Prednicarbate Biotransformation in Human Foreskin Keratinocytes and Fibroblasts

Purpose. Evaluation of skin layer-specific prednicarbate (PC) biotransformation, possibly explaining the improved benefit/risk ratio of this topical corticosteroid in atopic dermatitis (1,2). Methods. Metabolism of PC in keratinocyte and fibroblast monolayers derived from human juvenile foreskin was evaluated. Drug concentration was determined by HPLC/UV-absorption. Accompanying cell viability tests (MTT-tests) were performed to exclude toxic drug effects. Results. Keratinocytes hydrolyzed the double ester PC (2.5 × 10^–6 M) at position 21 to the monoester prednisolone 17-ethylcarbonate (P17EC) which nonenzymatically transformed to prednisolone 21-ethylcarbonate (P21EC). This metabolite was enzymatically cleaved to prednisolone (PD), the main biotransformation product at 24 hours. Fibroblasts, however, showed a distinctively lower enzyme activity. Both, PC and P17EC (or rather P21EC) were hydrolyzed to a minor extent only. The biotransformation pathway, however, was the same. When P17EC was added separately, it transformed to P21EC and again was cleaved by keratinocytes to a much higher extent. Despite of the rather high glucocorticoid concentration MTT-tests proved a non-disturbed cell viability and proliferation rate. Conclusions. Extrapolating our results to the in-vivo situation, topically applied PC may be metabolized by epidermal cells during skin penetration. A complex mixture of compounds reaches the dermis, whose fibroblasts are barely able to metabolize the steroids. Since skin atrophy is less pronounced with PC as compared to conventional halogenated glucocorticoids, less potent PC metabolites appear to be the dominant species in the dermis.     

Skin Penetration and Metabolism of Topical Glucocorticoids in Reconstructed Epidermis and in Excised Human Skin

Purpose. To investigate pharmacokinetic differences between the non-halogenated double ester prednicarbate (PC) and the fluorinated monoester betamethasone 17-valerate (BM17V) their metabolism in human keratinocytes and fibroblasts as well as their permeation and biotransformation in reconstructed epidermis and excised human skin was compared. Special attention was given to the 17-monoesters because of their high receptor affinity and antiproliferative effects. Methods. Glucocorticoid penetration was determined using Franz diffusion cells, quantifying metabolite concentrations by HPLC. Chemical stability and reactivity of the monoesters was determined by molecular modeling analysis. Results. PC accumulated in the stratum corneum. A considerable amount of penetrating PC was hydrolyzed by viable keratinocytes to prednisolone 17-ethylcarbonate (P17EC). P17EC permeated the skin very rapidly when compared to BM17V. Overall P17EC concentrations in viable tissue were low. Inside of the acceptor fluid, but not within the tissue, P17EC was converted to the more stable prednisolone 21-ethylcarbonate (P21EC). Conclusions. The inactivation of highly potent, but also cell toxic, 17-monoesters to almost inactive 21-congeners seen with isolated cell monolayers appears less important in the skin. In vitro determination of the dermal 17-monoesters concentrations may allow the prediction of the atrophogenic risk in man. BM17V levels exceeding P17EC concentrations about 6-fold may contribute to its lower tolerance when compared to PC.     

An In Vitro Method of Evaluating Tolnaftate Release from Topical Powder

A dissolution apparatus was constructed to evaluate tolnaftate release from topical powders. It consisted of a mesh unit to support the powder, a receptor phase, and a sink. This report describes three parameters that were used to evaluate this technique. First, three different areas of contact were examined using 52-, 41-, or 30-µm mesh supports. Second, the effect of the pH on the dissolution rate was studied, using aqueous buffers of pH 3, 5, 7, or 8 as the receptor phase. Finally, different topical powder formulations containing different amounts of tolnaftate were tested. The results obtained showed that the percentage of tolnaftate released from topical powders increased at low pH levels and with the larger mesh support. The percentage released was greater in a starch–talc preparation than in a talc-only preparation. The mesh was replaced by a semipermeable membrane (2.5- to 4 nm pore size) to function as an in vitro model for intradermal diffusion. The results showed that a cream initially released more drug than powder formulations.     

