
Archives of Dermatological Research  2009;301(8):609-613.
To disrupt the barrier function of the skin, different in vivo methods have been established, e.g., by acetone wiping or tape-stripping. In this study, the acetone-induced barrier disruption of hairless mice was investigated in order to establish a reliable model to study beneficial, long-term effects on barrier recovery after topical application. For both treatments (i.e., acetone treatment and tape-stripping) the transepidermal water loss directly after disruption and the subsequent barrier recovery profile were similar. Histological assessment showed significant lower number of corneocyte layers in acetone-treated and tape-stripped skin compared to untreated skin, while there was no statistical difference between the two treatments. Lipid analysis of acetone-treated skin revealed that only small fraction of lipids were extracted consisting of predominantly nonpolar lipids. Importantly, the ratio of the barrier lipids, i.e., cholesterol, free fatty acids and ceramides, remained similar between control and acetone-treated skin. This reflects the undisrupted lipid organization, as determined by small-angle X-ray diffraction measurements: the long-periodicity lamellar phase was still present after acetone treatment. Our results contradict earlier studies which reported no mechanical stratum corneum removal, a substantial extraction of lipids and disruption in lipid organization. In conclusion, our studies demonstrate that barrier disruption due to acetone treatment is mainly due to removal of corneocytes.
PMCID: PMC2728065  PMID: 19350255
Lipid organization; Skin barrier perturbation; Murine model
5.  Correlative Time-Resolved Fluorescence Microscopy To Assess Antibiotic Diffusion-Reaction in Biofilms 
The failure of antibiotics to inactivate in vivo pathogens organized in biofilms has been shown to trigger chronic infections. In addition to mechanisms involving specific genetic or physiological cell properties, antibiotic sorption and/or reaction with biofilm components may lessen the antibiotic bioavailability and consequently decrease their efficiency. To assess locally and accurately the antibiotic diffusion-reaction, we used for the first time a set of advanced fluorescence microscopic tools (fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence lifetime imaging) that offer a spatiotemporal resolution not available with the commonly used time-lapse confocal imaging method. This set of techniques was used to characterize the dynamics of fluorescently labeled vancomycin in biofilms formed by two Staphylococcus aureus human isolates. We demonstrate that, at therapeutic concentrations of vancomycin, the biofilm matrix was not an obstacle to the diffusion-reaction of the antibiotic that can reach all cells through the biostructure.
PMCID: PMC3370788  PMID: 22450986
6.  Enhanced transdermal delivery of evodiamine and rutaecarpine using microemulsion 
The purpose of this study was to improve skin permeation of evodiamine and rutaecarpine for transdermal delivery with microemulsion as vehicle and investigate real-time cutaneous absorption of the drugs via in vivo microdialysis.
Pseudoternary phase diagrams were constructed to evaluate microemulsion regions with various surfactants and cosurfactants. Nine formulations of oil in water microemulsions were selected as vehicles for assessing skin permeation of evodiamine and rutaecarpine in ex vivo transdermal experiments. With a microdialysis hollow fiber membrane implanted in the skin beneath the site of topical drug administration, dialysis sampling was maintained for 10 hours and the samples were detected directly by high performance liquid chromatography. Real-time concentrations of the drugs in rat skin were investigated and compared with those of conventional formulations, such as ointment and tincture. Furthermore, the drugs were applied to various regions of the skin using microemulsion as vehicle.
In ex vivo transdermal experiments, cutaneous fluxes of evodiamine and rutaecarpine microemulsions were 2.55-fold to 11.36-fold and 1.17-fold to 6.33-fold higher, respectively, than those of aqueous suspensions. Different drug loadings, microemulsion water content, and transdermal enhancers markedly influenced the permeation of evodiamine and rutaecarpine. In microemulsion application with in vivo microdialysis, the maximum concentration of the drugs (evodiamine: 18.23 ± 1.54 ng/mL; rutaecarpine: 16.04 ± 0.69 ng/mL) were the highest, and the area under the curve0–t of evodiamine and rutaecarpine was 1.52-fold and 2.27-fold higher than ointment and 3.06-fold and 4.23-fold higher than tincture, respectively. A greater amount of drugs penetrated through and was absorbed by rat abdominal skin than shoulder and chest, and a reservoir in the skin was found to supply drugs even after the microemulsion was withdrawn.
