PERCUTANEOUS ABSORPTION OF ADRENAL STEROIDS 149 Later studies by Livingood, et al. (17), in which radioactivity was meas- ured in urine specimens subjected to enzymatic hydrolysis, confirmed the percutaneous absorption of hydrocortisone-4-C TM in humans. Absorp- tion through normal skin sites was found to be somewhat higher from lotion bases than from ointment vehicles. Absorption of the hormone from sites of inflammatory dermatoses ranged as high as 15 per cent. An investigation of the pathways of absorption of hydrocortisone-4-C TM free alcohol was carried out with the technique of autoradiography by Scott and Kalz (18). These workers showed that hydrocortisone enters the skin through both the transepidermal and the pilosebaceous routes. Radioactive material absorbed via the transepidermal pathway reached the basal cell layer two hours after application and proceeded to diffuse through- out the corium during the next four hours after sixteen hours almost all of the hydrocortisone had presumably passed into the systemic circula- tion. Further studies of hydrocortisone absorption through the skin and also through certain mucous membrane surfaces were reported by Liddle (19). This inyestigator utilized the technique of reducing endogenous urinary 17-hydroxycorticoid levels almost to zero by the administration of A•, 9c•-fiuorohydrocortisone, a compound which even in small doses is extremely effective in suppressing corticotropin secretion. By maintaining such suppresslye therapy Liddie was then able to detect the penetration of hydrocortisone by a rise in the level of urinary 17-hydroxycorticoids. In a series of normal subjects average values for absorption of hydrocortisone acetate from ointment vehicles were 2 per cent from the skin, 29 per cent from the vagina, and 26 per cent from the rectum. Additional indirect evidence for the percutaneous absorption of hydro- cortisone in animals was obtained from the so-called "thymus involution test" (20). Hydrocortisone acetate or hydrocortisone free alcohol prepara- tions ranging in concentration from 0.25 to 1.0 per cent were applied once daily for three days to shaved sites on the skin of infantile female rats. Seventy-two hours after the first application the animals were sacrificed and the thymus glands removed and immediately weighed. In the treated animals the weights of the glands were reduced to a level of less than 30 per cent of that found in the control group. With this technique the use of a wide variety of vehicles produced no significant differences in absorption of the hormone. Malkinson, et aL (21), using a gas-flow cell to measure changes in surface radioactivity, studied the passage of hydrocortisone-4-C TM free alcohol through damaged human skin sites. At each site tested the superficial epidermal barrier was removed by successive applications of adhesive cello- phane tape (22), following which the radioactive steroid in an ointment base was applied to the stripped areas. Although it had been shown

150 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS earlier that hydrocortisone penetrates the superficial barrier (18), it was now found that absorption of the hormone is remarkably enhanced by removal of this layer. As measured locally by decreasing levels of residual radioactivity, significant amounts of hydrocortisone-4-C TM were absorbed within five minutes after application. Within an hour 63 to 84 per cent of the hormone had been absorbed and at the end of four to six hours 78 to 91 per cent of the hydrocortisone had entered the skin. The almost complete absorption of hormone from stripped areas six hours after applica- tion contrasts markedly with the 1 to 2 per cent absorption found at skin sites in which the barrier remains intact. CORTISONE The numerous studies which demonstrated conclusively that hydro- cortisone was absorbed in small amounts through intact skin and in large amounts through damaged skin raised certain questions concerning the percutaneous absorption of cortisone. In sharp contrast to the experience with hydrocortisone clinical evalua- tions of topical cortisone acetate preparations had shown this hormone to be without significant therapeutic effect (23, 24). These observations led to speculations that possibly cortisone was not absorbed by the skin or that perhaps the hormone was metabolized by different pathways or inactivated more rapidly than hydrocortisone after reaching the dermis. The earliest evidence for the percutaneous absorption of cortisone came from the observations of atrophic changes induced in rat skin following topical application of the hormone (9, 25, 26). These changes were analogous to those described for hydrocortisone (9). Speirs (27), working with hybrid adrenalectomized mice, studied changes in the eosinophile count after application of 0.1 mgm. of cortisone acetate in an ointment base to shaved skin sites. There was a marked eosinopenic response which began within one hour after inunction of the ointment, reached a maximum at four to six hours, and returned to normal levels after twenty hours. The magnitude of this response was comparable to that seen in animals receiving cortisone subcutaneously, intraperitoneally or orally. Additional experiments indicated that topical administration of as little as 3 t•g. of cortisone acetate in oil was effective in producing a marked eosinopenia in these animals. in a later study of normal human subjects by Danto and Maddin (28) local application of cortisone acetate ointment failed to induce any change in circulating eosinophile counts performed at four-hour, six-hour and twenty-four-hour intervals after inunction. Subsequently, Paulsen and Rerup (20) found indirect evidence for the absorption of cortisone in rats by use of the "thymus involution test," described above for hydrocortisone. Following topical applications of