Quantification of corticosteroid-induced skin vasoconstriction: visual ranking, chromameter measurement or digital imaging analysis.

Topical corticosteroid formulations have been evaluated by visual grading protocols for many years. Toward a more objective methodology, several instrumental methods have been evaluated for applicability in quantifying the vasoconstriction side-effect that follows corticosteroid application to the skin. Although the chromameter has been adopted by regulatory bodies throughout the world as the current standard for topical bioequivalence determinations, there is considerable criticism of this instrument from several quarters. A preliminary comparison reported here indicates that digital image analysis provides statistically significant results that are similar to those obtained by visual assessment techniques, and shows considerably greater precision than that obtained by the chromameter. Continued evaluation of objective assessment techniques, such as digital imaging, and continued modernisation of regulatory bioequivalence requirements will assist in protecting patients and optimising clinical results.

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 activity 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.

Analysis of chromameter results obtained from corticosteroid-induced skin blanching assay: comparison of visual and chromameter data.

In a Guidance document, the American FDA recommends the use of a Minolta chromameter rather than the human eye for the quantitative assessment of the pharmacodynamic blanching response produced by topical application of corticosteroids. The purpose of this study was to compare the appropriateness of the human eye and two models of chromameter for the estimation of skin blanching, in terms of the quality of the data generated by each method. The corticosteroid-induced skin blanching from four different betamethasone 17-valerate cream formulations was compared in a typical human skin blanching trial. The optimized assay methodology routinely practised in our laboratories was utilized. The blanching responses were assessed visually by three trained, independent observers and recorded by two chromameters (Minolta model CR-200 and model CR-300). The topical availability of the four creams was determined using visual scoring and chromameter measurements. All data were manipulated in such a manner as to produce a blanching response versus time profile from which AUBC analysis could be performed. Good correlation was observed between the visual assessments made by three independent observers. In contrast, moderate correlation was determined between visual, CR-200 and CR-300 measurements. Surprisingly, no direct linear relationship between the AUBCs produced by the two chromameters was observed indicating that the quality of the data obtained from the two instruments may not be equal. This investigation also indicated that the use of the chromameter is not completely objective. Visual scoring and chromameter measurement produce data sets that differ in quality. Each procedure needs to be validated and investigators have to be trained for both visual assessment and the operation of the chromameter, particularly with regard to the manipulation of the measuring head of the instrument.

Analysis of chromameter results obtained from corticosteroid-induced skin blanching. I: Manipulation of data.

PURPOSE: One of the unresolved issues in the FDA Guidance document for topical corticosteroid bioequivalence testing is the method of manipulation suggested for the chromameter data. The purpose of this study was to manipulate the instrumental data from a typical blanching study in a number of ways to investigate the appropriateness of these procedures for comparison with the subjective visually-assessed results. METHODS: The human skin blanching assay methodology routinely practiced in our laboratories was utilised and the vasoconstriction produced by two corticosteroid formulations of different potency was assessed visually and instrumentally by use of a Minolta chromameter. The instrumental data were corrected for zero-time and unmedicated site readings. In addition, Euclidean distances were calculated using all data generated by the instrument. RESULTS: Individually the a-, b- and L-scale chromameter values are imprecise and there is negligible vasoconstriction response recorded for the moderately potent formulation. Arithmetical manipulation of the data as suggested by the FDA does not appear to improve the quality of the data in any way. Euclidean distance analysis more closely resembles the visual data and appears to have better precision. CONCLUSIONS: It is clear that mathematical correction of chromameter data is unnecessary, especially since the instrumental data are extremely imprecise. Furthermore, the assessment of each individual chromameter index does not adequately characterise the blanching response profile. It is therefore suggested that Euclidean distance may be a better measure on which to base an analysis of bioequivalence than the truncated data set methodology currently suggested by the FDA.

Evaluation of the proposed FDA pilot dose-response methodology for topical corticosteroid bioequivalence testing.