Compared to conventional formulations, higher cutaneous fluxes of evodiamine and rutaecarpine were achieved with microemulsion. Based on this novel transdermal delivery, the transdermal route was effective for the administration of the two active alkaloids.
PMCID: PMC3205141  PMID: 22072882
microemulsion; evodiamine; rutaecarpine; transdermal delivery; microdialysis
7.  Quantitative Analysis of Microbicide Concentrations in Fluids, Gels and Tissues Using Confocal Raman Spectroscopy 
PLoS ONE  2013;8(12):e85124.
Topical vaginal anti-HIV microbicides are an important focus in female-based strategies to prevent the sexual transmission of HIV. Understanding microbicide pharmacokinetics is essential to development, characterization and implementation of efficacious microbicide drug delivery formulations. Current methods to measure drug concentrations in tissue (e.g., LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry) are highly sensitive, but destructive and complex. This project explored the use of confocal Raman spectroscopy to detect microbicide drugs and to measure their local concentrations in fluids, drug delivery gels, and tissues. We evaluated three candidate microbicide drugs: tenofovir, Dapivirine and IQP-0528. Measurements were performed in freshly excised porcine buccal tissue specimens, gel vehicles and fluids using two Horiba Raman microscopes, one of which is confocal. Characteristic spectral peak calibrations for each drug were obtained using serial dilutions in the three matrices. These specific Raman bands demonstrated strong linear concentration dependences in the matrices and were characterized with respect to their unique vibrational signatures. At least one specific Raman feature was identified for each drug as a marker band for detection in tissue. Sensitivity of detection was evaluated in the three matrices. A specific peak was also identified for tenofovir diphosphate, the anti-HIV bioactive product of tenofovir after phosphorylation in host cells. Z-scans of drug concentrations vs. depth in excised tissue specimens, incubated under layers of tenofovir solution in a Transwell assay, showed decreasing concentration with depth from the surface into the tissue. Time-dependent concentration profiles were obtained from tissue samples incubated in the Transwell assay, for times ranging 30 minutes - 6 hours. Calibrations and measurements from tissue permeation studies for tenofovir showed good correlation with gold standard LC-MS/MS data. These results demonstrate that confocal Raman spectroscopy holds promise as a tool for practical, minimally invasive, label-free measurement of microbicide drug concentrations in fluids, gels and tissues.
PMCID: PMC3875564  PMID: 24386455
8.  Cefodizime in serum and skin blister fluid after single intravenous and intramuscular doses in healthy volunteers. 
Antimicrobial Agents and Chemotherapy  1987;31(11):1822-1825.
In gonorrhea therapy, cephalosporins are conventionally administered by intramuscular (i.m.) injection, which rather frequently leads to local side effects. To investigate whether the well-tolerated intravenous (i.v.) injection of cephalosporins may be of comparable gonocidal effect, levels of cefodizime, a new broad-spectrum cephalosporin, in serum and tissue fluid (suction blister and cantharides blister fluid) were determined in six healthy men. Single doses of 1 g of cefodizime were injected i.v. and i.m. according to a randomized crossover design. On i.m. injection the drug was completely bioavailable, and the peak concentration in serum was 75 +/- 8 micrograms/ml. The terminal half-life of serum levels was 2.4 h. Cefodizime concentrations in the blister fluids increased for 1.5 to 3 h after the i.v. dose and for at least 3 h on i.m. administration. The concentrations of non-protein-bound cefodizime in blister fluid already exceeded the MIC for 90% of Neisseria gonorrhoeae strains 10 min after i.v. injection and 20 to 30 min after the i.m. dose. At 6 h after each dose, active concentrations were still present in serum. The results suggest that cefodizime administered i.v. and i.m. has equivalent high cure rates in uncomplicated gonorrhea. This hypothesis should be tested further by a controlled clinical trial. If equivalent, i.v. administration excels because it is better tolerated locally.
PMCID: PMC175046  PMID: 3435129
9.  Topical Apigenin Improves Epidermal Permeability Barrier Homeostasis in Normal Murine Skin by Divergent Mechanisms 
Experimental dermatology  2013;22(3):210-215.
