Arch Dermatol Res (1987) 279: 351- 356 Archives of N@8@mT@N (cid:14)9 Springer-Verlag 1987 In vivo relationship between transepidermal water loss and percutaneous penetration of some organic compounds in man: effect of anatomic site C. Lotte 1, A. Rougier 1, D. R. Wilson 2, and H. I. Maibach 2 1 D6partement de Biologic, Laboratoires de Recherche Fondamentale de l'Or6al, 1 Avenue Eugene Schueller, F-93601 Aulnay sous Bois, France 2 Department of Dermatology, University of California, San Francisco, California 94143, USA Summary. The relationship between the percutaneous penetration of four chemicals and transepidermal water loss (TEWL) was investigated in vivo in man as a func- tion of anatomic site. The findings showed an appreci- able difference in the permeability of the skin from one site to another with regard to both water loss and chemical penetration. In addition, independent of the physicochemical properties of the molecules adminis- tered, there was a linear relationship between TEWL and penetration. These data confirm both the impor- tance of anatomic site in the degree of permeability of the cutaneous barrier and the utility of determinations of TEWL and percutaneous absorption in the evaluation of its functional condition. Key words: Transepidermal water loss - Percu- taneous absorption - Anatomic site Introduction Barrier function is doubtless one of the most impor- tant skin functions. The stratum corneum restricts entry by obstructing the penetration of substances [19, 21] and is involved in the homeostasis of the body, in particular by limiting the loss of water [4, 17, 30], transport mechanisms being diffusional [21, 28]. To date, the relationship between these two aspects of the cutaneous barrier have aroused little investigative interest. In vivo and in vitro studies performed in man [ll] and in animals after UV irradiation [16] have demonstrated that when transepidermal water loss (TEWL) increases, percutaneous penetration in- creases. However, these studies are qualitative obser- vations and the mechanism linking these two parameters is still unknown. Offprint requests to: Andr4 Rougier (address see above) Apart from pathologic considerations, the func- tional state of the cutaneous barrier may vary con- siderably under physiologic conditions [39]. Thus, in man, cutaneous permeability with regard to applied compounds varies from one site to another [9, 18, 26]. The present study investigates the influence of anatomic site in man in relation to both TEWL and percutaneous absorption in order to establish the pre- cise relationship between these two indicators of the functional state of the cutaneous barrier. Material and methods Percutaneous absorption The percutaneous absorption of four radiolabelled compounds (New England Nuclear): acetylsalicylic acid (carboxyl-14C), benzoic acid (ring-14C), caffeine (1-methyl-14C), and benzoic acid sodium salt (ring-14C) was determined for four anatomic sites, the exact locations of which are shown in Fig. 1. For each molecule and each site, 6 to 8 male caucasian volunteers aged 28 + 2 years were studied. Application conditions In 20 gl of the appropriate vehicle, 1,000 nmol of each com- pound, with a specific activity adjusted to 10 -3 gCi/nmol, was applied to an area i cm 2. The composition of these vehicles, shown in Table 1, was selected according to the solubility of each compound. Triton XI00 was added as a surfactant to obtain smooth spreading of the vehicle over the treated area, the boundaries of which were circumscribed by an open circular cell fixed by silicone glue to prevent any chemical loss. After 30 min, excess substance was rapidly removed by washing twice (2 x 300 Ixl) with an ethanol-water mixture (95 : 5), followed by two rinses (2 x 300 gl) with distilled water and gentle drying with cotton-wool buds. Measurement conditions The molecules tested were selected on the basis of the rapidity and high level of their urinary excretion. In view of the literature concerning urinary excretion kinetics for these substances after administration via various routes in different species [5, 6, 8, 10, 24, 25], the total amounts which had penetrated during the 4
