Ungar, Kobrin and Sezesny (1959) devised an anti-inflammatory assay method the principle of which is to punch out the skin pieces from the inflamed and control sites and weigh them to obtain the net weight increase of the skin produced by phlogogenic substances. The present author carefully re-examined this method with special consideration on the experimental conditions and the reproducibility of the results. The inflammatory edema was induced in animals by the intracutaneous injection of anti-rat rabbit serum, serotonin, bradykinin, dextran,
carrageenin, formalin (in rats) and histamine (in guinea pigs). It was found that the weight increase (edema intensity) of the skin induced by phlogogenic substances is related with the body weight of animals, the caliber of the punch and the pH of the vehicle in which irritants are dissolved. Satisfactory results were obtained with the use of male rats weighing 150±10 g, the punch of 12 mm caliber, and 0.9% physiological saline adjusted to pH 7.2 as the vehicle. As the skin could be punched out from 4 test sites and 4 control sites of one animal, it was possible to estimate the edema intensity with good reproducibility of the results using a relatively small number of animals. There was found a linear correlation between log doses of the phlogogenic substance and the weight increase of skin. A linear correlation was also obtained between the log doses of the anti-inflammatory agents tested and the percentage inhibition of weight increase of inflamed skin. With respect to anti-rat rabbit serum edema, the anti-inflammatory effect of oxyphenbutazone was somewhat longer lasting than that of phenylbutazone. Peroral phenylbutazone was slightly more effective than intramuscular. These results reflect well the course of blood level of these agents reported in rats as well as in man. The author depicted log dose-effect curves of 7 kinds of glucocorticoids and of 12 non-steroid anti-inflammatory agents of current clinical use. The order of the anti-inflammatory potency of these compounds coincided with the order of the clinical effect surmised on the basis of doses. Phenylbutazone, oxyphenbutazone, hydrocortizone, prednisolone, dexamethazone, chloroquine, aminopyrine and aspirin also showed clear inhibitory effects on the inflammatory edema induced by histamine, serotonin, bradykinin, and dextran. Discussion was made on the simplified experimental design with this method, for the anti-inflammatory screening of newly synthesized compounds, with some concrete examples.