The beneficial effects of certain herbal medicines on cutaneous function have been appreciated for centuries. Among these agents, Chrysanthemum extract, apigenin, has been used for skin care, particularly in China, for millennia. However, the underlying mechanisms by which apigenin benefits the skin are not known. In the present study, we first determined whether topical apigenin positively influences permeability barrier homeostasis, and then the basis thereof. Hairless mice were treated topically with either 0.1% apigenin or vehicle alone twice-daily for 9 days. At the end of treatments, permeability barrier function was assessed with either an electrolytic water analyzer or a Tewameter. Our results show that topical apigenin significantly enhanced permeability barrier homeostasis after tape stripping, though basal permeability barrier function remained unchanged. Improved barrier function correlated with enhanced filaggrin expression and lamellar body production, which was paralleled by elevated mRNA levels for the epidermal ABCA12. The mRNA levels for key lipid synthetic enzymes also were up-regulated by apigenin. Finally, both CAMP and mBD3 immunostaining were increased by apigenin. We conclude that topical apigenin improves epidermal permeability barrier function by stimulating epidermal differentiation, lipid synthesis and secretion, as well as cutaneous antimicrobial peptide production. Apigenin could be useful for the prevention and treatment of skin disorders characterized by permeability barrier dysfunction, associated with reduced filaggrin levels, and impaired antimicrobial defenses, such as atopic dermatitis.
PMCID: PMC3626082  PMID: 23489424
Apigenin; Transepidermal Water Loss; Barrier; Differentiation; Antioxidant
10.  Preparation, characterization, and in vivo evaluation of a self-nanoemulsifying drug delivery system (SNEDDS) loaded with morin-phospholipid complex 
As a poorly water-soluble drug, the oral application of morin is limited by its low oral bioavailability. In this study, a new self-nanoemulsifying drug delivery system (SNEDDS), based on the phospholipid complex technique, was developed to improve the oral bioavailability of morin.
Morin-phospholipid complex (MPC) was prepared by a solvent evaporation method and characterized by infrared spectroscopy and X-ray diffraction. After formation of MPC, it was found that the liposolubility of morin was significantly increased, as verified through solubility studies. An orthogonal design was employed to screen the blank SNEDDS, using emulsifying rate and particle size as evaluation indices. Ternary phase diagrams were then constructed to investigate the effects of drug loading on the self-emulsifying performance of the optimized blank SNEDDS. Subsequently, in vivo pharmacokinetic parameters of the morin-phospholipid complex self-nanoemulsifying drug delivery system (MPC-SNEDDS) were investigated in Wistar rats (200 mg/kg of morin by oral administration).
The optimum formulation was composed of Labrafil® M 1944 CS, Cremophor® RH 40, and Transcutol® P (3:5:3, w/w), which gave a mean particle size of approximately 140 nm. Oral delivery of the MPC-SNEDDS exhibited a significantly greater Cmax (28.60 μg/mL) than the morin suspension (5.53 μg/mL) or MPC suspension (23.74 μg/mL) (all P < 0.05). Tmax was prolonged from 0.48 to 0.77 hours and to 1 hour for MPC and MPC-SNEDDS, respectively. In addition, the relative oral bioavailability of morin formulated in the MPC-SNEDDS was 6.23-fold higher than that of the morin suspension, and was significantly higher than that of the MPC suspension (P < 0.05).
The study demonstrated that a SNEDDS combined with the phospholipid complex technique was a promising strategy to enhance the oral bioavailability of morin.
PMCID: PMC3260034  PMID: 22267925
morin; phospholipid complex; self-nanoemulsifying drug delivery system; oral bioavailability
11.  Ocular Pharmacokinetics of Acyclovir Amino Acid Ester Prodrugs in the Anterior Chamber: Evaluation of Their Utility in Treating Ocular HSV Infections 
To evaluate in vivo corneal absorption of the amino acid prodrugs of acyclovir (ACV) using a topical well model and microdialysis in rabbits.
Stability of L-Alanine-ACV (AACV), L-Serine-ACV (SACV), L-Isoleucine-ACV (IACV), γ-Glutamate-ACV (EACV) and L-Valine-ACV (VACV) prodrugs was evaluated in various ocular tissues. Dose dependent toxicity of these prodrugs was also examined in rabbit primary corneal epithelial cell culture (rPCEC) using 96-well based cell proliferation assay. In vivo ocular bioavailability of these compounds was also evaluated with a combination of topical well infusion and aqueous humor microdialysis techniques.
