Evaluation of percutaneous absorption and skin irritation of ketoprofen through rat skin: in vitro and in vivo study.

The influences of different mechanisms of penetration enhancers (such as menthol, azone, ethanol and nonivarnide) regarding the percutaneous absorption and skin irritation of ketoprofen formulations through rat skin were investigated by in vitro and in vivo study. The skin irritation degree at the end of the experiment (10 h) was deterinined by pathologic biopsy and colorimetry methods. In vitro, the menthol showed the most potent enhancing effect. Furthermore, the enhancement effect of a combination of menthol and nonivamide was higher than that of their individual use alone. In vivo the formulation containing 0.05% nonivantide, 5% menthol and 20% ethanol showed a higher penetration rate and an acceptable degree of skin irritation compared to a commercial product (Formax plus gel containing 3% ketoprofen), indicating that it could be used in the clinical situation.     

Pimecrolimus: skin disposition after topical administration in minipigs in vivo and in human skin in vitro.

The dermal disposition of pimecrolimus, a non-steroid, anti-inflammatory calcineurin inhibitor used for the treatment of atopic dermatitis, was evaluated in minipigs in vivo and in human skin in vitro using tritium-radiolabeled compound, and in dermal toxicokinetic investigations in minipigs using unlabeled compound. Following topical application of pimecrolimus 1% market form (MF) cream to minipig skin, approximately 2% of the dose penetrated into the stratum corneum and part of it into deeper skin layers. The remainder of the dose was recovered non-absorbed on the skin surface. The total systemic absorption was or=94% of dose remained non-absorbed, 3.1% was found in the epidermis (including stratum corneum) and 2.9% in the dermis. There was no indication of metabolism of pimecrolimus in human skin in vitro or minipig skin in vivo. No drug accumulation was observed in minipig skin after up to 13 weeks of once daily topical application of 0.1% or 0.3% pimecrolimus cream.     

Pharmacokinetics of ketoprofen in man after repeated percutaneous administration

A topical formulation of ketoprofen, a widely used nonsteroidal antiinflammatory drug, has recently been developed. Ten healthy young subjects (mean age 23.5 +/- 2.5 years) received daily 15 g of 2.5% ketoprofen gel, corresponding to 375 mg on the skin of the back over an area of 750 cm2. Plasma samples were collected after the first dose and after 10 days of chronic treatment. Urine was also collected. Ketoprofen was assayed by HPLC. The peak plasma concentration was 144 +/- 91 ng/ml after the first administration with apparent absorption and elimination half-lives of 3.2 +/- 2.4 h and 27.7 +/- 18.0 h, respectively. The total quantities of ketoprofen eliminated in the urine represented about 2.6% of the first dose applied. At the end of the study, the apparent half-life of unchanged ketoprofen was 17.1 +/- 9.1 h, and there was no accumulation. In this study, no sign of local intolerance was seen.     

Comparison of tissue concentrations after intramuscular and topical administration of ketoprofen

Purpose. To assess whether topical ketoprofen, which has been reported to provide analgesic effects in clinical studies, reaches predictable tissue concentrations high enough to account for the reported analgesia. Intramuscular ketoprofen was used as positive control. Methods. Muscle and subcutaneous tissue concentrations were assessed by microdialysis. Plasma and tissue concentrations after intramuscular injection were described using a three-compartment population pharmacokinetic model. The prediction performance of the model was assessed by superimposing tissue concentrations of 12 subjects that did not participate in the present study. Results. Most dialysate concentrations after topical dosing of ketoprofen (100 mg) were below the quantification limit of 0.47 ng/ml. Plasma concentrations increased slowly and reached an apparent plateau of 7-40 ng/ml at 10-12h. No decline was observed up to 16 h. Tissue concentrations after intramuscular injection (100 mg) were about 10 times higher than those after topical dosing. Tissue concentrations measured in the majority of the 12 subjects that did not participate in the present study were found within the range of two-thirds of the predicted concentrations. Conclusion. Predictable and cyclooxygenase-inhibiting concentrations of ketoprofen were achieved in subcutaneous and muscle tissue after intramuscular but not after topical dosing. Thus, the tissue concentrations of ketoprofen after topical administration can hardly explain the reported clinical efficacy of topical ketoprofen.     

