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.

Magnetic resonance imaging after operative repair of achilles tendon rupture.

We performed a magnetic resonance imaging (MRI) study in 16 consecutive patients who had undergone open repair of a unilateral Achilles tendon rupture (ATR) at an average of 32.5 (SD 3.2) (range 29-36) months from the operation. We measured the widest antero-posterior diameter of the tendon, the longest distance between the insertion of the Achilles tendon on the calcaneum and the musculo-tendinous junction of the soleus muscle on the Achilles tendon, the distance between the insertion of the Achilles tendon on the calcaneum and the point of maximal width of the tendon. We also ascertained whether areas of altered signal were present in and around the tendon. The operated tendons were always significantly thicker than the non-operated ones. There was a non-significant trend for the other measurements to be greater in the operated tendons. In five patients, areas of dishomogeneous signal were present in the operated tendon. These areas were less than 25% of the antero-posterior diameter of the tendon, and were clinically silent. These findings probably represent normal features of long-term tendon healing following open repair of an ATR.

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.

Lactate distribution in the blood during steady-state exercise.

PURPOSE: The purpose of this investigation was to examine the plasma to red blood cell (RBC) lactate concentration ([La]) gradient and RBC:plasma [La] ratio during 30 min of steady-state cycle ergometer exercise at work rates below lactate threshold ( LT. Blood samples were taken from a heated forearm vein, immediately cooled to 4 degrees C in a dry-ice ethanol slurry, and centrifuged at 4 degrees C to separate plasma and RBCs. RESULTS: During >LT, plasma [La] rose to 8.8+/-1.1 mM after 10 min and remained above 6 mM. RBC [La] (4.9+/-0.7 mM) was significantly lower than plasma [La] at 10 min and remained lower throughout exercise. As a result, there was a sizable [La] gradient (approximately 3.5 mM) from plasma to RBC during most of >LT. In LT, the ratio of RBC [La]:plasma [La] was the same for both (0.58+/-0.02) and not significantly different from rest. CONCLUSIONS: These results refuted our hypothesis that the RBC:plasma [La] ratio would decrease at the onset of >LT exercise because of muscle lactate release exceeding the ability of RBCs to take up the lactate. Instead, there appears to be an equilibrium between plasma [La] and RBC [La] in arterialized venous blood from a resting muscle group as evidenced by the constant RBC [La]:plasma [La] ratio.

Lactate influx into red blood cells from trained and untrained human subjects.

PURPOSE: The purpose of this study was to compare the fractional contributions of the three pathways of lactate transport (band 3 system, nonionic diffusion, and monocarboxylate pathway) into red blood cells (RBC) from trained and untrained humans. METHODS: Blood samples were obtained from 19 male subjects: 5 untrained, 5 aerobically-trained, 5 competitive collegiate cross-country runners, and 4 competitive collegiate sprinters. The influx of lactate into the RBC was measured by a radioactive tracer technique using [14C]lactate. Discrimination of each pathway of lactate transport was achieved by using PCMBS (1 mM) to block the monocarboxylate pathway and DIDS (0.2 mM) to block the band 3 system. Nonionic diffusion was calculated as the difference between total lactate influx and the sum of band 3 and monocarboxylate lactate influx. RESULTS: Total lactate influx into the RBC from the more aerobic individuals (trained subjects and cross-country runners) was significantly faster at 1.6 mM lactate concentration ([La]) as compared with the influx into RBC of the untrained subjects. Total influx of lactate was significantly higher (P < 0.05) in the RBC from the sprinters as compared with that in the RBC from the untrained subjects at 41 mM [La]. There were no significant differences among the four groups with regard to the total influx of lactate at 4.1, 8.1, and 20 mM [La]. In general, the percentage of total lactate influx accounted for by each of the three parallel pathways at 1.6, 8.1, and 41.0 mM [La] was not different among the four groups of subjects. CONCLUSIONS: Overall, the groups were more similar than different with regard to RBC lactate influx.

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.

Lactate distribution in the blood during progressive exercise.

