Metabolism of ethyl 4-aminobenzoate in the isolated single-pass perfused rabbit ear.

Isolated rabbit ears were single-pass perfused with a protein-free Tris buffer solution. Ethyl 4-amino[U-ring-14C]benzoate or 4-amino[U-ring-14C]benzoic acid was applied to the epithelial surface in isopropyl myristate, or perfused arterially. Metabolites in the effusate were extracted and separated by high-performance liquid chromatography. Five metabolites were separated from ethyl aminobenzoate and identified as aminobenzoic acid, or acid-labile conjugates from either substrate. Their chemical structure has not yet been established. One metabolite, perhaps a glycolic acid N-conjugate of ethyl 4-aminobenzoate, was found at about 100 times higher concentration after arterial than after dermal drug application, indicating that the quantitative metabolic patterns in skin differ depending on whether the xenobiotic reaches the enzymes of the skin by cutaneous absorption or by the blood circulation. The metabolite pattern of the skin was compared with that from rabbit or rat liver cell preparations.

Ester hydrolysis and conjugation reactions in intact skin and skin homogenate, and by liver esterase of rabbits.

1. Procaine, 2-chloroprocaine, ethyl aminobenzoate and methyl salicylate were added at various concentrations to liver esterase, supernatant of skin homogenate, or single-pass perfused ears of rabbits. 2. Vmax of product formation by purified liver esterase correlated with the rank order of the distribution coefficients (n-octanol/buffer) of the substrates and ranged between 11 and 1100 pmol/min per micrograms protein. Km values were between 20 and 50 microM. 3. No correlation was observed when the apparent enzyme kinetics, calculated by non-linear adaptation, were compared with each other after substrate administration to skin, arterial influx, or incubation with skin homogenate. 4. An acid labile conjugate of ethyl 4-aminobenzoate was found, mainly during arterial perfusion and in supernatant of skin homogenate, after administration of ethyl 4-aminobenzoate. 5. Acetamidobenzoic acid was observed in quantities of about 10% of the free 4-aminobenzoic acid during dermal or arterial application of procaine. This metabolite was not found with ethyl 4-aminobenzoate. 6. The results from isolated rabbit ear perfusion differ quantitatively and qualitatively with those obtained from supernatant of skin homogenate or purified liver esterase.

Dermal metabolism of 4-nitrophenol and 4-nitroanisole in single-pass perfused rabbit ears.

1. 4-Nitrophenol and 4-nitroanisole were applied either dermally or arterially to isolated rabbit ears perfused under single-pass conditions with protein-free buffer solution. 2. 4-Nitroanisole yielded only phase II metabolites of 4-nitrophenol. 3. The apparent Vmax values for 4-nitrophenol glucuronidation and sulphation were about 20 pmol and 10 pmol/min per cm2, respectively. 4. The difference in apparent Km between dermal and arterial drug application is a measure of first-pass metabolism by the epidermal layer. 5. The amount of 4-nitrophenyl conjugate detected after 4-nitroanisole administration was assumed to represent O-dealkylation of 4-nitroanisole; the capacity of this reaction was one order of magnitude lower than the direct conjugation of 4-nitrophenol.