Among the amino acid ester prodrugs, SACV was most stable in aqueous humor. Enzymatic degradation of EACV was the least compared to all other prodrugs. Cellular toxicity of all the prodrugs was significantly less compared to trifluorothymidine (TFT) at 5mM. Absorption rate constants of all the compounds were found to be lower than the elimination rate constants. All the prodrugs showed similar terminal elimination rate constants (λz). SACV and VACV exhibited approximately two fold increase in area under the curve (AUC) relative to ACV (p < 0.05). Clast (concentration at the last time point) of SACV was observed to be 8 ± 2.6µM in aqueous humor which is two and three times higher than VACV and ACV, respectively.
Amino acid ester prodrugs of ACV were absorbed through the cornea at varying rates (ka) thereby leading to varying extents (AUC). The amino acid ester prodrug, SACV owing to its enhanced stability, comparable AUC, and high concentration at last time point (Clast) seems to be a promising candidate for the treatment of ocular HSV infections.
PMCID: PMC2528302  PMID: 18472234
Acyclovir; Amino acid ester prodrugs; Ocular absorption; Microdialysis
12.  In vivo pharmacokinetics and pharmacodynamics of topical ketoconazole and miconazole in human stratum corneum. 
A direct study evaluating whether differential drug uptake of topical 2% miconazole and 2% ketoconazole from cream formulations into human stratum corneum correlated with differential pharmacological activity against Candida albicans was investigated in healthy human subjects. A single 24-h topical dose of 2% ketoconazole cream or 2% miconazole cream was applied unoccluded, at the same dose (2.6 mg of formulation per cm2 of surface area), at four skin sites on both ventral forearms of six human subjects. At the end of the treatment, residual drug was removed with a tissue from all sites and the treated site was tape stripped 11 times, either 1, 4, 8, or 24 h later. The first tape disc was discarded. The remaining tape discs, 2 through 11, were combined and extracted for drug quantification by high-performance liquid chromatography and bioactivity against C. albicans growth in vitro. Topical 2% ketoconazole produced 14-, 10-, and 7-fold greater drug concentrations in stratum corneum than 2% miconazole at 1, 4, and 8 h after a single topical dose. Ketoconazole and miconazole concentrations in the stratum corneum were similar 24 h after drug removal. Tape disc extracts from 2% ketoconazole-treated skin sites demonstrated significantly greater bioactivity in the bioassay than 2% miconazole. The increased efficacy of 2% ketoconazole compared with that of 2% miconazole in vitro reflects their differential uptake into the stratum corneum and inherent pharmacological activity. Tape stripping the drug-treated site in conjunction with a bioassay is therefore a useful approach in the determination of bioavailability of topical antifungal agents.
PMCID: PMC284402  PMID: 8141586
13.  An Evaluation of Barrier Repair Foam on the Molecular Concentration Profiles of Intrinsic Skin Constituents Utilizing Confocal Raman Spectroscopy 
For decades, transepidermal water loss and corneometry have been accepted as measures of skin barrier function. However, these tests are not capable of informing clinicians of the biochemical constituents and biophysical status of the stratum corneum. Knowledge of how the stratum corneum reacts to topical agents is important, as it reveals significant detail regarding the composition and function of this vital skin layer. Furthermore, transepidermal water loss and corneometry serve only as surrogate markers of barrier function. A more precise method of assessing stratum corneum hydration and lipid levels is emerging; in vivo confocal Raman spectroscopy is able to detect and quantify specific biochemical constituents in skin. This information then allows for assessment of the actual physiological status of this vital layer of the skin. This pilot study sought to elucidate a biophysical rationale for the clinical improvement achieved by hyaluronic acid/ceramide barrier repair foam in prior studies as measured by in vivo confocal Raman spectroscopy. Study results include increased lipid and hydration levels in the stratum corneum to depths of 25µm and 40µm, respectively, at the 2-hour, 48-hour, and 7-day time points.
PMCID: PMC3424594  PMID: 22916310
14.  The prostaglandin D2 receptor CRTH2 is important for allergic skin inflammation following epicutaneous antigen challenge 
Cutaneous PGD2 levels increase following scratching. CRTH2 mediates chemotaxis to PGD2 and is expressed on Th2 cells and eosinophils, which infiltrate skin lesions in atopic atopic dermatitis (AD).
Examine the role of CRTH2 in a murine model of AD.