Assessment of Value and Applications of In Vitro Testing of Topical Dermatological Drug Products

The FDA recently issued a guidance covering practices of scaleup and post approval changes with semisolids (SUPAC-SS). This guidance outlines the steps that must be taken by a company to maintain certification of its semisolid dermatological products after quantitative changes have been made in their compositions and/or after changes have been made in the sourcing of their key ingredients, in their processing, in their batch sizes, and/or after their site of manufacture has been relocated. A key element within the guidance is a release test to be used to determine if the diffusional release of a drug found in a formulation is the same after changes have been made to the formulation as it was prior to implementing the changes. The AAPS-FDA sponsored workshop was set up to explore this qualifying test. The stated aims of the workshop were: a) to illustrate the methodology and techniques of in vitro release testing, b) to show the sensitivity of in vitro release with respect to manufacturing variables and to variations in components and composition (of specific formulations), c) to recognize in vitro release testing as a useful procedure for SUPAC documentation, d) to highlight and evaluate other applications of in vitro release testing, e) to explore the degree to which in vitro release testing and bioavailability may be related, and f) to evaluate the role of in vitro release testing of topical dosage forms as a tool to improve product quality.     

In Vitro Cutaneous and Percutaneous Delivery and in Vivo Efficacy of Tetracaine from Liposomal and Conventional Vehicles

The aim of this study was to quantitate drug delivery and correlate drug concentration in the skin with anesthetic effect. The rate of delivery of radiolabelled tetracaine from two different liposome formulas (1F2 and 23C2) and two conventional dosage forms (PEG Ointment USP and Glaxal^® base) was investigated in Flow-thru diffusion cells using human breast skin from mammoplasty. The results indicated a 1.5 and 4 times higher concentration of tetracaine within the skin when the liposomal formula (1F2) was used, compared to tetracaine in Glaxal^® base and PEG Ointment USP, respectively. The amount of drug delivered into the skin in 24 h from the liposomal formula was 5.3% of total applied whereas from Glaxal^® base it was 3.3% and from PEG Ointment base it was 1.2%. The amount of liposomal (1F2) phospholipids in the skin after 24h was 68.3 µg/cm² (0.2% of total applied). The steady state flux of tetracaine from liposomes (1F2) was 16.06 µg/cm²/h with a lag time of 3.1h, from Glaxal^® base 10.24 µg/cm/h with a lag time of 11.2h and from PEG Ointment it was 5.70 µg/cm²/h with a lag time of 9.0h. The second liposome formula (23C2) showed similar flux and permeability coefficient than the Glaxal^® base, however the lag time was about half. The results indicated that optimized liposome formulation is necessary to achieve maximum drug delivery. The concentration of drug within the skin and the flux measured in vitro showed correlation with in vivo efficacy. The in vivo data showed that liposomal (1F2) tetracaine produced the deepest anesthesia with shortest onset in volunteers, followed by Glaxal^® base, liposome formula 23C2 while tetracaine in PEG Ointment had a lack of effect.     

Implications for Clinical Pharmacodynamic Studies of the Statistical Characterization of an In Vitro Antiproliferation Assay

Modeling of nonlinear pharmacodynamic (PD) relationships necessitates the utilization of a weighting function in order to compensate for the heteroscedasticity. The structure of the variance was studied for concentration–effect data generated in an in vitro 96-well plate cell growth inhibition assay, where data are numerous (480 data points per experiment) and replication is easy. From the five candidate models that were considered, the power function , where is the sample mean and is the sample variance, was shown to be the most appropriate to describe the nonuniformity of the variance along the range of measured effect for 253 sets of data. The Hill model was fit to the concentration–effect data with weighted nonlinear regression, where the weights were equal to the reciprocal of the predicted variance. The examination of the distribution of the 253 sets of parameters of the PD model showed that IC₅₀ was lognormally distributed whereas the distribution of was normal. The characterization of the appropriate variance function and concentration–effect function in a simple in vitro experimental setting with a large number of experiments, with each experiment including a large number of data points, will be useful for guiding similar in vitro concentration–effect studies where data are plentiful and for guiding PD modeling in complex clinical settings in which extensive data for model characterization is impossible to obtain.     