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`352 C. Lotte et al.: Transepidermal water loss and percutaneous penetration ~174 \ / U Fig. 1. Anatomic sites tested: 1, Forehead; 2, postauricular; 3, arm (upper, outer); 4, abdomen days following application could be calculated, after scintillation counting, from the quantities found in the urines up to 24 h. The proportions of the total amounts of benzoic acid sodium salt, caffeine, benzoic acid, and acetylsalicylic acid excreted within 24 h were 75%, 50%, 75~ and 31%, respectively. ACETYL SALICYLIC ACID g3o-/ 8 / " ,/'- .__~10" ~// r=0.71 .,_u 5 1(3 15 T.E.W.L. (g.m-2. h -t ) CAFFEINE E r = 0.73 / I ,/" " E (cid:12)9 il (cid:12)9 ~" -~ I/ .~ ,/ ~o i ~ ..... , , 5 10 15 T.EW.L (g.m-2.h -1 ) BENZOIC ACID ~ 40 - "~020- r = 0.62 /IV d~' S.,~o ~(cid:12)9 (cid:12)9 TE.WL.(g.m-2J1-D 6" E (cid:12)9 ~ 20- 10- BENZOIC ACID SODIUM SALT I r=0.68 . /I ! I'','.< 5 10 15 T.E.W.L. (g.m-2.h -1 ) Fig. 2. In vivo relationship between transepidermal water loss (TEWL) and percutaneous absorption of different compounds according to the anatomic site in man Transepidermal water loss (TEWL) After topical administration of the tested compound, TEWL was measured with an evaporimeter EPIC (Servo Med, Sweden) from a contralateral site (same anatomic region) in each subject. The hand-held probe was fitted with a l-cm tail cimmey extension to reduce air turbulence around the hydrosensors and the metallic shield (supplied by Servo Med) eliminated the possibility of sensor contamination. Measurements (g-m -2 (cid:12)9 h-1) stabilized within 30-45 s. Since the room environment was comfortable (20 _ 1 ~ C) and the subjects physically inactive, the TEWL should closely reflect stratum corneum water flux without gross interference from sweating. Results The amounts present in the 24-h urine sample and the total amounts which had penetrated over a 4-day period for each of the anatomic sites investigated (see Fig. 1) are in Table 1. This table also reports the TEWL values obtained on each site tested. Figure 2 presents the relationship between TEWL and total percutaneous absorption of each of the molecules studied for each individual separately but combining the findings for all the sites. Discussion The results show (Table l) that the percutaneous pene- tration of the test molecules varied with the anatomic location. The areas behind the ear and the forehead were the most permeable, regardless of the physico- chemical properties of the compound tested. In general, the order of cutaneous permeability was as follows: arm ~< abdomen < postauricular < forehead. Although the literature provides few details about the influence of anatomic site on the absorption of molecules, the rank order obtained here agrees with studies performed with other chemicals 9, 10, 18, 35. Unfortunately, reviews dealing with percutaneous absorption 1, 13, 29 frequently deduce contradictory explanations for the variations in permeability from one anatomic site to another. Mathematical expressions of the laws of diffusion emphasize the thickness of the membrane crossed. In our opinion, general laws of this type can provide only a theoretical approach to the problem; experience has revealed their deficiencies when applied to discontinu- ous membranes with the physicochemical complexity of the stratum corneum. Thus, our results show that skin permeability is about two to three times higher on the forehead than on the forearm or the abdomen for both water (TEWL) and molecules (percutaneous absorption, although the thickness and number of cell layers of the stratum corneum of these sites are similar (on average 15 gm and 18 cell layers) 12, 22. There- fore, the observed differences in permeability on the sites that we have studied have to be explained by additional criteria, such as structural and physico-
