Naruhito Higo

Validation of Reflectance Infrared Spectroscopy as a Quantitative Method to Measure Percutaneous Absorption In Vivo

Attenuated total-reflectance infrared (ATR-IR) spectroscopy has been used to follow the penetration of a model compound (4-cyanophenol; CP) across human stratum corneum (SC) in vivo, in man. CP was administered for periods of 1, 2, or 3 hr, either (a) as a 10% (w/v) solution in propylene glycol or (b) in an identical vehicle which also contained 5% (v/v) oleic (cis-9-octadecenoic) acid. At the end of the treatment periods, SC at the application site was progressively removed by adhesive tape-stripping. Prior to the removal of the first tape-strip, and after each subsequent tape-strip, an ATR-IR spectrum of the treated site was recorded. The presence of CP, as a function of position in the SC, was monitored spectroscopically via the intense C≡N stretching absorbance at 2230 cm−1. The absolute amount of CP, as a function of SC depth, was determined by spiking” the applied solutions with 14C-labeled compound and subsequent liquid scintillation counting of the removed tape-strips. The presence of oleic acid in the applied formulation significantly increased the rate and extent of CP delivery as evaluated by either spectroscopy or radiochemical analysis. Furthermore, the ATR-IR and direct 14C analysis of CP as a function of SC position were highly correlated. These data strongly support, therefore, the validation of ATR-IR as a quantitative tool to assess percutaneous penetration in vivo.

Cutaneous Metabolism of Nitroglycerin in Vitro. II. Effects of Skin Condition and Penetration Enhancement

The effects of skin storage, skin preparation, skin pretreatment with a penetration enhancer, and skin barrier removal by adhesive tape-stripping on the concurrent cutaneous transport and metabolism of nitroglycerin (GTN) have been studied in vitro using hairless mouse skin. Storing the skin for 10 days at 4°C did not alter barrier function to total nitrate flux [GTN + 1,2-glyceryl dinitrate (1,2-GDN) + 1,3-glyceryl dinitrate (1,3-GDN)]. However, metabolic function was significantly impaired and suggested at least fivefold loss of enzyme activity. Heating skin to 100°C for 5 min appreciably damaged hairless mouse skin barrier function. The ability to hydrolyze GTN was still present, however, and remained constant over the 10-hr experimental period, in contrast to the “control,” which showed progressively decreasing enzymatic function with time. Pretreatment of hairless mouse skin in vivo (prior to animal sacrifice, tissue excision, and in vitro transport/metabolism studies) with 1-dodecylazacyclo-heptan-2-one (Azone), a putative penetration enhancer, significantly lowered the skin barrier to nitrate flux (relative to the appropriate control). Again, barrier perturbation resulted in essentially constant metabolic activity over the observation period. The ratio of metabolites formed (1,2-GDN/1,3-GDN) was increased from less than unity to slightly above 1 by the Azone treatment. Adhesive tape-stripping gradually destroyed skin barrier function by removal of the stratum corneum. The effects of 15 tape-strips were identical to those of Azone pretreatment: a greatly enhanced flux, a constant percentage formation of metabolites over 10 hr (once again), and an increase in the 1,2-GDN/1,3 GDN ratio. Overall, the experiments caution that, for transdermal drug delivery candidates susceptible to skin metabolism, the status of barrier function (enhancer pretreated, skin damage or disease, etc.) may significantly affect systemic availability.

Cutaneous Metabolism of Nitroglycerin in Vitro. I. Homogenized Versus Intact Skin

The metabolism of nitroglycerin (GTN) to 1,2- and 1,3-glyceryl dinitrate (GDN) by hairless mouse skin in vitro has been measured. In the first set of experiments, GTN was incubated with the 9000g supernatant of fresh, homogenized tissue in the presence and absence of glutathione (GSH), a cofactor for glutathione-S-transferase. After 2 hr of incubation with GSH, 30% of the initially present GTN had been converted to 1,2- and 1,3-GDN; without GSH, less than 5% of the GTN was metabolized. The ratio of 1,2-GDN to 1,3-GDN produced by the homogenate was 1.8– 2.1. In the second series of studies, GTN was administered topically to freshly excised, intact hairless mouse skin in conventional in vitro diffusion cells. The concurrent transport and metabolism of GTN was then monitored by sequential analysis of the receptor phase perfusing the dermal side of the tissue. Three topical formulations were used: a low concentration (1 mg/ml) aqueous solution, a 2% ointment, and a transder-mal delivery system. Delivery of total nitrates (GTN + 1,2-GDN + 1,3-GDN) into the receptor phase was similar for ointment and patch formulations and much greater than that from the solution. The percentage metabolites formed, however, was greatest for the solution (61% and 2 hr, compared to 49% for the patch and 35% for the ointment). As has been noted before, therefore, the relative level of skin metabolism is likely to be greatest when the transepidermal flux is small. Distinct from the homogenate experiments, the 1,2/1,3-GDN ratios in the penetration studies were in the range 0.7– 0.9. It would appear that homogenization of the skin permits GTN to be exposed to a different distribution of enzymes than that encountered during passive skin permeation.