Development of Liposomal Systems of Finasteride for Topical Applications: Design,Characterization, and In Vitro Evaluation

Finasteride (FNS) is a “drug of choice” for benign prostate hypertrophy and prostate cancer. The drug has also been reported to be useful orally in the treatment of some difficult-to-treat androgen-dependent skin disorders, such as seborrhea, acne, hirsutism, and androgenetic alopecia. However, the ideal route for its administration (i.e., topical) remains unexplored. This has logically suggested the search for strategic formulation approaches to make the drug effective on topical applications, hitherto unexplored. The present study targets the encasement of drug molecules in the interiors of vesicular compartments (liposomes) made up of hydrogenated phospholipids, as an attempt toward the development of a trans-epidermal therapeutic system of FNS. Multilamellar drug-loaded liposomes were prepared by thin-film hydration with sonication method and optimized with respect to drug payload, entrapment efficiency, and size by formulating different vesicular compositions under different process conditions. The vesicular systems consisting of saturated phospholipid (100 mg), cholesterol (50 mg), and FNS (5 mg) showed highest drug payload (2.9 mg/100 mg of total lipids), and drug entrapment efficiency (88.6%). Mean (± SD) vesicle size of the prepared liposomes was found to be 3.66 ± 1.6 μm. Significantly higher skin permeation of FNS through excised abdominal mice skin of FNS was achieved from the liposomal formulations vis-à-vis corresponding solution and conventional gels. Liposomal FNS formulations also showed more than fivefold higher deposition of drug in skin than the corresponding plain drug solution and conventional gel. Stability studies indicated that the liposomal formulations were quite stable in the refrigerated conditions for 2 months with negligible drug leakage or vesicle size alteration during the storage period. Results of the current studies with FNS-loaded vesicular systems project the high plausibility of a topical liposomal formulation for effective localized delivery of Finasteride.     

Effect of Concentration and Degree of Saturation of Topical Fluocinonide Formulations on In Vitro Membrane Transport and In Vivo Availability on Human Skin

Purpose. The thermodynamic acitvity of drugs in topical vehicles is considered to significantly influence topical delivery. In vitro diffusion across a synthetic membrane was shown to be correlated to the degree of saturation of the drug in the applied vehicle and therefore offers a potential for increased topical drug delivery. Fluocinonide a topical corticosteroid, was chosen as a model compound to investigate in vitro and in vivo availability from formulations with different degrees of saturation. Methods. Sub-, as well as, supersaturated drug solutions were prepared using PVP as an antinucleant agent. In vitro membrane diffusion experiments across silicone membrane and in vivo pharmacodynamic activity assessments, using the human skin blanching assay, were carried out. Results. Over the concentration range studied, the in vitro membrane transport of fluocinonide was proportional to the degree of saturation of the respective formulations. The in vivo pharmacodynamic response in the human skin blanching assay was related to the concentration of the drug in the vehicle irrespective of the degree of saturation. Conclusions. From the membrane permeation experiment it can be concluded, that the drug flux might be increased supra-proportionally with increasing donor concentration, drug (super-)saturation (proportional), beyond what would be anticipated based on ideal donor concentration and partition coefficient considerations only. These findings could not be confirmed in the in vivo investigation, probably due to additional vehicle effects (e.g., enhancement, irritation, drug binding) which have to be expected and could have altered the integrity of the stratum corneum and therewith topical bioavailability of the drug.     