Ex vivo calcium percutaneous eggression in normal and tape-stripped human skin

Ions play a crucial role in skin homeostasis. Calcium, a participant in wound healing and keratinization, is localized at an increasing gradient from the stratum basal to the stratum granulosum. In vivo and in vitro studies show disturbances in this gradient in damaged skin. We developed here a model to study ex vivo calcium outward flux from normal and tape-stripped human skin. We measured here, with a calcium specific electrode, ex vivo calcium percutaneous eggression from dermis to epidermis in normal and tape-stripped skin places in franz cells, with a calcium source or not in the dermis compartment. Tape-stripped skin released a greater calcium concentration and had a higher rate over time than normal skin in both cases. The rate went from 8.1.10(-3) ± 8.9.10(-3) nmol.cm(-2).min(-1) to 4.8.10(-2)?±?1.8.10(-2)?nmol.cm(-2).min(-1) with no calcium in the dermis compartment and from 1.7.10(-1) ± 1.2.10(-1) nmol.cm(-2).min(-1) to 5.9?±?3.4 nmol.cm(-2).min(-1) with calcium. Also calcium uptake from the dermis is greater in tape-stripped skin during the first 5?h of the experiment. This ex vivo method reproduced the increased of calcium skin permeability with a disrupted barrier. This approach aims to develop a non-invasive method to measure calcium flux concentration in normal and damaged skin that might be reproduced and validated in vivo.     

Comparative in vitro and in vivo studies on the permeation and penetration of ketoprofen and ibuprofen in human skin

The aim of the present in vitro and in vivo studies was to compare the permeation and penetration of a 2.5% ketoprofen (CAS 22071-15-4) gel [Phardol Schmerz-Gel (Test-D)] with the permeation and penetration of two other ketoprofen gels (Ref-I, Ref-E) and an ibuprofen (CAS 15687-27-1) gel (Ref-D) on excised human skin. Furthermore, in vivo studies were performed. The permeation studies utilizing static Franz diffusion cells allow the determination of the transdermal (systemic) transport, whereas the penetration studies in vitro (according to the Saarbrücker model) and in vivo permit setting up a concentration-depth profile. For this purpose the permeation kinetics of ketoprofen from three different gels (each containing 2.5% ketoprofen) over a period of two days were determined at heat-separated human skin of different donors. The in vitro permeability coefficients for Test-D (6.50 x 10(-7) cm x s(-1)) and Ref-I (5.72 x 10(-7) cm x s(-1)) were comparable and the transport occurred for both by a factor of 8-9 faster than with Ref-E (0.78 x 10(-7) cm x s(-1)). In parallel to the permeation studies with ketoprofen, the permeability coefficient of caffeine from an ointment was assessed using the skin biopsies of the same donors as a quality assurance. In a second part of the studies, the in vitro penetration of ketoprofen from Test-D was determined over a period of 3 h at three different skin biopsies in comparison to a commercially available 5% ibuprofen gel (Ref-D). As a main result a concentration-depth profile for ketoprofen and ibuprofen could be issued. The ketoprofen (37.7 +/- 12.1 microg/cm2) and the ibuprofen (30.1 +/- 6.0 microg/cm2) penetrate to the same order of magnitude into the upper part of the Stratum corneum, whereas ibuprofen stronger accumulates in the deeper layers (ketoprofen: 27.3 +/- 8.5 microg/cm2; ibuprofen: 73.7 +/- 31.1 microg/cm2). An additional in vivo penetration study was performed with Test-D to set up an in vitro-in vivo (IVIV) correlation. Over a period of 3 h, the amount of ketoprofen in the Stratum corneum in vivo was 78.4 +/- 19.1 microg/cm2 being comparable to the in vitro data.     