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`C. Lotte et al. : Transepidermal water loss and percutaneous penetration Table. 1. Percutaneous absorption and transepidermal water loss (TEWL) values according to anatomic site 353 n Anatomic site Amount in urine Total amount penetrated Transepidermal water after 24 h a within 4 days b loss c Relative permeability to arm Penetration TEWL Compound: benzoic acid sodium salt, vehicle A 6 Arm (upper, outer) 3.02 d (0.34) e 4.02 (0.45) 6.06 (0.36) 6 Abdomen 5.73 (0.54) 7.65 (0.72) 5.37 (0.46) 6 Postauricular 7.54 (0.62) 10.06 (0.82) 7.72 (0.64) 8 Forehead 9.31 (1.76) 12.32 (2.30) 12.29 (0.96) 1 i 1.9 0.9 2.5 1.3 3.1 2 b Calculated from urinary excretion: (b) - (a) 0.75 Compound: caffeine, vehicle B 7 Arm (upper, outer) 6.04 (0.92) 12.09 (1.84) 7.04 (0.95) 6 Abdomen 3.76 (0.67) 7.53 (1.34) 6.05 (0.43) 7 Postauricular 5.87 (0.52) 11.72 (1.05) 8.74 (0.62) 6 Forehead 11.17 (1.20) 22.35 (2.39) 12.77 (1.05) 1 1 0.6 0.9 1 1.2 1.9 1.8 b Calculated from urinary excretion: (b) - (a) 0.5 Compound: benzoic acid, vehicle A 8 Arm (upper, outer) 6.87 (0.75) 9.15 (1.01) 4.24 (0.35) 7 Abdomen 10.88 (1.23) 14.52 (1.64) 4.40 (0.51) 8 Postauricular 16.87 (3.85) 22.49 (5.14) 8.35 (0.41) 7 Forehead 20.10 (2.39) 26.80 (3.19) 10.34 (0.70) 1 1 1.6 1 2.5 1.9 2.9 2.4 b Calculated from urinary excretion: (b) - (a) 0.75 Compound: acetylsalicylic acid, vehicle A 7 Arm (upper, outer) 5.27 (0.18) 17.00 (0.37) 5.08 (0.79) 6 Abdomen 5.34 (t.03) 17.20 (3.35) 5.16 (0.43) 6 Postauricular 11.04 (2.50) 29.17 (5.37) 9.04 (0.84) 6 Forehead 10.89 (1.02) 35.14 (3.29) 11.22 (0.96) 1 1 1 1 2.1 1.8 2.1 2.2 (a) b Calculated from urinary excretion: (b) - 0.31 Measured just before the application, expressed in g - m- 2. h- 1 d Expressed in nmol - cm-2 e SD Vehicle A: (ethyleneglycol/triton XI00) (90/10); Vehicle B: [(ethyleneglycol/triton X100) (90/10)]/(H20)(50/50) chemical properties of the horny layer, whose partici- pation may be relevant to the apparent discrepancies between our present data and previous findings. For, example, no differences in the absorption ofhydrocor- tisone [9], parathion and malathion [18] were found between palms and soles and other sites such as forearm and abdomen, where the horny layer is about 40 to 50 times thinner. On the other hand, it is well established that TEWL on these sites varies greatly. It is about 20- 40 g/m 2 per h on palms and soles, while being 4-7 g/m 2 per h for arm and abdomen. These data apparently do not fit with the linear-like relation- ship between TEWL and percutaneous absorption that we have found in the work reported here (Fig. 2). It is nevertheless possible that, apart from stratum corneum thickness, various morphologic differences of the skin of these sites may partly explain such a contradiction. For example, the human palm and sole are anatomic sites where the highest distribution of sweat glands can be found. However, at the present time there is no evidence that sweat glands contribute in enhancing or hindering percutaneous absorption of chemicals. On the contrary, it is evident that the water loss on palms and soles is largely overestimated, even in nonsweating subjects, when determined at room
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`354 C. Lotte et al. : Transepidermal water loss and percutaneous penetration temperature. According to the work of Thiele and Realy 36, sweat glands have two modes of function- ing; first, the well-known one, which responds to stress, and, second, a continuous one, where the secretory duct acts as a "heat pipe" which is a high- efficiency calorie exchanger. It can be assumed that at the upper part of the duct, where water vapor condenses before running down the duct walls, some water vapor may escape and thus be taken into account in TEWL. It is worth noting that the second mechanism should be the more important one since it would constantly control the normal thermoregula- tion. Palms and