Assessment and Prediction of the Cutaneous Bioavailability of Topical Terbinafine,In Vivo,in Man

Pharmaceutical Research -     

Optimizing Metrics for the Assessment of Bioequivalence Between Topical Drug Products

PURPOSE: Stratum corneum tape stripping post-application of a drug product followed by analysis of the active agent in this tissue layer is an approach being seriously considered for the comparative assessment of topical bioavailability. Key issues revolve around how best to perform this experiment and interpret the data. METHODS: Using previously published results from a comparative study of three 0.025% tretinoin gel products, alternative data analysis approaches are presented that may render the technique more accessible to the evaluation of new and generic topical dosage forms. RESULTS: For the tretinoin gel study, the conclusions for bioequivalence from measurements of drug levels at only one uptake and one clearance time were the same as those from the original study, which required measurements at eight different treatment times. Furthermore, comparisons of drug levels at one uptake and one clearance time discriminated differences in bioequivalence for clearance and uptake, which had previously been missed. Half-life estimates, derived from time course data of drug clearance, can be related to lag time for drug penetration through the SC. CONCLUSIONS: This new data analysis demonstrates that comparative bioequivalence might be assessed more easily.     

Dermatopharmacokinetics: Factors Influencing Drug Clearance from the Stratum Corneum

Purpose  The dermatopharmacokinetic methodology, in which tape stripping of the stratum corneum (SC) is used to access the amount of drug accumulated in the skin barrier, has been proposed for the quantification of topical drug bioavailability. This investigation examined the clearance phase of a model drug from the SC after a short application of an infinite dose. Methods  A saturated solution of ibuprofen in propylene glycol/water was applied to the forearm of human volunteers for 30 min. The formulation was then removed and the drug profile across the SC was assessed immediately, and over the next 4 h. Results  The clearance phase depends only on drug diffusivity in the SC. However, the expected, progressive “flattening” of the concentration profiles with increasing time post-formulation removal was not observed. It was subsequently deduced, using infrared spectroscopy, that the rapid percutaneous diffusion of propylene glycol, relative to ibuprofen, resulted in the transient maintenance of a saturated drug concentration at the SC surface even after removal of the original formulation. Conclusions  The important role of formulation excipients in topical delivery is demonstrated, and the local disposition of cosolvents within the SC may impact significantly on drug dermatopharmacokinetics and local bioavailability.     

Improved Bioequivalence Assessment of Topical Dermatological Drug Products Using Dermatopharmacokinetics

Purpose  A dermatopharmacokinetic (DPK) approach, in which drug levels in the stratum corneum (SC) are measured as a function of time post-application and post-removal of the product using tape-strip sampling in vivo in humans, has been considered for the comparative assessment of topical bioavailability. Its application to-date has been limited by contradictory results and concerns that variability in the method necessitates large numbers of treatment sites and volunteers. The objective of this study was to test whether a revised protocol could better assess bioequivalence. Methods  A blinded study of three 1% econazole nitrate cream products, for which the SC is the site of action, was conducted to examine several modifications to the DPK methodology. In addition to protocol changes designed to reduce experimental variability, bioequivalence was assessed at a single uptake time and a single clearance time measured in duplicate in each subject. Results  Conclusive determinations of bioequivalence were achieved with only four treatment sites per product in each of 14 volunteers, which was less than one-third the number required in a previous DPK investigation. Conclusions  Comparative bioequivalence can be assessed conclusively with fewer treatment sites in fewer subjects with robust methods that should be less sensitive to inter-laboratory differences.     