PURPOSE: The American FDA has recently released a Guidance document for topical corticosteroid bioequivalence testing. The purpose of this study was to evaluate the recommendations of this document for appropriateness. The new specifications require a dose-vasoconstriction response estimation by the use of a Minolta chromameter in a preliminary pilot study to determine the parameters for use in a pivotal bioequivalence study. METHODS: The visually-assessed human skin balancing assay methodology routinely practiced in our laboratories was modified to comply with the requirements of the pilot study so that visual and chromameter data could be compared. Two different cream formulations, each containing 0.12% betamethasone 17-valerate, were used for this comparison. RESULTS: Visual data showed the expected rank order of AUC values for most dose durations whereas the chromameter data did not show similar results. The expected rank order of AUC values for both chromameter and visual data was not observed at very short dose durations. In fitting the data to pharmacodynamic models, equivalent goodness of fit criteria were obtained when several different parameter estimates were used in the model definition, however the visual data were best described by the sigmoid Emax model while the chromameter data were best described by the simple Emax model. CONCLUSIONS: The Emax values predicted by the models were close to the observed values for both data sets and in addition, excellent correlation between the AUC values and the maximum blanching response (Rmax) (r > 0.95) was noted for both methods of assessment. The chromameter ED50 values determined in this study were approximately 2 hours for both preparations. At this dose duration the instrument would not be sensitive enough to distinguish between weak blanching responses and normal skin for bioequivalence assessment purposes.

Sensitivity of different areas of the flexor aspect of the human forearm to corticosteroid-induced skin blanching.

The intensity of corticosteroid-induced blanching has been found to vary at different areas of the flexor aspect of the human forearm. A retrospective analysis of 38,880 observations of skin blanching in 56 volunteers was conducted to assess the sensitivity of forearm skin to betamethasone 17-valerate. The mid-forearm appears to be more sensitive to the blanching response than do the areas close to the wrist or elbow. These results indicate that each preparation under evaluation should be applied to several sites along the forearm when using the human skin blanching assay in order to obtain an accurate comparative assessment of corticosteroid release from topical delivery vehicles.

In vitro diffusion cell design and validation. II. Temperature, agitation and membrane effects on betamethasone 17-valerate permeation.

An in vitro permeation cell has been designed and validated for use in monitoring the transmembrane permeation of betamethasone 17-valerate. The design utilizes common laboratory equipment and incorporates as many beneficial features as possible from other designs. The importance of fully validating the hydrodynamic performance of the cell prior to experimentation is stressed. The cell was validated by monitoring the diffusion of betamethasone 17-valerate in isopropyl myristate solution into purified isopropyl myristate receptor phase at different temperatures, different agitation rates and through different synthetic and biological membranes. The results of the hydrodynamic validation agree with data from other researchers and show that the permeation cell is adequately sensitive to these experimental parameters. The results of the membrane evaluation allow appropriate selection of the barrier material for representative transdermal experiments to be conducted. While human and porcine stratum corneum/epidermis are similar in diffusive properties, hairless mouse skin appears to be the most convenient animal membrane for these studies. Although silicone and cellulose membranes appear to be useful in this application, porous filter membranes and egg-shell membranes are insufficiently discriminatory to betamethasone 17-valerate diffusion to provide useful in vitro permeation data.

Blanching activities of betamethasone formulations. The effect of dosage form on topical drug availability.

The blanching activities of Betnovate cream, lotion, ointment and scalp application (each containing 0.1% betamethasone (as the 17-valerate] were determined using healthy human subjects over a 32 h period in both the occluded and unoccluded modes. Considering that all four formulation types contained the same label concentration of corticosteroid, it may be presumed that the formulations would show similar topical drug availability: this was, however, not found to be the case. The scalp application demonstrated the highest topical availability in both the occluded and unoccluded modes. The lotion formulation showed the greatest increase in topical availability on occlusion and the ointment formulation was the least sensitive to the effects of occlusion. These differences, due solely to the effects of the vehicle, may have important clinical implications.

Potency ranking of two new topical corticosteroid creams containing 0.1% desonide or 0.05% halometasone utilising the human skin blanching assay.