Bioequivalence determination of topical ketoprofen using a dermatopharmacokinetic approach and excised skin penetration

Ketoprofen is a photolabile drug. The aim of the present study was to compare the bioavailability of ketoprofen in a photo-stabilised formulation with a gel without photoprotection using a new dermatopharmacokinetic tape-stripping model and an established ex vivo penetration method using human skin. Analyses of the stratum corneum showed that during the first 45 min about 12 microg/cm2 ketoprofen was absorbed into the skin from the formulations. The area under the ketoprofen content-time curve (AUC0-6 h) for the ratio photo-stabilised gel/transparent gel was 73% with a 90% confidence interval (CI) 65-83. The rate of penetration of ketoprofen through isolated skin was approximately 0.2 microg/cm2 h for both formulations. AUC0-36 h for the ratio was 84% with 90% CI 64-105. Thus, the two methods did not disagree in terms of relative efficacy of the two gels. However, the difference obtained in vivo was statistically significant, whereas no significant data arise from the ex vivo study. Comparing the amount of ketoprofen in the skin after 45 min with the amount penetrated through the excised skin during 36 h, suggests a change in the thermodynamic activity of ketoprofen during the exposure. A supersaturated formulation may well have been formed initially due to evaporation of ethanol.     

Simultaneous absorption of vitamins C and E from topical microemulsions using reconstructed human epidermis as a skin model

Antioxidants provide the mainstay for skin protection against free radical damage. The structure of microemulsions (ME), colloidal thermodynamically stable dispersions of water, oil and surfactant, allows the incorporation of both lipophilic (vitamin E) and hydrophilic (vitamin C) antioxidants in the same system. The objective of this work was to investigate the potential of non-thickened (o/w, w/o and gel-like) and thickened (with colloidal silica) ME as carriers for the two vitamins using reconstructed human epidermis (RHE). The amounts of these vitamins accumulated in and permeated across the RHE were determined, together with factors affecting skin deposition and permeation. Notable differences were observed between formulations. The absorption of vitamins C and E in RHE layers was in general enhanced by ME compared to solutions. The incorporation of vitamins in the outer phase of ME resulted in greater absorption than that when vitamins were in the inner phase. The location of the antioxidants in the ME and affinity for the vehicle appear to be crucial in the case of non-thickened ME. Addition of thickener enhanced the deposition of vitamins E and C in the RHE. By varying the composition of ME, RHE absorption of the two vitamins can be significantly modulated.     

Penetration and metabolism of topical retinoids in ex vivo organ-cultured full-thickness human skin explants

The human epidermis contains endogenous retinoids [retinol (vitamin A) and retinyl esters] and carotenoids (mostly beta-carotene). Previous studies in the mouse have shown that the enzymes involved in retinoid metabolism are present in the epidermis. In this study, we wanted to assess the skin penetration and metabolism of topical retinoids in the human. To do this, fresh surgically excised human abdominal skin was mounted on Franz perfusion cells. Topical retinoic acid, retinal, retinol and retinyl palmitate were applied at 2.5 mg/cm(2) in oil-in-water creams containing 0.05% retinoids on the donor compartment, while the receptor compartment was filled with culture medium. The skin was incubated for 24 h at 37 degrees C, then epidermal retinoid concentrations were determined by HPLC. The same experiment was performed with mouse back skin mounted on Franz cells. Finally, topical retinoids were applied on the back of hairless mice for 24 h; then the mice were sacrificed and retinoid concentrations were assayed in the epidermis. In all three models, retinol and its esters were found to be endogenous, as was the case in previous studies in the mouse in vivo. The four applied retinoids penetrated well into the epidermis. Topical retinoic acid did not increase endogenous retinoids, whereas the latter were greatly increased following topical retinal in the mouse. Retinal was also metabolized into retinoic acid, unlike topical retinol and retinyl palmitate, which only increased endogenous retinoids. Topical retinal and retinol did undergo a higher metabolism in both mouse models than in human skin. In summary, the penetration and metabolism patterns of topical retinoids were quite similar in the two mouse models used, indicating that the Franz cells appear to be a good model to predict in vivo metabolism of topical retinoids. When applying this concept to our results obtained in Franz cells with human skin, we conclude that topical retinol and retinal load human skin with both storage and functional vitamin A.     

In vitro topical delivery of non-steroidal anti-inflammatory drugs through human skin.

The objective of the present study was to evaluate in vitro the percutaneous absorption, across human skin, of 5 non-steroidal anti-inflammatory drugs (NSAIDs) formulated as gels: ketoprofen (CAS 22071-15-4), epolamine diclofenac (CAS 15307-86-5), piroxicam (CAS 36322-90-4) and niflumic acid (CAS 4394-00-7) or as emulgel: diclofenac sodium (CAS 15307-79-6) and to compare the different formulations as drug delivery systems. Because the concentrations of the NSAIDs in the different excipients were not identical, the comparison of their diffusional properties was expressed in term of release efficiency (or diffusion efficacy). The results obtained show that, across human skin under standardized experimental conditions, ketoprofen and piroxicam have the best rank order followed by niflumic acid, diclofenac sodium and epolamine diclofenac.     