Feasibility of Measuring the Bioavailability of Topical Betamethasone Dipropionate in Commercial Formulations Using Drug Content in Skin and a Skin Blanching Bioassay

An in vivo technique has been developed which simultaneously compares a skin blanching bioassay with drug content in human stratum corneum following topical application of four 0.05% beta-methasone dipropionate formulations. Bioavailability of drug from commercial cream and ointment formulations was assessed by quantification of drug content in tape-stripped stratum corneum and skin blanching in the treated skin site under occluded conditions. Tape-stripping removed stratum corneum to a varying degree between individuals but was consistent (35%) within an individual with all formulations, day to day. A correlation (r = 0.9935) between the amount of drug in the treated stratum corneum normalized for surface area and the corresponding skin blanching score was observed with four 0.05% betamethasone dipropionate formulations. Increasing the amount of drug in the tape-stripped stratum corneum correlated with an increased skin blanching score. Ointment formulations delivered more drug to the skin and produced greater blanching scores than the cream formulations. Topical corticosteroid content in the treated skin site can therefore be quantified and correlates well with the resulting pharmacodynamic activity.     

Epidermal Differentiation and Permeability in Fetal Pig Skin

Pharmaceutical Research -     

Determining Dermal Absorption Parameters in Vivo From Tape Strip Data

Purpose: Tape stripping the outermost skin layer, the stratum corneum (sc), is a popular method for assessing the rate and extent of dermal absorption in vivo. Results from tape strip (TS) experiments can be affected significantly by chemical diffusion into the sc during the time required to apply and remove all of the TSs, t_TS. Here, we examine the effects of this problem on the interpretation of TS experimental results. Methods: Dermal absorption of 4-cyanophenol (4CP) in humans was studied using TS experiments to assess conditions in which diffusion alters TS results. Mathematical models were developed to assess the effects of diffusion on parameter estimation. Results: For an experiment with t_TS > t_lag (i.e., the lag time for a chemical to cross the sc), the permeability coefficient for 4CP, P_sc,v, calculated including t_TS, was consistent with values from the literature (i.e., 0.0019 cm/h). When diffusion during stripping was not included in the model, P_sc,v was 70% smaller. Conclusions: Calculations show that chemical concentrations in TSs can be affected by diffusion during tape stripping, but if t_TS < 0.2 t_lag and the exposure time is > 0.3 t_lag, TS concentrations are not significantly affected by t_TS.     

Porcine Ear Skin as a Model for the Assessment of Transdermal Drug Delivery to Premature Neonates

PURPOSE: The purpose of this study was (i) to validate differentially tape-stripped, porcine skin as an in vitro model for the evaluation of transdermal drug delivery (TDD) to premature neonates, (ii) to determine whether the model could estimate neonatal skin permeability as a function of postconceptional age (PCA), and (iii) to demonstrate that iontophoretic delivery permits precise control of drug input independent of skin barrier function. METHODS: Passive permeation of caffeine, phenobarbital, and lidocaine across tape-stripped porcine skin barriers was measured. Iontophoretic delivery of lidocaine across skins with different barrier competencies was also evaluated. RESULTS: For all drugs, passive permeation correlated with skin barrier function; that is, with transepidermal water loss (TEWL): Jss = A x exp[B x TEWL]. Combining this result with a previously derived dependence of TEWL upon the PCA of premature neonates in vivo allowed a relative value of Jss to be predicted for a given PCA. Comparison of these predictions showed excellent agreement with experimental data reported for diamorphine. Iontophoretic lidocaine delivery was precisely controllable independent of barrier competency. CONCLUSIONS: Porcine skin, in vitro, differentially tape-stripped to specific barrier competencies, is a useful model to explore TDD in premature neonates. The potential for iontophoresis to provide improved dose control and adjustment, irrespective of skin barrier maturity, is established.     

Skin disposition of menthol after its application in the presence of drug substances

Many drug products that are applied onto the skin contain menthol. Menthol plays a dual role in the analgesic and anti-inflammatory drugs: it causes cooling and local anesthetic effects and, being a penetration enhancer, it increases the skin permeation of the drug substances. However, there are no data concerning the skin penetration of menthol after its application in the most commonly used vehicles and in the presence of drug substances. Therefore, this study evaluated the ex vivo skin disposition of menthol after application of the commercially available drug products containing aluminum acetotartrate, methyl salicylate, ibuprofen and naproxen, using full human-skin mounted in flow-through diffusion cells. After 15, 30 and 60 min of application, the skin was progressively tape-stripped into three fractions of stratum corneum and the remaining epidermis with dermis. The content of menthol in the skin layers was determined by GC method. Varying degrees of penetration of menthol into the skin layers was observed, depending on its amount in the vehicle and the presence of drug substance. In the presence of aluminum acetotartrate, the skin penetration of menthol was limited only to the outer fraction of the stratum corneum. In the case of drug products containing naproxen, the concentration of the drug substance significantly influenced the skin penetration of menthol.     