Evaluation of Monooleine Aqueous Dispersions as Tools for Topical Administration of Curcumin: Characterization,In Vitro and Ex-Vivo Studies

Curcumin (CUR) is a well-known natural compound showing antioxidant, anti-inflammatory, and antitumor abilities but characterized by poor bioavailability and chemical instability, which drastically reduce its application in the treatment of chronic diseases such as osteoarthritis. The aim of the present study is the design and evaluation of monooleine aqueous dispersion (MAD) as novel carriers for the topical administration of CUR. CUR-loaded MAD was formulated using two different emulsifier systems, namely poloxamer 407 (MAD-A) and sodium cholate-sodium caseinate (MAD-B). These vehicles were characterized, and their influence on in vitro percutaneous absorption of CUR was also evaluated. Furthermore, an oxygen radical absorbance capacity assay was used to determine their antioxidant activity, and a Western blot analysis was performed to evaluate the inhibitory effect of the formulations on inducible nitric oxide synthase and cyclooxygenase 2 expressions. From the obtained results, CUR encapsulation efficiency was higher than 98% for MAD-A and 82% for MAD-B. Shelf-life studies showed that MAD-A maintains CUR stability better than MAD-B, and both vehicles demonstrated, in vitro, control of drug diffusion through the skin. Finally, MAD-A and MAD-B were able to extend the antioxidant/anti-inflammatory effects of CUR, also confirming the protective effect toward CUR chemical stability. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2349–2361, 2013     

Design of Chronomodulated Drug Delivery System of Valsartan: In Vitro Characterization

The aim of the present study was to design and evaluate a chronomodulated time-clock pulsatile tablets of valsartan to release it after a certain lag time, independent of the gastrointestinal pH, in its absorption window to cope with the circadian rhythm of human body for blood pressure elevation. Core tablets were prepared by direct compression of a homogenous mixture of valsartan, Avicel PH101, croscarmellose sodium, magnesium stearate and Aerosil. The core tablets were then sprayed coated with a sealing layer formed of ethyl cellulose that was subsequently coated with a release-controlling layer. Three different aqueous dispersions namely; carnauba wax or beeswax or a mixture in a ratio of 2.5:1, respectively, were used to form five time-clock tablet formulations having the release controlling layer with different thickness {B5, B10, B20, BW5 and CW5}. Quality control testing were carried out to the core tablets. Differential scanning calorimetry was also performed to detect the possible drug excipient interaction in the core tablet formulation. The release was carried out, for the prepared time-clock tablet formulations, in 0.1 N hydrochloric acid for the first 2 h, followed by phosphate buffer (pH 6.8) for 4.5 h. The effect of pH on valsartan release was studied through a release study in 0.1 N hydrochloric acid for 6.5 h. Two phase dissolution study was performed to the selected time-clock tablet formulation to predict the drug permeation through the gastrointestinal tract. Stability study of the selected formula was performed at 25°/60% RH and at 40°/75% RH for 3 months. Results showed that a release-controlling layer composed of a mixture of carnauba wax and beeswax in a ratio of 2.5:1 showed a reasonable release lag time. The release lag time of the tablets increased with the increase of the coat thickness, thus B20>B10>B5 with corresponding lag time values of 4.5, 3 and 2.5 h, respectively. Selected B5 tablet formula exhibited a reasonable lag time after which the highest, complete % drug release at pH 6.8 was obtained. In addition, a good partitioning of valsartan, between the aqueous and organic phases in a ratio of 1:7, was observed. The selected formula was stable for at least 3 months under standard long-term and accelerated storage conditions. In conclusion, in vitro studies revealed that the novel time-clock system could be used successfully to deliver valsartan in a pulsatile pH-independent manner. It provided a desirable lag time followed by a rapid and complete drug release accompanied by an expected effective permeation through the biological membranes upon release in the duodenum; the window of absorption, as indicated by the two phase release study.     