酮洛芬及其异丙酯对HaCaT细胞构建的组织工程皮肤的渗透作用

目的体外构建适用于经皮给药研究的组织工程皮肤。方法以表皮角质形成细胞系HaCaT细胞及人真皮成纤维细胞为细胞来源，用I型牛胶原蛋白为真皮基质包埋成纤维细胞，其上接种HaCaT细胞，采用气-液界面方式培养，观察不同的培养介质对组织工程皮肤的影响，HE染色切片观察培养皮肤结构形态。以酮洛芬及其异丙酯为模型药物研究经皮渗透和代谢特性。结果HaCaT细胞经气-液界面培养可形成类表皮层，但不能形成完整的角质层。维生素C可明显促进细胞增殖，维生素D₃可促进细胞分化，雌二醇对此组织工程皮肤没有明显的影响。同皮肤细胞匀浆代谢相似，酮洛芬异丙酯被代谢成原药酮洛芬。结论HaCaT细胞在气-液界面培养条件下可形成多层分化不完全的表皮层，保留了一定的酶活性，可用于药物的经皮渗透和代谢等研究。     

In vitro and in vivo percutaneous absorption of topical dosage forms: case studies

This article evaluated the influence of vehicle compositions on topical drug availability. In vitro drug release and in vivo experiments were performed in case of the hydrophilic ketamine hydrochloride and the lipophilic piroxicam. Ketamine hydrochloride is a NMDA receptor antagonist that has been useful for anesthesia and analgesia. The study of transdermal ketamine delivery is a novelty, because nobody has investigated the hypnotic effects of ketamine after this administration route. In vitro measurements gave a good basis for screening among the developed products. The physiological changes after ketamine administration showed, that there were significant differences among the parameters tested (breathing rate, duration of sleep) from the developed products (hydrogel, lyotropic liquid crystal and o/w cream) compared to the reference product (Carbopol gel). The in vivo feedback for piroxicam was the measurement of the anti-inflammatory activity by edema inhibition percentage. Significant differences were measured in case of the developed systems compared to the reference.     

Spatial distribution of 8-methoxypsoralen penetration into human skin after systemic or topical administration

AIMS: Photochemotherapy employing psoralens combined with UVA irradiation (PUVA) is a standard therapy for a variety of dermatoses. Psoralens can be administered orally or topically in the form of bath or cream preparations. Recommendations for the time of UVA irradiation are mainly based on the time course of minimal phototoxic doses. However, the time course and depth of skin penetration of psoralens is not well characterized. METHODS: We assessed the time course of 8-MOP concentrations in horizontal epidermal and dermal skin sections in 10 patients undergoing oral (n = 3), cream (n = 4) and bath (n = 3) PUVA therapy. Punch biopsies (4 mm) were taken from "healthy" skin sites. A highly sensitive LC-MS-MS method was employed for 8-MOP analysis. RESULTS: Epidermal concentrations following cream or bath were highest at the end of the application period (time zero) when irradiation is performed. At this time, 8-MOP cream provided significantly higher epidermal concentrations (mean +/- s.e. mean 128.0 +/- 22.6 pg mm-3; 95% CI: 77.6, 178.4) than oral 8-MOP (27.0 +/- 25.3 pg mm-3; 95% CI: 29.3, 83.3 at 1 h; P = 0.025). Conversely, concentrations in the papillary dermis were significantly higher with oral 8-MOP (20.2 +/- 3.1 and 16.2 +/- 2.2 pg mm-3 at 1 and 2 h, respectively) than with 8-MOP cream (7.1 +/- 2.8 and 8.4 +/- 2.0 pg mm-3 time zero and 0.5 h, respectively; P = 0.020 and 0.045, respectively) or bath (8.8 +/- 3.1 and 7.7 +/- 2.2 pg mm-3; P = 0.050 and 0.039, respectively). The observed time courses of 8-MOP concentrations correspond to time courses of photosensitivity found previously with the different treatment modalities. CONCLUSIONS: The higher epidermal 8-MOP concentrations found after topical 8-MOP may explain the lower UVA doses needed with the topical route. These results suggest that topical 8-MOP may be superior in patients where the pathology is localized in the epidermis. In sclerosing diseases, which mainly affect the dermis oral PUVA might be advantageous because dermal concentrations are highest with this route of administration.     