soles, therefore, may be considered as completely distinct from other skin sites. From a theoretical viewpoint, it is relatively easy to imagine a molecule's penetration by simultaneously adopting the follicular, sweat, and transcorneal routes. It is unfortunately difficult to evaluate the relative extent of each route. It is conceivable that the higher penetration of areas such as the forehead, where there are more sebaceous glands, might be explained by an increase in transfollicular in preference to trans- epidermal absorption. However, how can one explain the considerable disproportion (a factor of 50 to 100) in the number of sebaceous glands between the abdomen and the forehead 2, 34 and the relatively slight difference (on average, a factor of 2) detected for the penetration levels of these two areas? In addi- tion, of the total surface area of the appendages, sweat and sebaceous glands account for between 0.1% and 1% of the total skin area, depending on the site in- volved 3. Thus, the total volume available for the transport of molecules across structures other than the continuous stratum corneum is probably slight, of the order of 0.01% to 0.1% of the total volume of the skin 3. It would be hasty and hazardous to conclude that the transfollicular route does not exist on the sole basis of these morphologic considerations and our findings. However, in agreement with several in- vestigations 3, 14, 27, 37, 38 it would seem that the importance of this route in percutaneous absorption phenomena may have been overestimated. By virtue of its density of active sebaceous glands, the forehead is the richest of the sites studied in terms of sebum. This forms an irregular film of a thickness ranging from 0.4 to 4 l.tm 15. It is reasonable to question to what extent the physicochemical nature of the molecule applied interacts with this film and to what extent this initial contact influences the absorp- tion. However, although the real influence of this film on percutaneous penetration requires elucidation, it is of note that its removal or its artificial thickening have no effect on TEWL 7, 15. In adults, the fiat surface of the forehead stratum corneum cells is 30% less than that from other sites, such as the arm or the abdomen 20, 23. There is an inverse relationship between the area of the cor- neocytes and the TEWL value 20. Our results (Fig. 2) show a direct correlation between TEWL and penetra- tion. It is possible that the degreee of percutaneous absorption may itself be inversely linked to the size of the corneocytes. One of the most important of many current applications of the investigation of the cutaneous permeability in function of anatomic site is the selec- tion of areas most suitable for systemic medication via the transdermal route. The scopolamine transdermal drug, delivery systems make use of the postauricular area as the administration site 31, 32. The wisdom of this choice is confirmed by our findings. The permeability of this area with regard to both water and chemicals is similar to that of the forehead (Table 1). From the morphologic standpoint, the postauricular region presents one characteristic which may partially explain its high permeability. According to Taskovich and Shaw 35, the pronounced insertions of the dermal papilla into the epidermis may favor molecular resorption since the capillaries are nearer the skin's surface. Every anatomic site has, therefore, its particular features and we have only mentioned some of them. These features, some known, others unknown, combine together in varying degrees to produce the different experimental values of TEWL and penetra- tion observed here. Although most authors recognize the importance of anatomic site with regard either to the degree of absorption of molecules or the TEWL, the literature does not include any quantitative data on the relationship which may exist in man between these two parameters. Cutaneous permeability is generally considered a mirror of the integrity