In Vitro Cutaneous Disposition of a Topical Diclofenac Lotion in Human Skin: Effect of a Multi-Dose Regimen

Purpose. This study determines comparative bioavailability of diclofenac sodium lotion compared to an aqueous solution after topical application to viable human skin in vitro. In addition, the difference between a single dose and multiple doses (8 times) was also determined. Methods. An in vitro flow-through diffusion cell system was employed, using radiolabelled diclofenac sodium. Results. Multiple doses of lotion (2 l/cm² and 5 l/cm²) delivered a total of 40.1 ± 17.6 g and 85.6 ± 41.4 g diclofenac, respectively, at 48 h, compared to only 9.4 ± 2.9 g and 35.7 ± 19.0 g absorbed after topical application of diclofenac as an aqueous solution (P < 0.05). A single dose study showed no statistical difference between diclofenac delivered in lotion or an aqueous solution. Over 48 h the total absorption from lotion was 10.2 ± 6.7 g and 26.2 ± 17.6 g (2 l/cm² and 5 l/cm², respectively), compared to 8.3 ± 1.5 g and 12.5 ± 5.7 g from an aqueous solution. Both single doses of lotion and aqueous diclofenac showed decreased diclofenac absorption into the receptor fluid between 12 and 24 h. However, when applied multiple times, absorption from lotion was continually increasing up to 48 h. The total dose accountability ranged from 76.8 ± 8.2% to 110.6 ± 15.1% of the applied dose. Conclusions. Diclofenac lotion exhibited enhanced diclofenac percutaneous absorption rate through human skin (mass, flux and partition coefficient) when applied a multiple number of times and this enhanced absorption was maintained over 48 h. This suggests that a constituent of the lotion (DMSO) will enhance human skin absorption of diclofenac when used in a multi-dose regimen, but not after a single dose.     

Matrix-Type Transdermal Patches of Verapamil Hydrochloride: In Vitro Permeation Studies Through Excised Rat Skin and Pharmacodynamic Evaluation in Rats

The objectives of this study were to develop matrix-type transdermal patches of verapamil hydrochloride (VRP) with pectin as a matrix polymer to investigate the influence of several terpenes on in vitro permeation of VRP through rat skin and to evaluate pharmacodynamic activity of transdermal formulations in rats. Matrix-type transdermal patches containing VRP were prepared using pectin as a matrix agent and propylene glycol as a plasticizer agent. Terpenes such as nerolidol, d-limonene, eucalpytol, menthone, and menthol were also used as a chemical enhancer to improve the skin penetration of VRP. The permeation studies were perfomed using Franz-type diffusion cells and full-thickness excised abdominal rat skin. Effects of terpenes on the permeation parameters of VRP were evaluated. In vitro skin permeation studies showed that nerolidol was the most promising enhancer among the enhancers examined in the present study, followed by d-limonene. Pharmacodynamic activity of the transdermal patches containing nerolidol or d-limonene was evaluated in rats by the measurement of systolic blood pressure for 360 min with the use of the tail cuff method. VRP transdermal patches significantly decreased the systolic blood pressure after 30 min and transdermal patches containing nerolidol and d-limonene maintained the decrease in blood pressure during the observation of 360 min.     

Ciprofloxacin Loaded Bovine Serum Albumin Microspheres： Prepa-ration and Drug Release Characterization In Vitro

Ciprofloxacin loaded microspheres were prepared by spray drying technique, with bovine serum albumin as the natural biodegradable wall materials. The obtained microspheres, using aqueous system, were organic solvent-free. The diameters of the spherical microspheres were in the range of 1-5 1:4. The drug entrapment of microspheres, formulated with different ciprofloxacin/albumin ratios as 1:1, 1:2 and 1:4, were 46.93%, 32.96% and 20.56% (n=3). And the encapsulation efflciencies for ciprofloxacin during spray drying were higher than 90%. Thermal denaturation programs at different temperatures (100-120℃) for different time intervals (3-6-12 h) were further processed to stabilize the spray-dried microspheres. The higher the extent for thermal denaturation, the slower the rate of ciprofloxacin released from microspheres in vitro. So the release rate of ciprofloxacin from microspheres can be controlled by modifing the conditions of thermal denaturation.     