CRTH2−/− mice and WT controls were epicutaneously (EC) sensitized by repeated application of ovalbumin (OVA) to tape-stripped skin for 7 weeks, then challenged by OVA application to tape-stripped previously unsensitized skin for one week. Skin histology was assessed by H&E staining and immunohistochemistry. Cytokine mRNA expression was examined by quantitative RT-PCR. Levels of PGD2, antibody and cytokines were measured by ELISA.
PGD2 levels significantly increased in skin 24 hrs after tape-stripping, but not in skin subjected to repeated sensitization with OVA. Allergic skin inflammation developed normally at sites of chronic EC sensitization with OVA in CRTH2−/− mice, but was severely impaired in previously unsensitized skin challenged with OVA, as evidenced by significantly decreased skin infiltration with eosinophils and CD4+ cells and impaired Th2 cytokine mRNA expression. Impaired skin inflammation at sites of acute OVA challenge in CRTH2−/− mice was not due to impaired systemic response to EC sensitization, as OVA specific IgG1 and IgE antibody levels and OVA driven splenocyte secretion of cytokines in these mice were comparable to those of WT controls.
CRTH2 promotes allergic skin inflammation in response to cutaneous exposure to antigen in previously sensitized mice.
PMCID: PMC2950231  PMID: 20713302
CRTH2; PGD2; atopic dermatitis
15.  Microdialysis as a tool in local pharmacodynamics 
The AAPS Journal  2006;8(2):E222-E235.
In many cases the clinical outcome of therapy needs to be determined by the drug concentration in the tissue compartment in which the pharmacological effect occurs rather than in the plasma. Microdialysis is an in vivo technique that allows direct measurement of unbound tissue concentrations and permits monitoring of the biochemical and physiological effects of drugs throughout the body. Microdialysis was first used in pharmacodynamic research to study neurotransmission, and this remains its most common application in the field. In this review, we give an overview of the principles, techniques, and applications of microdialysis in pharmacodynamic studies of local physiological events, including measurement of endogenous substances such as acetylcholine, catecholamines, serotonin, amino acids, peptides, glucose, lactate, glycerol, and hormones. Microdialysis coupled with systemic drug administration also permits the more intensive examination of the pharmacotherapeutic effect of drugs on extracellular levels of endogenous substances in peripheral compartments and blood. Selected examples of the physiological effects and mechanisms of action of drugs are also discussed, as are the advantages and limitations of this method. It is concluded that microdialysis is a reliable technique for the measurement of local events, which makes it an attractive tool for local pharmacodynamic research.
PMCID: PMC3231563  PMID: 16796373
Microdialysis; pharmacodynamics (PD); neurotransmitter; glucose; hormone
16.  Preparation and Evaluation of Dermal Delivery System of Griseofulvin Containing Vitamin E-TPGS as Penetration Enhancer 
AAPS PharmSciTech  2011;13(1):67-74.
Griseofulvin, an antifungal agent, is a BCS class II drug slowly, erratically, and incompletely absorbed from the gastrointestinal tract in humans. The clinical failure of the conventional oral therapy of griseofulvin is most likely attributed to its poor solubility and appreciable inter- and intra-subject variation in bioavailability from different commercial products. Moreover, the conventional oral therapy is associated with numerous adverse effects and interactions with other drugs. The purpose of the study was to formulate a topical application of griseofulvin which would deliver the drug locally in a therapeutically effective concentration. Griseofulvin was solubilized in ethanol, d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), and combinations of ethanol with varying amounts of TPGS; then, it was incorporated in the Carbopol (980 NF) base. The formulations were characterized and evaluated ex vivo using Laca mice skin, microbiologically against Microsporum gypseum and Microsporum canis and clinically in a small group of patients. The current study suggested that TPGS and ethanol synergistically enhanced the drug permeation and drug retention in the skin. The selected formulation F VII was found to be effective against M. gypseum and M. canis, non-sensitizing, histopathologically safe, stable at 4°C, 25°C, and 40°C with respect to percent drug content, permeation characteristics, pH, transparency, feel, viscosity, and clinically effective in a small group of subjects. The proposed topical formulation of griseofulvin may be an effective and convenient alternative to the currently available oral therapy for the treatment of superficial fungal infections.
PMCID: PMC3299441  PMID: 22130790
griseofulvin; penetration enhancer; permeation; solubility; vitamin E-TPGS
17.  In vivo microdialysis for PK and PD studies of anticancer drugs 
The AAPS Journal  2005;7(3):E659-E667.