The human blanching assay was used to assess the potency of two new proprietary corticosteroid creams. The blanching abilities of 0.1% desonide cream and 0.05% halometasone cream were evaluated relative to the blanching elicited by 0.05% clobetasol 17-propionate cream, 0.1% betamethasone 17-valerate cream and 0.05% clobetasone 17-butyrate cream. The results of the trial indicated that the 0.1% desonide cream falls into the potent group of topical corticosteroid preparations and the 0.05% halometasone cream falls into the moderately potent group.

High-performance liquid chromatographic analysis of oleandomycin in serum and urine.

The determination of oleandomycin in serum and urine by high-performance liquid chromatography using erythromycin as internal standard is described. The separation was achieved on a reversed-phase C18 column employing acetonitrile-0.05 M phosphate buffer (30:70), adjusted to pH 7.0, as the mobile phase with UV detection at 200 nm. A solid-phase extraction procedure, combined with a simple phase-separation step was used prior to chromatographic analysis. Linear calibration curves were obtained in the concentration ranges 0.25-5.0 micrograms/ml (serum) and 1.0-25.0 micrograms/ml (urine). Precise quantitative analysis has been achieved at these levels with relative standard deviations of less than 5%.

Determination of erythromycin in serum and urine by high-performance liquid chromatography with ultraviolet detection.

A high-performance liquid chromatographic analysis of erythromycin in human serum and urine with UV detection at 200 nm is presented. The method involves a solid-phase extraction procedure followed by a simple phase separation step and chromatography on a reversed-phase column. The method has sensitivity limits of 0.25 and 1.0 micrograms/mL in serum and urine, respectively, and is sufficiently sensitive to monitor concentrations of erythromycin in human serum and urine after the administration of a single 500-mg erythromycin stearate tablet.

Determination of phenylpropanolamine in serum and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic analysis of phenylpropanolamine in human serum and urine without prior derivatization is presented. Using direct UV detection the method is sufficiently sensitive to detect 25 ng of drug/ml of serum or urine; the coefficients of variation at 25 ng/ml and 500 ng/ml were 5. 16 and 2.12, respectively, in serum. The method involves serum and urine extraction at a basic pH with chloroform, a single back-extraction, and chromatography on a reverse-phrase column. Serum and urine data following administration of a single 150-mg sustained-release tablet of phenylpropanolamine hydrochloride in six healthy volunteers demonstrates the suitability of the analytical method.

Possible dosage regimens for topical steroids, assessed by vasoconstrictor assays using multiple applications.

The bioavailabilities and activities of three amcinonide preparations and Betnovate cream were assessed using three multiple-dosage regimen vasoconstrictor assays in 10 volunteers. Applications were made once daily, twice daily and every alternate day with an initial three times daily loading dose applied on the first day only. Blanching responses first increased and then decreased due to tachyphylaxis. It is proposed that clinically the most advantageous dosage regimen is a once daily application with no loading dose.

Simultaneous determination of trimethoprim, sulphamethoxazole and N4-acetylsulphamethoxazole in serum and urine by high-performance liquid chromatography.

The simultaneous determination of trimethoprim, sulphamethoxazole and N4-acetyl-sulphamethoxazole in serum and urine by high-performance liquid chromatography using sulphafurazole as internal standard is described. The separation was achieved on a reversed-phase column employing acetic acid-methanol as the mobile phase with spectrophotometric detection at 230 nm. Precise simultaneous quantitative analysis of the relative components has been achieved at levels of 0.1 microgram/ml for trimethoprim and 1.0 microgram/ml for both sulphamethoxazole and its N4-acetyl metabolite using 1 ml of serum of urine.

Release of betamethasone 17-valerate from extemporaneous dilutions of a proprietary topical cream.

Six different vehicles for topical use were used to prepare 50% dilutions of Betnovate (betamethasone 17-valerate, 0.1%) cream. Blanching assessment as undertaken immediately after preparing the various dilutions and at 1 and 3 months thereafter. Few statistically significant differences were noted between any of the preparations tested indicating hat the rate of release of betamethasone 17-valerate is relatively unaffected by dilution. All preparations were assayed by a stability indicating high pressure liquid chromatographic technique for corticosteroid content. A diminution in the content of betamethasone 17-valerate in the E45 dilution was found 14 months after preparation. All other formulations tested were found to comply with label claim specifications.