QSAR Analysis of Skin Permeability of Various Drugs in Man as Compared to in Vivo and in Vitro Studies in Rodents

A general mathematical model involving partition coefficient, molecular weight and hydrogen bonding has been formulated for correlating the structures and skin permeability of a wide range of compounds through human skin and through hairless mouse skin. The correlations obtained are dependent not only on the biological system but also on the vehicle used. Without the use of lipophilic vehicle, the ideal lipophilicity for maximum permeability through human skin as measured by log P_o(oct/w)ranges from 2.5 to 6 (extrapolated value). When a lipophilic vehicle was used in hairless mouse skin study, the log P_o(oct/w) was lowered to around 0.4 ~ 0.6. While increased M.W. always has a negative effect on the permeability, increased H-bond can have either a slight positive or a slight negative effect, depending on the experiments (absorption vs. permeability constant). Cross validations with previously unanalyzed data as well as other biological systems support the usefulness of the general model developed for passive diffusion.     

Stratum corneum reservoir as a predictive method for in vitro percutaneous absorption

Interaction between drug and proteins and lipids in stratum corneum (SC) is an important pharmacokinetic parameter in early steps of absorption. Previous in vivo studies showed that the total amount of compound, regardless of properties, penetrating over a 96 h period could be predicted by the amount present in SC 30 min after application by a linear relationship. Validating this linear relationship through in vitro study would facilitate testing of transdermal drug delivery platforms. We aimed to determine in vitro penetration behavior across SC of humans by determining the relationship between quantity present in SC reservoir 30 min after application with 24 h skin absorption and penetration. In this study, use of the SC reservoir effect to predict absorption and penetration of topical compounds is reaffirmed with in vitro models involving human skin. These results indicate the amount in short‐term (30 min) SC reservoir predict long‐term (24 h) skin absorption and penetration, as characterized by statistically significant linear relationships determined via regression. This may be explained by the fact that SC is a rate‐limiting barrier to percutaneous drug transport. After molecules diffuse through SC barrier, passage into deeper dermal layers and systemic uptake occur relatively quickly. These results enable one to measure quantity in SC reservoir shortly after topical application as a proxy for absorption and penetration over longer periods. With respect to drug development and risk assessment of toxic substances, this may simplify assays attempting to quantitate penetration capacity. Further investigation with a larger range of compounds is needed to clarify the observations recorded here. Copyright © 2015 John Wiley & Sons, Ltd.     

In Vivo Skin Penetration of Acitretin in Volunteers Using Three Sampling Techniques

Etretinate and acitretin are given orally to treat psoriasis and various keratinization disorders. Acitretin, the main active metabolite of etretinate, has the pharmacokinetic advantage of being rapidly eliminated, but it shares etretinate's toxicologic profile. Thus a topical delivery of acitretin with no or reduced systemic adverse effects is desirable. To characterize the therapeutic potential of topically delivered acitretin, we quantitatively assessed its percutaneous penetration in healthy human volunteers. Additionally, three skin sampling techniques, the punch biopsy, the shave biopsy, and the suction blister technique, were validated to quantitate acitretin in the skin. The results suggest that topical delivery of acitretin renders skin concentrations which exceed those reported after oral administration of etretinate or acitretin. However, because of possible interlaminate drug contamination, drug localization within a particular skin compartment cannot be determined.     

