Hydrogel increases diclofenac skin permeation and absorption.

PURPOSE: Topical nonsteroidal anti-inflammatory drug formulations are used commonly to treat musculoskeletal pain and inflammation. Drug properties and formulation composition are the primary determinants of the transdermal drug delivery rate. The ex vivo transdermal flux through human skin of three topical diclofenac formulations was compared. METHODS: The formulations tested were hydrogel 1% diclofenac sodium and two emulsion gels (1.16%/2.32% diclofenac diethylamine, equivalent to 1%/2% diclofenac sodium). Human abdominal skin obtained during unrelated surgical procedures was stored at -20 °C until use. Skin specimens were thawed, prepared and placed in Franz diffusion cells (stratum corneum facing donor cell). The test formulation (~200 mg) was applied to the donor cell skin surface, and the receptor compartment was periodically sampled over 48 hours. The drug concentration in the receptor medium was determined by a validated HPLC method. Raman spectral imaging was performed to visualize the location and distribution of diclofenac. RESULTS: After 5 hours, the cumulative amount of hydrogel diclofenac transiting the skin was about 10 times that of the emulsion gel 1.16% (P=0.0004) and about twice that of the emulsion gel 2.32% (P=0.022). Similar results were seen after 9 hours. Raman spectroscopy showed that the hydrogel formulation was a homogeneous mixture of its various components, including diclofenac. The emulsion gels were non-homogeneous, with diclofenac in close proximity to the lipophilic (paraffin) phase. CONCLUSIONS: The transdermal transit of diclofenac from the hydrogel demonstrated a faster onset and a greater absorption rate than either emulsion gel formulation, suggesting that the hydrogel formulation may have a faster onset of action in underlying tissues vs. the emulsion gel products.

Ex vivo transdermal absorption of a liposome formulation of diclofenac.

Topical formulations of non-steroidal anti-inflammatory drugs are often used to provide effective local drug concentration while limiting systemic exposure and associated adverse events. Formulation composition has great influence on the rate of transdermal drug transport through human skin. This study was performed to compare the ex vivo transdermal transport of diclofenac from three topical formulations, a 1% liposomal gel formulation of diclofenac sodium and two emulsion gel formulations, 1.16% and 2.32% diclofenac diethylamine (equivalent to 1% and 2% diclofenac sodium). Human skin was obtained during unrelated surgical procedures and frozen at -20 °C until use. Three skin specimens were thawed, prepared for testing, and placed in a Franz cell with the stratum corneum facing the donor compartment. About 200 µl of each formulation was placed on the skin, and the receptor compartment fluid (phosphate buffered saline, pH 7.4, 32 °C) was sampled over the next 48 h. Diclofenac concentration was measured with a validated HPLC method. The transdermal permeability coefficient for the liposome 1% gel was 69.3 ± 14.4 10-8 cm•s-1, compared with 34.9 ± 9.1 10-8 cm•s-1 (P = 0.001) and 47.1±9.5 10-8 cm•s-1 (P = 0.005) for the emulsion gel 1.16% and emulsion gel 2.32%, respectively. A statistically significant difference between transdermal transport of diclofenac from the liposome gel 1% and the emulsion gel 1.16% was evident after 9 h, a clinically relevant result because these products are typically applied 2 to 4 times daily. Based on these observations, liposome gel 1% formulation of diclofenac may have a clinical advantage compared with the emulsion gel 1.16% formulation.

In Vitro Bioavailability Study of an Antiviral Compound Enisamium Iodide.

An investigation into the biopharmaceutics classification and a study of the in vitro bioavailability (permeability and solubility) of the antiviral compound enisamium iodide (4-(benzylcarbamoyl)-1-methylpyridinium iodide) were carried out. The solubility of enisamium iodide was determined in four different buffers. Apparent intestinal permeability (Papp) of enisamium iodide was assessed using human colon carcinoma (Caco-2) cells at three concentrations. The solubility of enisamium iodide in four buffer solutions from pH 1.2 to 7.5 is about 60 mg/mL at 25 °C, and ranges from 130 to 150 mg/mL at 37 °C, depending on the pH. Based on these results, enisamium iodide can be classified as highly soluble. Enisamium iodide demonstrated low permeability in Caco-2 experiments in all tested concentrations of 10-100 µM with permeability coefficients between 0.2 × 10-6 cm s-1 and 0.3 × 10-6 cm s-1. These results indicate that enisamium iodide belongs to class III of the Biopharmaceutics Classification System (BCS) due to its high solubility and low permeability. The bioavailability of enisamium iodide needs to be confirmed in animal and human studies.

Comparative permeability and diffusion kinetics of cyclosporine A liposomes and propylene glycol solution from human lung tissue into human blood ex vivo.

Aerolized cyclosporine A (CsA) has been successfully used for prevention of organ rejection in lung transplant recipients. Various formulations of CsA are available and so far no direct comparison of their pharmacokinetics has been performed. Since clinical studies are elaborate, we sought a way to predict the kinetic behaviour of a propylene glycol solution of CsA (CsA-PG) and a liposomal formulation (L-CsA). The permeability across the human bronchial cell line Calu-3 revealed low permeability for CsA with the apparent permeability for CsA-PG being twice as high as for L-CsA. Employing a previously described dialysis model, the diffusion of CsA from human lung tissue into human blood was determined ex vivo. Consistent with the cell culture model results, we observed that the degree and rate of drug transfer into human blood was more pronounced for CsA-PG than for L-CsA with the area under the curve (AUC) of CsA-PG being about 1.6 times higher than for the L-CsA formulation. The diffusion rate was more than 50% higher from CsA-PG than from the liposomes. To conclude, both model systems consistently revealed that L-CsA displayed clearly a prolonged release effect and favourable longer tissue retention than CsA-PG.