In vivo microdialysis technique has become one of the major tools to sample endogenous and exogenous substances in extracellular spaces. As a well-validated sampling technique, microdialysis has been frequently employed for quantifying drug disposition at the desired target in both preclinical and clinical settings. This review addresses general methodological considerations critical to performing microdialysis in tumors, highlights selected preclinical and clinical studies that characterized drug disposition in tumors by the use of microdialysis, and illustrates the potential application of microdialysis in the assessment of tumor response to cancer treatment.
PMCID: PMC2751268  PMID: 16353942
microdialysis; in vivo sampling; tumors; drug distribution; pharmacokinetics
18.  Evaluation of Paeonol Skin-Target Delivery from Its Microsponge Formulation: In Vitro Skin Permeation and In Vivo Microdialysis 
PLoS ONE  2013;8(11):e79881.
The aim of the present study was to design a novel topical skin-target drug-delivery system, the paeonol microsponge, and to investigate its drug-release patterns in dosage form, both in vitro and in vivo. Paeonol microsponges were prepared using the quasi-emulsion solvent-diffusion method. In vitro release studies were carried out using Franz diffusion cells, while in vivo studies were investigated by microdialysis after the paeonol microsponges were incorporated into a cream base. In vitro release studies showed that the drug delivered via microsponges increased the paeonol permeation rate. Ex vivo drug-deposition studies showed that the microsponge formulation improved drug residence in skin. In addition, in vivo microdialysis showed that the values for the area under the concentration versus time curve (AUC) for the paeonol microsponge cream was much higher than that of paeonol cream without microsponges. Maximum time (Tmax) was 220 min for paeonol microsponge cream and 480 min for paeonol cream, while the half-life (t1/2) of paeonol microsponge cream (935.1 min) was almost twice that of paeonol cream (548.6 min) in the skin (n = 3). Meanwhile, in the plasma, the AUC value for paeonol microsponge cream was half that of the paeonol cream. Based on these results, paeonol-loaded microsponge formulations could be a better alternative for treating skin disease, as the formulation increases drug bioavailability by lengthening the time of drug residence in the skin and should reduce side-effects because of the lower levels of paeonol moving into the circulation.
PMCID: PMC3835837  PMID: 24278204
19.  Dynamics of the cellular and humoral components of the inflammatory response elicited in skin blisters in humans. 
Journal of Clinical Investigation  1992;89(6):1734-1740.
Skin blisters induced by suction on the forearm of normal volunteers provide a convenient model to study the inflammatory response in vivo in man. In our study, after removal of the roof of the blister, i.e., the epidermis, the exposed floor of the blister (dermal-epidermal interface) was bathed with 70% autologous serum using a multiwell skin chamber. Migration of leukocytes (90-95% neutrophils) into the chamber fluid was detectable within 3 h, and appeared to plateau at 16-24 h. Sampling of the dermal-epidermal interface revealed primarily mononuclear cells during the first 8 h of the inflammatory response; however, their prevalence at 24 h was greatly diminished due to neutrophil infiltration. Accompanying the cellular immune response was the accumulation of inflammatory mediators in the bathing medium. The accumulation of IFN-gamma reached a plateau within 3 h; significant accumulations of the complement fragment, C5a, and of leukotriene B4 were also detected at 3 h. The accumulation of C5a did not peak until 5 h, whereas leukotriene B4 continued to accumulate through 24 h. IL-6 and IL-8 concentrations were minimal at 3-8 h but dramatic by 24 h while IL-1 beta, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor were undetectable within 3-8 h, but markedly elevated by 24 h. There was little accumulation of IL-4 and no accumulation of IL-1 alpha or IL-2 during the 24-h period. The sequential appearance of mediators at an inflammatory focus suggests that a carefully regulated dynamic system is responsible for controlling the evolution of the inflammatory response.
PMCID: PMC295861  PMID: 1601984
20.  Mechanical injury polarizes skin dendritic cells to elicit a Th2 response by inducing cutaneous TSLP expression 
Biochemical pharmacology  1990;39(1):19-26.
Atopic dermatitis (AD) is characterized by scratching and by Th2-dominated immune response to cutaneously introduced antigens. Antigen application to skin mechanically injured by tape stripping results in Th2-dominated skin inflammation.
To examine the effect of tape stripping on the capacity of skin dendritic cells (DCs) to polarize T cells towards a Th2 phenotype.