环丙沙星白蛋白微球的制备及体外释药特性(英文)

以牛血清白蛋白为囊材,采用喷雾干燥工艺将环丙沙星制成微球。以水溶液系统制备的白蛋白微球不含任何有机溶剂,外观呈圆球型,微球粒径在1～5 (m之间,符合干粉末肺部吸入给药的要求。以不同的环丙沙星/白蛋白比例（1:1、1:2及1:4）配制喷雾干燥样品,制得微球的载药量分别为46.93%、32.96% 和20.56% (n=3),药物的包封产率均在90% 以上。采用不同的粉末加热变性温度 (100～120 ℃) 和时间 (3～6～12 小时),对喷雾干燥制得的粉末白蛋白微球进行热变性处理,考察热变性后微球的体外释药情况,热变性程度越大,药物释放愈缓慢,说明可通过改变热变性条件调控微球中药物的释放。     

Characterization of Methacrylated Inulin Hydrogels Designed for Colon Targeting: In Vitro Release of BSA

Purpose. To characterize methacrylated inulin hydrogels with respect to their release properties. Methods. Proteins (bovine serum albumin or lysozyme) were used as model drugs and were loaded during or after hydrogel formation. Parameters such as the drug loading method, the molecular weight of the proteins, the initial drug loading concentration, the hydrogel feed composition, degree of substitution, and size of the hydrogel were investigated by determining the release of the model proteins from the hydrogels in a phosphate buffer solution. The biodegradable properties were investigated by studying the release of bovine serum albumin in a solution of inulinase. Results. In vitro protein release from methacrylated hydrogels was influenced by factors such as the drug loading procedure and the molecular weight and loading concentration of the proteins. The feed composition and degree of substitution of inulin seem to be crucial in controlling both the extent and the rate of release. Protein release was clearly enhanced in the presence of inulinase, indicating the biodegradable properties of methacrylated inulin hydrogels. Conclusions. Several hydrogels show interesting properties with respect to the development of a colon-specific drug delivery system.     

Aryl Ester Prodrugs of Cyclic HPMPC. I: Physicochemical Characterization and In Vitro Biological Stability

Purpose. The chemical, enzymatic, and biological stabilities and physical properties of a series of salicylate and aryl ester prodrugs of the antiviral agent, cyclic HPMPC, were evaluated to support the selection of a lead compound for clinical development. Methods. Chemical stabilities of the prodrugs in buffered solutions at 37°C were determined. Stability was also studied in the presence of porcine liver carboxyesterases (PLCE) at pH 7.4 and 25°C. Tissue stabilities were examined in both human and dog intestinal homogenates, plasmas and liver homogenates. Prodrug and product concentrations were determined by reverse phase HPLC. Results. Chemical degradation of the prodrugs resulted in the formation of both cyclic HPMPC and the corresponding HPMPC monoester. Chemical stability was dependent on the orientation of the exo-cyclic ligand; the equatorial isomers were 5.4- to 9.4-fold more reactive than the axial isomers. In the presence of PLCE, the salicylate prodrugs cleaved exclusively to give cyclic HPMPC and not the HPMPC monoester. In plasma, but not intestinal or liver homogenates, the salicylate esters of cyclic HPMPC cleaved readily with a rate dependent on the chain length of the alkyl ester substituent. Conclusions. The carboxylate function on the salicylate prodrugs of cyclic HPMPC provides an additional handle to chemically modify the lipophilicity, solubility and the biological reactivity of the prodrug. In tissue and enzymatic studies, the major degradation product is cyclic HPMPC. The salicylate ester prodrugs are attractive drug candidates for further in vivo evaluation.     