The physicochemical parameters of marker compounds and vehicles for use in in vitro percutaneous absorption studies.

In order to prepare for a validation study to compare percutaneous absorption through reconstructed human epidermis with ex vivo skin absorption through human and animal skin, nine test compounds, covering a wide range of physicochemical properties were selected, namely: benzoic acid; caffeine; clotrimazole; digoxin; flufenamic acid; ivermectin; mannitol; nicotine; and testosterone. The donor and receptor media for the test substances, the addition of a solubiliser for the lipophilic compounds, as well as the stability and solubility of the test substances in the vehicles, were systematically analysed. Hydrophilic molecules, being freely soluble in water, were applied in buffered saline solutions. In order to overcome solubility restrictions for lipophilic compounds, the non-ionic surfactant, Igepal CA-630, was added to the donor vehicle, and, in the case of clotrimazole and ivermectin, also to the receptor fluid. The model molecules showed a suitable solubility and stability in the selected donor and receptor media throughout the whole duration of the test.

The use of reconstructed human epidermis for skin absorption testing: Results of the validation study.

A formal validation study was performed, in order to investigate whether the commercially-available reconstructed human epidermis (RHE) models, EPISKIN, EpiDerm and SkinEthic, are suitable for in vitro skin absorption testing. The skin types currently recommended in the OECD Test Guideline 428, namely, ex vivo human epidermis and pig skin, were used as references. Based on the promising outcome of the prevalidation study, the panel of test substances was enlarged to nine substances, covering a wider spectrum of physicochemical properties. The substances were tested under both infinite-dose and finite-dose conditions, in ten laboratories, under strictly controlled conditions. The data were subjected to independent statistical analyses. Intra-laboratory and inter-laboratory variability contributed almost equally to the total variability, which was in the same range as that in preceding studies. In general, permeation of the RHE models exceeded that of human epidermis and pig skin (the SkinEthic RHE was found to be the most permeable), yet the ranking of substance permeation through the three tested RHE models and the pig skin reflected the permeation through human epidermis. In addition, both infinite-dose and finite-dose experiments are feasible with RHE models. The RHE models did not show the expected significantly better reproducibility, as compared to excised skin, despite a tendency toward lower variability of the data. Importantly, however, the permeation data showed a sufficient correlation between all the preparations examined. Thus, the RHE models, EPISKIN, EpiDerm and SkinEthic, are appropriate alternatives to human and pig skin, for the in vitro assessment of the permeation and penetration of substances when applied as aqueous solutions.

Establishment and validation of an ex vivo human cervical tissue model for local delivery studies.

The objective of this study was to establish and validate an ex vivo human cervical tissue model appropriate for transport studies of molecular and especially nucleic acid based drugs. For that purpose conditions had to be established for a standardized tissue handling and preparation following hysterectomy to allow an immediate experimental use of fresh tissue samples. Samples of the ectocervical, endocervical and the transition zone representing the entire cervix organ were characterized in Franz diffusion cells by the determination of the in vitro permeation of low and high molecular weight markers (propanolol, mannitol, dextran 4000, 10,000, 20,000 and 40,000Da). Additionally, the permeability of mannitol and dextran 4000 across fresh and frozen cervical tissue was compared. The apparent permeability coefficients (P(app)) of the various markers demonstrated (i) that with increasing molecular weight the marker permeability decreases, (ii) an upper permeability limit between 10,000 and 20,000Da, (iii) no significant difference of the permeability across the three cervical tissue zones, (iv) a statistically significant but effectively small variation of the permeability among different patient samples. A continuous difference of approximately two log values between the P(app) values of mannitol and dextran 4000 makes them suitable as an internal marker control pair for each biopsy. Moreover, the P(app) values of both markers across fresh and frozen tissue are comparable. According to the presented data we conclude that the human cervical tissue model has been well characterized and is therefore suitable for local delivery and permeation studies.

Permeability of the reconstructed human epidermis model Episkin in comparison to various human skin preparations.

The objective of this work was to compare the barrier function of the small diameter reconstructed human epidermis model Episkin (d=12 mm) to human skin in vitro. For that purpose a modification for the Franz diffusion cell (d=15mm) had to be developed so as to allow direct comparison with the following human skin preparations: Full thickness skin (FTS), split thickness skin (STS), heat-separated epidermis (HSE), and trypsin isolated stratum corneum (TISC). Among the tested preparations, HSE appeared to be the most preferable due to its clear morphological structure and ease of preparation. The lipid profile of HSE and Episkin was analyzed and showed significant differences in terms of cholesterol, ceramides and triglycerides contents, whereas cholesterol esters and fatty acids were not different. Permeation data with HSE and Episkin were then gathered using caffeine and testosterone. Both test compounds permeated much faster through Episkin than through HSE. Moreover, opposed to Episkin, HSE differentiated between the two test compounds. In spite of the remarkable progress in developing RHEs in the past years at this time Episkin can obviously not yet fully replace human skin for in vitro permeability experiments.

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study.

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.