The purpose of this study was to examine the effect of increment durations of 1-min and 4-min during progressive incremental exercise tests on: 1) the distribution of lactate between plasma and red blood cells (RBCs), and 2) lactate threshold (LT) detection via three conventional methods using whole blood lactate concentration ([La]) or plasma [La]. Eight males (age, 22.5 +/- 0.6 yr: height, 170.6 +/- 2.3 cm, weight, 76.0 +/- 3.1 kg, and VO2peak, 42.8 +/- 2.0 mL.kg-1.min-1) performed two progressive load tests to volitional fatigue on a cycle ergometer. Work rate was increased 30 W at 1-min or 4-min intervals. All data were normalized to individual LT work rates. For both protocols, whole blood [La], plasma [La], RBC [La], and [La] gradient increased significantly (P < 0.05) after exercise intensity exceeded LT. However, the RBC:plasma [La] ratio remained at the resting value throughout the progressive exercise tests. The increase in [La] gradient after LT, with no change in the RBC:plasma lactate ratio, suggests that given an incremental work rate increase of 30 W, 1 min is adequate for equilibration of lactate between the plasma and RBCs. Also, under the conditions of this investigation, neither blood fraction analyzed nor exercise protocol had any effect on estimations of LT (in terms of VO2) by the Visual and Log-Log methods. However, LT determined by a fixed [La] of 2 mM may underestimate LT when plasma samples are used.

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.

Lactate influx into red blood cells of athletic and nonathletic species.

Transport of lactate across the erythrocyte membrane proceeds by three distinct pathways: 1) nonionic diffusion of lactic acid, 2) inorganic anion exchange (band 3), and 3) a monocarboxylate-specific (MC) carrier mechanism. This study determined the contributions of these three pathways in the red blood cells (RBCs) of "athletic" and "nonathletic" species. Blood samples were obtained from four male animals of each species: 1) Canis familiaris (dogs), 2) Capra hircus (goats), 3) Equus caballus (horses), and 4) Bos taurus (cattle). Contribution of each pathway to total lactate influx was determined by measuring L-[14C]lactate influx into lactate-depleted control RBCs, p-chloromercuribenzenesulfonic acid (PCMBS)-treated (1 mM) RBCs, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-treated (0.2 mM) RBCs at three lactate concentrations ([La] values 1.6, 8.1, and 41 mM). PCMBS blocked MC transport and DIDS blocked the band 3 pathway. Lactate influx into the RBCs of the athletic species was 4-160 times faster (P < 0.05) than influx into the RBCs of the nonathletic species at 8.1 and 41 mM [La] values. Nonionic diffusion was greater in the RBCs of nonathletic animals (approximately 7-25%) than in the RBCs of athletic animals (approximately 4%). A significantly higher percentage of the total lactate influx occurred via the band 3 system in the RBCs from the nonathletic animals (approximately 56-83%) vs. the RBCs from the athletic animals (approximately 6-7%) at all [La] values.(ABSTRACT TRUNCATED AT 250 WORDS)

Clothing and exercise. I: Biophysics of heat transfer between the individual, clothing and environment.

Despite large environmental variations, the human body maintains a tightly regulated core temperature. Effective thermoregulation must balance the interaction between skin surface, clothing and ambient air. Indices of thermal stress (wet bulb globe temperature, heat stress index, maximum evaporation rate, required evaporative rate and wind chill) provide valuable information concerning the heat exchange between the individual and the environment, and serve as protective guidelines while working in environmental extremes. The role of clothing, as an interactive barrier, greatly affects thermal balance. Clothing is varied according to prevailing environmental conditions, metabolic heat production, gender and age differences, fabric thermal properties, garment design and intended use. Models (static, dynamic and human) have investigated the biophysical transfer of heat between the skin surface area, clothing and ambient air. Additionally, the role of metabolic heat production during exercise can greatly influence tolerance to thermal stress during a variety of environmental conditions.

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.

The local side effects of transdermally absorbed nicotine.

Percutaneous nicotine administration induces predominant sudorific and rubiform responses in the skin which may be accompanied by subtle piloerection, hyperalgesia and pruritus (although these signs are not overtly manifest). These dermal responses are complex and mechanisms have been proposed for the direct nicotine-stimulation of sweat glands, piloerection and vasoconstriction. These reactions are accompanied by secondary activation and release of vasodilator peptides which produce a predominating vasodilator tone following topical administration, this response masking the direct axon reflex-mediated vasoconstriction.