CD11c+ DCs were isolated from skin of BALB/c or C57BL/6 mice. FITC+ and FITC− DCs were isolated from draining lymph nodes (DLN) 24 hrs after painting the skin with FITC. DCs were assessed for their ability to induce cytokine secretion by OVA stimulated naïve CD4+ T cells from TCR-OVA transgenic DO11.10 mice. Cytokine mRNA levels were examined by quantitative PCR.
DCs isolated from the skin of wild-type (WT), but not TSLPR−/− or IL-10−/−, mice 6 hrs after tape stripping elicited significantly more IL-4 and IL-13, and significantly less interferon-γ production by CD4+ cells, than DCs isolated from unmanipulated skin, and expressed significantly more mRNA for the Th2 skewing molecules IL-10, Jagged1, and Jagged2, but significantly less mRNA for the Th1 skewing cytokine IL-12. CD11c+FITC+ cells isolated from DLN of shaved and tape stripped skin of WT, but not TSLPR−/− or IL-10−/−, mice polarized T cells significantly more towards Th2 and expressed significantly more IL-10, Jagged1 and Jagged2 mRNA than CD11c+FITC+ cells isolated from DLN of shaved skin. Tape stripping significantly increased TSLP levels in the skin, and TSLP was shown to play an essential role in the Th2 polarization of skin DCs by tape stripping.
Tape stripping upregulates TSLP levels in the skin, which polarizes skin DCs to elicit a Th2 response via the induction of IL-10.
Clinical Implications
Mechanical injury inflicted by scratching in patients with AD may cause skin DCs to polarize T cells to secrete Th2 cytokines.
Capsule Summary
Mechanical injury to the skin, such as that caused by scratching in AD, may cause skin DCs to polarize T cells to secrete more allergy-mediating Th2 cytokines.
PMCID: PMC2998067  PMID: 2105094
Atopic dermatitis; scratching; mechanical injury; thymic stromal lymphopoietin; IL-10; Th2 cytokines
21.  A Human Challenge Model for Mycobacterium tuberculosis Using Mycobacterium bovis Bacille Calmette-Guérin 
The Journal of Infectious Diseases  2012;205(7):1035-1042.
(See the editorial commentary by Dockrell, on pages 1029–31.)
Background. There is currently no safe human challenge model of Mycobacterium tuberculosis infection to enable proof-of-concept efficacy evaluation of candidate vaccines against tuberculosis. In vivo antimycobacterial immunity could be assessed using intradermal Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination as a surrogate for M. tuberculosis infection.
Methods. Healthy BCG-naive and BCG-vaccinated volunteers were challenged with intradermal BCG. BCG load was quantified from skin biopsy specimens by polymerase chain reaction (PCR) and culture colony-forming units. Cellular infiltrate was isolated by suction blisters and examined by flow cytometry. Prechallenge immune readouts were correlated with BCG load after challenge.
Results. In BCG-naive volunteers, live BCG was detected at the challenge site for up to 4 weeks and peaked at 2 weeks. Infiltration of mainly CD15+ neutrophils was observed in blister fluid. In previously BCG-vaccinated individuals, PCR analysis of skin biopsy specimens reflected a degree of mycobacterial immunity. There was no significant correlation between BCG load after challenge and mycobacterial-specific memory T cells measured before challenge by cultured enzyme-linked immunospot assay.
Conclusions. This novel experimental human challenge model provides a platform for the identification of correlates of antimycobacterial immunity and will greatly facilitate the rational down-selection of candidate tuberculosis vaccines. Further evaluation of this model with BCG and new vaccine candidates is warranted.
PMCID: PMC3295601  PMID: 22396610
22.  Dermal microdialysis technique to evaluate the trafficking of surface modified lipid nanoparticles upon topical application 
Pharmaceutical research  2012;29(9):2587-2600.
The central objective of the current study was to evaluate the skin pharmacokinetics and tissue distribution of cell penetrating peptides (CPP) modified nano-structured lipid carrier (NLC) using an in vivo dermal microdialysis (MD) technique.
Celecoxib (Cxb) encapsulated NLCs (CXBN), CPP modified CXBN (CXBN-CPP) and Cxb-Solution (CXBS) formulations were prepared and tested for in vitro skin distribution. MD was used to assess pharmacokinetic parameters of Cxb after topical application of Cxb formulations. The effect of pre-treatment with Cxb formulations was evaluated for expression of prostaglandin-E2 (PGE2) and Interleukin-6 (IL-6) after exposure of xylene using MD. Allergic contact dermatitis (ACD) model was used to confirm in vivo therapeutic response of Cxb formulations.
