370 JOURNAL OF COSMETIC SCIENCE the donor formulation to allow the top surface of the nails to dry out. The experi- ment was started by dosing the cells with the donor formulation [infinite dosing (200 lad for aqueous gels], and the cells were sealed with laboratory film (Parafilm©). Samples were drawn every hour for six hours by removing the entire contents of the receptor compartment and replenishing it with an equal volume of fresh receptor fluid. The samples were then analyzed for amount of water permeated. After completion of the study, the donor formulation adhering to the nail was wiped off. The nails were rinsed repeatedly (five to seven times for two-hour periods) in 10 ml of DI water by shaking in a mechanical shaker bath at 25øC to extract the label from the nails. Each of the extractions was also analyzed for 3H20 content. The label was considered to be extracted completely when the radioactive counts from the extractions approached blank values. Analysis of samples. Each of the samples (receptor solution and extractions) obtained during the permeation study was mixed with 10 ml of Scintiverse I (scintillation cocktail) and analyzed by liquid scintillation counting (LSC, Beckman model LS 5000T, Beckman Instruments, Somerset, NJ). Each sample was counted for at least five min- utes, and the counts in disintegrations per minute (dpm) were converted to amount of active, using the conversion factor 1 laCi = 2.2 x 10 6 dpm and the specific activity of 3H20. Data treatment. The cumulative amount of water permeated per unit area was plotted as a function of time. The flux, J, (mg cm-2h -1) was determined from the slope of the steady-state (straight-line) portion of the plot. The permeability coefficient, P, (cm h-1) was calculated by dividing the flux by the donor concentration of water. Due to the large differences in thickness between nails, the permeation parameters were normalized for nail thickness as shown below: J* = J x h (1) where J* = normalized flux (mg cm-lh -1) and h = nail thickness (cm) and P* = P x h (2) where P* = normalized permeability coefficient (cm2h-1). EFFECT OF ENVIRONMENTAL FACTORS (pH AND TEMPERATURE) ON NAIL PERMEATION Donor formulations. For pH studies, buffers of acidic, neutral, and basic pH values were prepared from standard tables (8). Specifically, a glycine-hydrochloric acid buffer (acidic, pH -2), monobasic potassium phosphate-dibasic sodium phosphate buffer (neutral, pH -7), and glycine-sodium hydroxide buffer (basic, pH -12) were prepared. HEC was added in a concentration of 1.5% to each of the buffer systems, and gel formation was allowed by shaking on a mechanical shaker bath for up to 12 hours. Each of the gels was spiked with 3H20 to obtain a hot:cold drug ratio of 1:1000. For temperature studies, aqueous gels spiked with 3H20 were used. Experimental design and method (a) pH studies. Two different studies were conducted to investigate the effect of pH. In the first study, the standard receptor was used. In the second, the receptor had the same com-

NAIL PERMEATION 371 position as the donor. In both studies, excised human toenails were used, and prior to the start of the study, a baseline water permeation profile for each toenail was established. A total of six nails were used, and these were assigned to two treatment groups (comprising three nails each) such that the mean normalized water flux for each group was approximately equal. In both studies, nails of both groups (n -- 6) were treated with the gel of pH 7. The method for the permeation studies and washout procedure was the same as that described above. The temperature was maintained at 37øC for all pH studies. Subsequently, nails of one group (n = 3) were treated with the gel of pH 2, and nails of the other group (n = 3) were treated with the gel of pH 12, also at 37øC. The permeation experiment and washout procedure was once again repeated. (b) Temperature studies. Three temperature settings were investigated: 37% 47 ø, and 57øC. As before, a total of six toenails were used, and these were again assigned to two treatment groups of varying temperature comprising three nails each. In order to achieve the different temperature settings, the diffusion cells were enclosed in an oven, and magnetic stirrer plates were placed below the cells to allow stirring. The oven temperature was monitored throughout the study (by placing thermometers in various parts of the oven) to ensure that it remained constant (+0.2øC). The donor gels and receptor solution were also equilibrated at the appropriate temperature before and during use. Initially, the water permeation studies were performed on all the nails at 37øC, followed by a washout period. After this, the permeation experiments were carried out on nails of one group (n = 3) at 47øC, and those of the other group (n = 3) at 57øC. A washout period followed once again. (c) Test for barrier integrity of nails after pH and temperature studies. In order to assess whether the barrier integrity of the toenails had been retained after pH and heat treatments, water permeation studies using aqueous gels spiked with 3H20 were performed on all the nails after pH/ternperature treatment and washout. The water permeation parameters so obtained were compared with the baseline parameters (which had been previously determined). Minimal changes in permeation parameters before and after pH/ternperature treatments indicated that the barrier integrity of the nails had been maintained. RESULTS AND DISCUSSION DEPENDENCE OF PERMEABILITY ON SOURCE OF NAILS Figure 2 depicts the permeation profiles for toenail pairs from different donors (desig- nated 1 and 2) different nails from the same donor are designated A and B. Profiles for nails of Pair 1 are illustrated by open symbols, while closed symbols are used for nails of Pair 2. Water permeation parameters for these two nail pairs (normalized for nail thickness) are shown in Table I. Repeated measurements on the same nail result in less than 5% variability in the data (as evidenced by the small error bars in Figure 2), indicating that the technique is highly reproducible. Figure 2 also shows that permeation patterns for two nails of the same pair are very similar however, these patterns differ considerably between pairs (water per- meation through nails of Pair 2 is much higher than that through nails of Pair 1). Thus,