of the horny layer. Even in normal skin, the efficacy of this barrier is not constant. Thus, as shown in Table 1, for a given anatomic site, the permeability varies widely in relation to the nature of the molecule administered, since this is related to the physicochemical interactions which may occur be- tween the molecule, the vehicle, and the stratum cor- neum. For the anatomic sites investigated and for the range of TEWL and penetration observed, there was a linear relationship between the permeability of the skin to the outward movement of water and the inward uptake of molecules (Fig. 2). Table 1 shows that the relationship between the mean values of TEWL and the mean values of percutaneous absorption fit with all the compounds tested, caffeine being an apparent exception (decreased penetration on abdomen and postauricular regions as compared to arm; while TEWL showed a slight decrease on the abdomen and
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`C. Lotte et al. : Transepidermal water loss and percutaneous penetration 355 increase in the postauricular region). It is, however, worth noting that the same relation, when expressed with individual values (Fig. 2), shows correlation coefficients between 0.62 and 0.73 (P = 0.05), the latter (and better one) corresponding to caffeine. With the four molecules investigated, a mean in- crease of 2.7 in percutaneous absorption corresponded to an increase of 3 in the TEWL value. This fact supports the hypothesis that the efficiency of the barrier is dependent of the physicochemical properties of the molecule administered, but its functional state is independent of it. Consequently, as with deter- minations of TEWL, percutaneous absorption measu- rement provides a good marker of the cutaneous barrier integrity. Acknowledgements. The authors would like to thank A. M. Cabaillot and J. McMaster for their excellent technical assis- tance. References t. Barry BW (1983) Dermatological formulations: percu- taneous absorption. In: Swarbrick J (ed) Drugs and the pharmaceutical sciences, vol 18. Dekker, New York Basel 2. Benfenati A, Brillanti F (1939) Sulla distribuzione delle ghiandole sebacee nella cute del corpo umano. Arch Ital Dermatol 15 : 33 - 42 3. Blank IH, Scheuplein RJ (1969) Transport into and within the skin. Br J Dermatol [Suppl 4] 81:4-10 4. Blank IH, Moloney J, Emslie AG, Simon I, Apt CH (1984) The diffusion of water across the stratum corneum as a function of its water content. J Invest Dermatol 82:188- 194 5. Bridges JW, French MR, Smith RL, Williams RT (1970) The fate of benzoic acid in various species. Biochem J 188:47-51 6. Bronaugh RL, Stewart RF, Congdon ER, Giles AL (1982) Methods for in vitro percutaneous absorption studies. I. Comparison with in vivo results. Toxicol Appl Pharmacol 62:474- 480 7. Burch GE, de Pasquale NP (1962) Hot climates, man and his heart. Thomas, Springfield Illinois 8. Dupuis D, Rougier A, Roguet R, Lotte C, Kalopissis G (1984) In vivo relationship between horny layer reservoir effect and percutaneous absorption in human and rat. J Invest Dermatol 82:353-356 9. Feldmann RJ, Maibach HI (1967) Regional variations in percutaneous penetration of 14C cortisol in man. J Invest Dermatol 48:181 -- 183 10. Feldmann RJ, Maibach HI (1970) Absorption of some organic compounds through the skin in man. J Invest Dermatol 54: 399-- 404 11. Guillaume JC, de Rigal J, Leveque JL, Galle P, Touraine R, Dubertret L (1981) Etude compar6e de la perte insensible d'eau et de la p6n+tration cutan6e des corticoides. Dermatological 162: 380- 390 12. Holbrook KA, Odland GF (1974) Regional differences in the thickness (cell layers) of the human stratum corneum: an ultrastructural analysis. J Invest Dermatol 62:415- 422 13. Idson B (1975) Percutaneous absorption. J Pharm Sci 64: 901 - 924 14. Kligman AM (1983) A biological brief on percutaneous absorption. Drug Dev Industr Pharm 9:521 -560 15. Kligman AM (1983) The use of sebum. Br J Dermatol 75:307--319 16. Lamaud E, Lambrey