Procedure for the evaluation of the bioavailability of topical formulations in vivo as the rate and extent to which the active drug product is transferred from the vehicle to the skin

A procedure was developed for the in vivo evaluation of the bioavailability of topical formulations containing a limited amount of the active drug product estimated as the rate and extent to which the drug is released from the vehicle to the skin. It is assumed that drug transfer follows Fick's law of diffusion. The formulation tested was a cream containing 40 μmol/ml (20 mg/ml) of sertaconazole, a novel imidazole antifungal agent, applied to healthy volunteers. Eight 9 cm² squares were marked in the volar forearm surface and 0.1 ml of the cream was applied to each square by gentle rubbing with a plastic thimble. The actually applied dose was estimated considering the drug amount that adhered to the thimble. Then one area at a time was cleansed at 0, 1, 2, 4, 6, 8, 12, and 24 h and the amount of sertaconazole transferred to the skin was estimated by determination of the amount of drug removed by cleansing. Drug release from the vehicle to the skin increased with time, but then reached a plateau. The maximal amount of drug transferred to the skin was 85.5 ± 3.3% (mean ± S.D.) of the actually applied dose, and this value was considered an expression of the extent of drug release. The rate of release was estimated as the half-life of the transfer process, which was 1.83 ± 0.76 h. This procedure is suitable for the evaluation of topical formulations containing a limited amount of the active drug product. © 1995 Wiley-Liss, Inc.     

Noninvasive Characterization of Regional Variation in Drug Transport into Human Stratum Corneum in Vivo

Purpose. To investigate the mechanisms underlying the regional variations in drug transport into human stratum corneum (SC) of two model compounds of different lipophilicity and molecular size, 4-cyanophenol (CP) and cimetidine (CM), in vivo by non-invasive, quantitative attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Methods. Saturated solutions of CP and CM were applied to the skin surface of eleven Chinese men, at five anatomical sites, including forearm, back, thigh, leg, and abdomen, for 10-15 min and 3-5 h, respectively. After the skin surface was cleansed of remaining chemicals, the SC was tape-stripped sequentially up to 20 times, and the drug concentration profiles in the tape-stripped SC were determined using ATR-FTIR spectroscopy. Thickness of the SC was estimated simultaneously using two-point measurements of transepidermal water loss before and after completion of tape stripping. Estimation of partition, diffusion, and permeability coefficients was achieved by analysis of the data using the unsteady-state diffusion equation. Results. The rank orders of regional variation in partition and diffusion coefficients of CP and CM were different. The rank order of regional variation in permeability coefficients was similar for both drugs and decreased in the order of back > forearm > thigh > leg abdomen, but the variation was more prominent for CM. Conclusions. Regional variation in SC transport of CP was mainly influenced by its intrinsic diffusivity across the SC, whereas variation in transport of CM could be attributed to both thermodynamic and kinetic differences among different anatomical skin sites.     

Evaluation of a Human Bio-Engineered Skin Equivalent for Drug Permeation Studies

Purpose. To test the barrier function of a bio-engineered human skin (BHS) using three model drugs (caffeine, hydrocortisone, and tamoxifen) in vitro. To investigate the lipid composition and microscopic structure of the BHS. Methods. The human skin substitute was composed of both epidermal and dermal layers, the latter having a bovine collagen matrix. The permeability of the BHS to three model drugs was compared to that obtained in other percutaneous testing models (human cadaver skin, hairless mouse skin, and EpiDerm). Lipid analysis of the BHS was performed by high performance thin layered chromatrography. Histological evalulation of the BHS was performed using routine H&E staining. Results. The BHS mimicked human skin in terms of lipid composition, gross ultrastructure, and the formation of a stratum corneum. However, the permeability of the BHS to caffeine, hydrocortisone, and tamoxifen was 3-4 fold higher than that of human cadaver skin. Conclusions. In summary, the results indicate that the BHS may be an acceptable in vitro model for drug permeability testing.     