Lecithin microemulsions in dimethyl ether and propane for the generation of pharmaceutical aerosols containing polar solutes

Water soluble compounds have been incorporated into solution phase metered dose inhalers (MDIs) utilizing lecithin inverse microemulsions in dimethyl ether (DME) and propane. DME and propane acted as both solvent and propellant. Experiments utilizing model propellants (dimethylethyleneglycol (DMEG) and hexane) were used to investigate microemulsion physicochemical phenomena, and the results were used to design and interpret the technically more challenging MDI experiments. NMR and viscosity experiments with model propellants were consistent with a "sphere-to-string" micellar shape change as the solvent was varied from pure DMEG to pure hexane. Water soluble solutes, including selected peptides and fluorescently labeled poly-alpha, beta-[N-(2-hydroxyethyl) D,L-aspartamide] (fPHEAs), dissolved in DME/propane dependent on lecithin and water content. MDIs containing microemulsions generated aerosols with mass median aerodynamic values ranging from 2.7 to 3.1 microns, within the range of commercially available formulations. Fine particle fraction values (50-70%) exceeded those of commercial formulations. fPHEA up to 18 kDa did not adversely affect the aerosol characteristics. Deposition of the aerosol onto a water surface resulted in the formation of liposomes with partially entrapped solute.     

Rate and extent of percutaneous absorption of sertaconazole nitrate after topical administration

The purpose of this open study was to evaluate the rate and extent of the penetration of sertaconazole nitrate (CAS 99592-32-2, Zala?n) penetration into the stratum corneum/lucidum of the human skin. Selected areas of 9 cm2 each of the back skin of 12 healthy volunteers were exposed over 8 different time intervals (between 0 and 48 h) to 100 mg of a 2% cream preparation of the compound or to placebo. Using a HPLC-assay the relative amounts of the applied dose of sertaconazole nitrate were determined in the residual cream of the skin surface as well as in 3 layers of the epidermis obtained by the stripping technique. Sertaconazole nitrate was shown to penetrate into the stratum corneum shortly after application, disappearing from the application areas with a mean apparent half-life of approximately 60 h. Immediately after topical application the residual amount of the applied mean dose of 2103 +/- 146.3 microg on the skin's surface was 88.9 +/- 2.3%, decreasing steadily to 52.4 +/- 8.5% after 48 h. A relevant amount of the applied dose (5.3 +/- 3.0%) was recovered from the stratum corneum already 30 min after application, and 3 h after administration a plateau was reached (6.9 +/- 3.2) which could be maintained until 48 h. A gradient from the site of application to the epidermis was apparent since the amounts recovered in The estimated average level of sertaconazole nitrate for a volume of 1 mL of stratum corneum after application of 100 mg cream was 1409 microg immediately after application and reached a plateau at 3 h with 9029 microg. Although not directly measured, the results also gave information about the mean amount of sertaconazole nitrate that penetrated through the stratum corneum and deeper layers allowing an estimate of the total mean amount of compound penetrating into the skin. The relative portion of this amount steadily increased from 1.1% of the applied dose at 0 h to 24.1% at 12 h, 34.2% at 24 h and finally to 37.6% of dose after 48 h of exposure. In view of the high target organ levels of the compound maintained over days, its rapid appearance in the stratum corneum after application and the earlier finding that Sertaconazole nitrate is not distributed into blood in substantial quantities the pharmacokinetic properties of this antifungal preparation therapy can be regarded as favourable.     

Diclofenac metabolic profile following in vitro percutaneous absorption through viable human skin.