The cumulative permeation of Cxb in MD dialysate after 24 h for CXBN-CPP was significantly higher (p<0.001) than CXBN and CXBS. Further, pre-treatment with CXBN-CPP significantly inhibited PGE2 and IL-6 expression compared to CXBS and CXBN (p<0.001). In ACD model, CXBN-CPP showed significant reduction (p<0.001) in ear thickness compared to controls.
Surface modification of NLC with CPPs can enhance the skin permeation of Cxb and MD can be used to investigate pharmacokinetics of Cxb nanoparticles in the skin.
PMCID: PMC3423188  PMID: 22644591
Nanoparticles; Dermal Pharmacokinetics; Cell Penetrating Peptides; Microdialysis; Polyarginine peptide
23.  Raman Spectroscopy and Related Techniques in Biomedicine 
Sensors (Basel, Switzerland)  2010;10(3):1871-1889.
In this review we describe label-free optical spectroscopy techniques which are able to non-invasively measure the (bio)chemistry in biological systems. Raman spectroscopy uses visible or near-infrared light to measure a spectrum of vibrational bonds in seconds. Coherent anti-Stokes Raman (CARS) microscopy and stimulated Raman loss (SRL) microscopy are orders of magnitude more efficient than Raman spectroscopy, and are able to acquire high quality chemically-specific images in seconds. We discuss the benefits and limitations of all techniques, with particular emphasis on applications in biomedicine—both in vivo (using fiber endoscopes) and in vitro (in optical microscopes).
PMCID: PMC3000600  PMID: 21151763
Raman spectroscopy; CARS microscopy; Raman imaging
24.  Raman Spectroscopy and Related Techniques in Biomedicine 
Sensors (Basel, Switzerland)  2010;10(3):1871-1889.
In this review we describe label-free optical spectroscopy techniques which are able to non-invasively measure the (bio)chemistry in biological systems. Raman spectroscopy uses visible or near-infrared light to measure a spectrum of vibrational bonds in seconds. Coherent anti-Stokes Raman (CARS) microscopy and stimulated Raman loss (SRL) microscopy are orders of magnitude more efficient than Raman spectroscopy, and are able to acquire high quality chemically-specific images in seconds. We discuss the benefits and limitations of all techniques, with particular emphasis on applications in biomedicine—both in vivo (using fiber endoscopes) and in vitro (in optical microscopes).
PMCID: PMC3000600  PMID: 21151763
Raman spectroscopy; CARS microscopy; Raman imaging
25.  Transdermal therapeutic system of carvedilol: Effect of hydrophilic and hydrophobic matrix on in vitro and in vivo characteristics 
AAPS PharmSciTech  2007;8(1):E13-E20.
The purpose of this research was to develop a matrix-type transdermal therapeutic system containing carvedilol with different ratios of hydrophilic and hydrophobic polymeric combinations by the solvent evaporation technique. The physicochemical compatibility of the drug and the polymers was studied by infrared spectroscopy and differential scanning calorimetry. The results suggested no physicochemical incompatibility between the drug and the polymers. In vitro permeation studies were performed by using Franz diffusion cells. The results followed Higuchi kinetics (r=0.9953−0.9979), and the mechanism of release was diffusion mediated. Based on physicochemical and in vitro skin permeation studies, patches coded as F3 (ethyl cellulose: polyvinylpyr-rolidone, 7.5∶2.5) and F6 (Eudragit RL:Eudragit RS, 8∶2) were chosen for further in vivo studies. The bioavailability studies in rats indicated that the carvedilol transdermal patches provided steady-state plasma concentrations with minimal fluctuations and improved bioavailability of 71% (for F3) and 62% (for F6) in comparison with oral administration. The antihypertensive activity of the patches in comparison with that of oral carvedilol was studied using methyl prednisolone acetate—induced hypertensive rats. It was observed that both the patches significantly controlled hypertension from the first hour (P<.05). The developed transdermal patches increase the efficacy of carvedilol for the therapy of hypertension.
PMCID: PMC2750430
Transdermal; antihypertensive; carvedilol; polyvinylpyrrolidone; ethyl cellulose; Eudragit

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