A Critical Comparison of Some Raman Spectroscopic Techniques for Studies of Human Stratum Corneum

This study evaluates a variety of techniques and sampling conditions for Raman spectroscopic investigations of human stratum corneum. Using a Fourier-transform Raman system and samples of stratum corneum in vitro, we demonstrated minimal inter- and intracadaver variations in molecular vibrations. We have also shown Raman spectroscopy to be relatively insensitive to the hydration state of human stratum corneum, indicating that the technique should be valuable for monitoring the transdermal delivery of drugs from aqueous solutions. The stability of human stratum corneum to near-infrared laser excitation was verified by spectral collection for approximately 1 hr. We have also compared FT-Raman spectra from human stratum corneum in vitro and in vivo. Of the different types of Raman instruments used in this study (visible-light excitation microprobe, visible-light excitation macroscopic sampling, and Fourier-transform Raman), the FT-Raman system provided good-quality spectra with high sample throughput, but systems using visible-light excitation should provide unique information for use in specialist applications.     

Use of Shed Snake Skin as a Model Membrane for in Vitro Percutaneous Penetration Studies: Comparison with Human Skin

The potential usefulness of shed snake skin as a model membrane for transdermal research was examined. There are similarities between shed snake skin and human stratum corneum in terms of structure, composition, lipid content, water permeability, etc. The permeability of various compounds and the contribution of several functional groups to the permeability were also found to be similar between shed snake skin and human skin. Moreover, the permeability of compounds through shed snake skin was increased by Azone, one of the most extensively studied transdermal penetration enhancers. Considering the similarities between shed snake skin and human skin, ease of storage and handling, and low cost, shed snake skin may offer a good model membrane for transdermal research.     

Drug-Metabolizing Enzymes in the Skin of Man,Rat, and Pig

The mammalian skin has long been considered to be poor in drug metabolism. However, many reports clearly show that most drug metabolizing enzymes also occur in the mammalian skin albeit at relatively low specific activities. This review summarizes the current state of knowledge on drug metabolizing enzymes in the skin of human, rat, and pig, the latter, because it is often taken as a model for human skin on grounds of anatomical similarities. However only little is known about drug metabolizing enzymes in pig skin. Interestingly, some cytochromes P450 (CYP) have been observed in the rat skin which are not expressed in the rat liver, such as CYP 2B12 and CYP2D4. As far as investigated most drug metabolizing enzymes occur in the suprabasal (i.e. differentiating) layers of the epidermis, but the rat CYP1A1 rather in the basal layer and human UDP-glucuronosyltransferase rather in the stratum corneum. The pattern of drug metabolizing enzymes and their localization will impact not only the beneficial as well as detrimental properties of drugs for the skin but also dictate whether a drug reaches the blood flow unchanged or as activated or inactivated metabolite(s).     

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.     

TARGETED DRUG DELIVERY TO THE SKIN AND DEEPER TISSUES: ROLE OF PHYSIOLOGY, SOLUTE STRUCTURE AND DISEASE

1. Drug delivery through the skin has been used to target the epidermis, dermis and deeper tissues and for systemic delivery. The major barrier for the transport of drugs through the skin is the stratum corneum, with most transport occurring through the intercellular region. The polarity of the intercellular region appears to be similar to butanol, with the diffusion of solutes being hindered by saturable hydrogen bonding to the polar head groups of the ceramides, fatty acids and other intercellular lipids. Accordingly, the permeability of the more lipophilic solutes is greatest from aqueous solutions, whereas polar solute permeability is favoured by hydrocarbon-based vehicles. 2. The skin is capable of metabolizing many substances and, through its microvasculature, limits the transport of most substances into regions below th. dermis. 3. Although the flux of solutes through the skin should be identical for different vehicles when the solute exists as a saturated solution, the fluxes vary in accordance with the skin penetration enhancement properties of the vehicle. It is therefore desirable that the regulatory standards required for the bio-equivalence of topical products include skin studies. 4. Deep tissue penetration can be related to solute protein binding, solute molecular size and dermal bloo. flow. 5. Iontophoresis is a promising area of skin drug delivery, especially for ionized solutes and when a rapid effect i. required. 6. In general, psoriasis and other skin diseases facilitate drug delivery through th. skin. 7. It is concluded that the variability in skin permeability remains an obstacle in optimizing drug delivery by thi. route.