The extent of metabolism of diclofenac sodium in excised viable human skin was investigated using combination HPLC and radioactivity assay. In an earlier diffusion experiment using an in vitro flow-through diffusion system, radiolabelled diclofenac sodium in either lotion (Pennsaid) or aqueous solution was applied to viable human skin, either as single dose or multiple dose (8 times over 2 days). In this study, the receptor fluid samples from the diffusion experiment were subjected to extraction and the aliquot was analysed using HPLC to separate diclofenac and authentic metabolites. Based on the radioactivity of each HPLC fraction, the collection time of the fractions was compared with the retention time of diclofenac and metabolites in standard solutions. The samples from a single or multiple dose application of lotion showed radioactivity in mainly one fraction, whose retention time corresponded with diclofenac. Other HPLC fractions showed none or only small amounts of radioactivity within the error range of the assay. The same results were obtained with the pooled samples from the application of the lotion or of aqueous solution. The results suggest that diclofenac sodium does not undergo metabolism in viable human epidermis during percutaneous absorption in vitro. Hence, with topical application to human skin in vivo, diclofenac will be delivered with minimal, if any, metabolism.     

Prediction of drug disposition kinetics in skin and plasma following topical administration

The development of a physically based pharmacokinetic model for percutaneous absorption is described. The simulation includes four first-order rate constants assigned the following significance: (a) absorption across the stratum corneum; (b) diffusion through the viable tissue; (c) a retardation process which retains penetrant in the stratum corneum (and hence provides a means to mathematically produce a "reservoir" effect, for example); and (d) uptake from the skin into the systemic circulation and subsequent elimination from the body. The kinetic equations of the model are solved and expressions are obtained for the concentration of penetrant within the stratum corneum (and available to subsequently partition into the viable epidermis) and the plasma concentration of the administered substance, as a function of time. Using example values for the four rate parameters, disposition profiles for the penetrant in skin and plasma were derived. The cases considered cover slow and fast stratum corneum penetrants, substances which are excreted rapidly or slowly from the body, and absorbing molecules with a variety of relative stratum corneum-viable tissue affinities. The results suggest a framework for the prediction of pharmaceutically and clinically relevant information following the topical administration of therapeutic agents for local or systemic effect.     

Nanoemulsions as vehicles for topical administration of glycyrrhetic acid: Characterization and in vitro and in vivo evaluation

Nano-emulsions are innovative colloidal systems characterized by high kinetic stability, low viscosity, and optical transparency, which make them very attractive in many dermatological applications. Furthermore their small size seems to favor the topical administration of actives which scarcely cross the skin. In the light of these interesting features, the present study was aimed to the evaluation, in vitro and in vivo, of glycyrrhetic acid (GA) release through the skin from the nanoemulsion system. GA-loaded nanoemulsion (GA_N) was prepared by phase inversion temperature (PIT) method, and was characterized in order to determine mean droplet size and its stability during a well-defined storage period. Further Cryo-TEM studies were performed to obtain information regarding nanoemulsion structure. The GA release pattern from nanoemulsion was evaluated in vitro, to determine its percutaneous absorption through excised human skin (stratum corneum and epidermis, SCE), and in vivo evaluating GA topical anti-inflammatory activity on healthy human volunteers by the UVB-induced erythema model. Nanoemulsions prepared by PIT method showed a mean droplet diameter of 210?nm that drastically changed during a storage of 5 weeks at room temperature. In vitro and in vivo evidence showed that the nanoemulsion system significantly increased the transdermal permeability of GA in comparison to a control O/W emulsion (GA_O/W) containing the same amount of active compound.     

The percutaneous absorption of hydroquinone (HQ) through rat and human skin in vitro

Because of the potential for human contact with photographic developer solutions containing hydroquinone (HQ), the rates of percutaneous absorption of HQ through human stratum corneum and full-thickness rat skin have been measured in vitro using 5% aqueous solutions of HQ as the donor solutions. The studies were performed using infinite doses of aqueous solutions containing ¹⁴C-labeled HQ in Franz-type diffusion cells. The measured absorption rate (mean ± S.D.) of HQ through human stratum corneum was 0.52 ± 0.13 μg/cm²/h, while that for full-thickness rat skin was 1.1 ± 0.65 μg/cm²/h. The ratio (rat/human) of the permeability constants (K_p) was 2.4. Using the definitions suggested by Marzulli et al. (1969) Toxicol. Appl. Pharmacol. Suppl. 3, 76–83, HQ would be classified as ‘slow’ with respect to its absorption through human stratum corneum.