B, Schalla W, Schaefer H (1984) Cor- relation between transepidermal water loss and penetration of drugs. J Invest Dermatol 82:556 17. Maibach HI, Bronaugh R, Guy R, Turr E, Wison D, Jacques S, Chaing D (1984) Noninvasives techniques for determining skin function. In: Drill VA, Lazar P (eds) Cutaneous toxicity. Raven Press, New York, pp 63-97 18. Maibach HI, Feldmann RJ, Milby T, Serat W (1971) Re- gional variation in percutaneous penetration in man. Arch Environ Health 23 : 208- 211 19. Malkinson FD (1958) Studies on percutaneous absorption of 14C labelled steroids by use of" the gas flow cell. J Invest Dermatol 31 : 19-28 20. Marks R, Nicholls S, King CS (1981) Studies on isolated corneocytes. Int J Cosmet Sci 3:251-258 21. Marzulli FN (1962) Barriers to skin penetration. J Invest Dermatol 39 : 387- 393 22. Pathiak MA, Fitzpatrick TB (1974) The role of natural photoprotective agents in human skin. In: Fitzpatrick TB (ed) Sunlight and man. University of Tokyo Press, Tokyo, pp 725- 750 23. Plewig G, Marples RR (1970) Regional differences of cell sizes in human stratum corneum, part I. J Invest Dermatol 54:13-18 24. Rougier A, Dupuis D, Lotte C, Roguet R, Schaefer H (1983) In vivo correlation between stratum corneum reservoir func- tion and percutaneous absorption. J Invest Dermatol 81:275-278 25. Rougier A, Dupuis D, Lotte C, Roguet R (1985) The mea- surement of the stratum corneum reservoir. A predictive method for in vivo percutaneous absorption studies: in- fluence of application time. J Invest Dermatol 84:66- 68 26. Rougier A, Dupuis D, Lotte C, Roguet R, Wester R, Maibach HI (1987) Regional variation in percutaneous absorption in man: measurement by the stripping method. Arch Derm Res 278:465-469 27. Scheuplein RJ (1967) Mechanism of percutaneous absorp- tion. II. Transient diffusion and relative importance of various routes of skin penetration. I Invest Dermatol 48:79-88 28. Scheuplein RJ (1979) The skin as a barrier. In: Jarret A (ed) The physiology and pathophysiology of the skin, vol 5. Academic Press, London, pp 1669 - 1692 29. Scheuplein RJ (1979) Site variations in diffusion and permeability. In: Jarret A (ed) The physiology and patho- physiology of the skin, vol 5. Academic Press, New York, pp 1731-1752 30. Scheuplein RJ, Blank IH (1971) Permeability of the skin. Physiol Rev 51:702-747 31. Shaw JE, Chandrasekaran SK, Michaels AS, Taskovitch L (1975) Controlled transdermal delivery, in vitro and in vivo. In: Maibach HI (ed) Animal models in human dermatology. Churchill Livingstone, Edinburgh London, pp 138-146 32. Shaw JE, Chandrasekaran SK, Campbell PS, Schmitt I_G (1977) New procedures for evaluating cutaneous absorp- tion. In: Drill VA, Lazar P (eds) Cutaneous toxicity. Academic Press, New York, pp 83- 94 33. Smith JG, Fischer RW, Blank IH (1961) The epidermal barrier: a comparison between scrotal and abdominal skin. J Invest Dermatol 36:337-343 34. Szabo G (1958) The regional frequency and distribution of hair follicles in human skin. In: Montagna W, Ellis RA (eds) The biology of hair growth. Academic Press, New York, pp 33-38
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`356 C. Lotte et al.: Transepidermal water loss and percutaneous penetration 35. Taskovitch L, Shaw JE (1978) Regional differences in the morphology of human skin: correlation with variations in drug permeability. J Invest Dermatol 70:217 36. Thiele FAF, Realy DA (1977) Heat transport mechanisms in the eccrine sweat gland. Congress of the Society of Cosmetic Chemists, Brussels 37. Tregear RT (1961) Relative permeability of hair follicles and epidermis. J Physiol 156: 707 - 713 38. Treherne JE (1956) Permeability of the skin to some nonelectrolytes. J Physiol 133:171 - 180 39. Wilson DR, Maibach HI (1982) A review of transepidermal water loss: physical aspects and measurements as related to infants and adults. In: Maibach HI, Boisits EK (eds) Neonatal skin. Dekker, New York, p 83 Received June 27, 1986
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