DRV Liposomal Bupivacaine: Preparation,Characterization, and In Vivo Evaluation in Mice

Purpose. To evaluate the dehydration-rehydration technique to prepare a formulation of liposomal bupivacaine, and to assess its analgesic efficacy. Methods. Bupivacaine hydrochloride (BUP) was encapsulated into dehydration-rehydration vesicles (DRV) of varying phospholipid (PL) compositions. Two bilayer-forming phospholipids were used, the fluid dimyristoyl-phosphatidylcholine and the solid dis- tearoyl-phosphatidylcholine (DSPC), with 20 or 40 mol% cholesterol, in the presence of bupivacaine at a 1.28 or 0.64 BUP/PL mole ratio. After rehydration, drug/lipid ratios were determined. The formulation with the highest drug/lipid ratio (DSPC/cholesterol in an 8:2 mole ratio prepared in the presence of bupivacaine in a 1.28 BUP/PL mole ratio) was adjusted to a final bupivacaine concentration of 3.5% or 0.5%. The duration of skin analgesia after subcutaneous injection in mice produced by these formulations was compared with the conventional administration of a plain 0.5% solution of BUP. In addition, the concentration of residual bupivacaine at the injection site was followed for 96 h. Results. The relatively low organic solvent/aqueous phase and membrane/aqueous phase partition coefficients, together with liposomal trapped volume and BUP/PL mole ratio, indicated that most of the drug was encapsulated in the intraliposome aqueous phase of the DRV. The DSPC/cholesterol 8:2 mole ratio had the best drug encapsulation (BUP/PL = 0.36). Compared to plain BUP, these BUP-DRV produced significant prolongation of analgesia, which is explained by longer residence time of the drug at the site of injection. Conclusions. Bupivacaine-DRV may have a role in achieving safe, effective, and prolonged analgesia in humans.     

Novel Animal Model for Evaluating Topical Efficacy of Antiviral Agents: Flux Versus Efficacy Correlations in the Acyclovir Treatment of Cutaneous Herpes Simplex Virus Type 1 (HSV-1) Infections in Hairless Mice

This report describes the study of a novel animal model for the topical treatment of cutaneous herpes virus infections, with a focus upon the relationship between the dermal flux of the antiviral agent and the effectiveness of the topical therapy. A recently developed (trans)dermal delivery system (TDS) for controlling acyclovir (ACV) fluxes was employed in the treatment of cutaneous herpes simplex virus type 1 (HSV-1) infections in hairless mice. The TDS's were fabricated with rate-controlling membranes to provide nearly constant fluxes of ACV for up to 3 to 4 days. At the end of each experiment an extraction procedure was used to determine the residual ACV, validating the drug delivery performance of the TDS. Virus was inoculated into the skin of the mice at a site distant from the TDS area, and the induced lesion development was evaluated to distinguish between topical and systemic effectiveness of the therapy. In the main protocol, ACV therapy was initiated 0, 1,2, and 3 days after virus inoculation and the lesion development scored on Day 5. The topical efficacies of 1- and 2-day-delayed treatments were essentially the same as that of a 0-day-delayed treatment, while the topical efficacy of a 3-day-delayed treatment was much poorer. Also, in the cases of 0-, 1-, and 2-day-delayed treatments, topical efficacy increased with increasing flux in the range of 10 to 100 µg/cm²-day. When the ACV flux was 100 µg/cm²-day or greater, a maximum 100% topical efficacy was obtained. The results for systemic efficacy were shifted to higher fluxes: approximately 10-fold greater ACV fluxes were necessary to provide efficacy equal to the topical efficacy results. The animals treated with a high ACV flux (350–500 µg/cm²-day) lived significantly longer than those treated with a low ACV flux (10–125 µg/cm²-day) and those of untreated (placebo) animals. Further, their mean survival time decreased with an increase in the time delay for ACV treatment. In contrast, the mean survival time for the animals which received a low ACV flux was similar to that of the control animals and remained unaltered with an increase in the time delay for ACV treatment. The approach developed in this study should be valuable in (a) the screening of new antiviral agents for the topical treatment of cutaneous herpes virus infections and (b) in the optimization of drug delivery systems (i.e., topical formulations).     

The Human Keratinocyte Cell Line HaCaT: An In Vitro Cell Culture Model for Keratinocyte Testosterone Metabolism

Pharmaceutical Research -