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Methods for In Vitro Percutaneous Absorption Studies V: Permeation Through Damaged Skin
Methods for In Vitro Percutaneous Absorption Studies V: Permeation Through Damaged Skin ROBERTL. BRONAUGH' AND RAYMOND F. STEWART Received April 19, 1985, from the Division of Toxicology, Food and Drug Administration, Washington, DC 20204. 12. 1985.
Abstract 0 The permeation of compounds through skin damaged by
different methods was compared because agents that are absorbed through skin are sometimes applied to a damaged barrier. The removal of the stratum corneum by stripping the skin with cellophane tape was the most effective method for enhancing absorption. A minimal increase in water permeation was obtained when one abrasion line was made with a hypodermic needle, but the absorption increased substantially when three to six lines were made across the site of application. Similar values were obtained with in vivo and in vitro techniques for penetration of cortisone and nicotinic acid through normal and abraded rat skin. Severe damage by UV irradiation to rats in vivo resulted in nicotinic acid absorption similar to that obtained in vitro through abraded or tapestripped skin. Damage from mild irradiation could not be accurately duplicated by in vitro methods. The magnitude of the increase in absorption of seven chemicals through abraded human and rat skin was related to the extent to which the molecules were absorbed by the skin. The greatest increases in penetration were obtained with the compounds that were most poorly absorbed.
Reliable in vitro techniques have been developed to measure permeation through excised skin in diffusion cells.'" Topical exposure to chemicals, however, sometimes occurs on skin that is damaged or diseased and therefore is a potentially deficient barrier. In fact, drugs are often topically applied to skin which is damaged in some way. Ingredients in cosmetics may be absorbed more readily by skin that is damaged by dryness, irritation, allergic reactions, and abrasions such as those from shaving. Little information exists on the effect of a damaged barrier on the percutaneous absorption of chemicals. In a few studies, the stratum corneum has been removed by repeated stripping with cellophane tape and the effect on absorption observed. Blank5 reported that, with excised human skin, tape-stripping of the membrane resulted in increased permeation of water approaching two orders of magnitude. In the clinical study by Feldmann and Maibach,6 however, an increase of only twofold in the skin permeation of hydrocortisone was obtained through tape-stripped skin. In the most comprehensive tape-stripping study, Flynn and co-workers7 removed the stratum corneum of hairless mouse skin and found that the amount of increased absorption of each of a homologous series of alcohols depended on the chemical's solubility properties. Felsher and Rothman8 found an increase of three- to tenfold in transepidermal water loss in patients with psoriasis and exfoliative dermatitis. Skin was abraded with a hypodermic needle to measure the increased absorption of lead through damaged skin in human volunt e e r ~When . ~ a single line was made a t the site of application, an increase of slightly more than twofold in total body lead was obtained. To facilitate accurate determination of the potential for increased absorption through damaged skin, we compared the effect on absorption of different procedures for damaging the skin barrier: (a)abrasion with a hypodermic needle, (b) UV irradiation (erythema and inflammation), and (c) remov1062 /Journal of Pharmaceutical Sciences Vol. 74, No. lo,October 1985
Accepted for publication July
al of the stratum corneum by repeated tape-stripping. To determine the value of in vitro studies, we compared in vivo measurements through damaged skin with those made with skin in a diffusion cell. We measured the skin permeation of a series of compounds (with different rates of absorption and solubility properties) through excised normal and abraded skin of the human and rat.
Experimental Section Percutaneous absorption was quantitated by measuring the permeation of radiolabeled compounds. The following compounds were obtained from New England Nuclear Corp., Boston, MA: tritiated water (specific activity, 1.0 mCilg); [1,2-3Hlcortisone (43.0 Ci/mmol); [7-'4C]benzoic acid (29.4 mCi/mmol); [l4C1urea (57.0 mCi/mmol); and [l-rnethyZ-14C]caffeine(49.3 mciimmol). [Carboxyl-'4Clnicotinic acid (61.0 mCi/mmol) was obtained from Amersham Corp., Arlington Heights, IL. Pathfinder Laboratories, St. Louis, MO, supplied [U-14C]phenol (11.6 mCilmmol), and ICN Pharmaceuticals, Irvine, CA, was the source of [2-3H]propylene glycol (400 mCi/mmol). All compounds were determined to have a radiochemical purity of 297%. Experimental samples were counted in a Beckman LS9000 scintillation counter with Beckman Ready-Solv MP scintillation fluid. Vehicle Preparation-Tritiated water was added to distilled water to give a final concentration of 3.4 pCi/cm2 of skin a t application. In the preparation of the petrolatum and cosmetic lotion (Vaseline Intensive Care) solutions, small portions of each radiolabeled compound in methanol were added to vials and dried. The vehicle was then added with mixing (on a hot plate to liquify petrolatum solutions). An amount of radioisotope was added to give a concentration of approximately 4 pg/cm2 (a typical level of exposure to many chemicals) a t application. For tritiated compounds, the addition of unlabeled material in methanol was also required. In Vitro Studies-Skin permeation was determined by the flowthrough cell techniques previously de ~c ribe d.~ Human abdominal skin was obtained at autopsy within 24 h of death. To be usable, water permeation measurements through the skin were required to yield a permeability constant (steady state ratelapplied concentrac d h . If the skin sample had been frozen for tion) of (2.5 x storage, it was rechecked for the rate of water permeation before use. Skin was not stored for longer than 2 months. Rat skin was freshly obtained from the backs of 3- to 6-month-old female Osborne-Mendel animals. Split-thickness 350-pm sections of both human and rat skin were prepared with a dermatome (Padgett, Kansas City, MO). Circles of skin were placed in the diffusion cells, and the normal saline receptor fluid was pumped underneath the skin a t a rate of approximately 1.5 mL/h. The effluent containing the absorbed radioactivity was collected in scintillation vials in a fraction collector. Skin surface temperature was maintained a t 32°C by heating the aluminum holding block to 35°C. In experiments in which I3H1water permeation was measured, 300 pL of the compound in water was applied to the surface of the skin and the cell tops were covered with Parafilm to prevent evaporation. When compounds were applied in a thin layer of petrolatum or cosmetic lotion, the surface of the skin was washed at 24 h as described in the next section. In Vivo Studies-Values for cortisone and nicotinic acid absorption were obtained by the method previously described.' Rats were partially restrained with rubber tubing tied around the body behind 0022-3549/85/10001062$0 1 .OO/O 0 1985,American Pharmaceutical Association
the front legs and in front of the rear legs. The back of each rat was lightly shaved with electric clippers. A nylon ring (area = 1.25 cm2) was attached to the back with a cyanoacrylate adhesive. The vehicle containing the test compound was applied in a thin layer to the back skin, and the site of application was covered with a screen. Urine samples were collected at 24-h intervals for 5 d, at which time background levels of radioactivity were approached. In separate experiments, approximately 1 pCi of each test compound was injected intraperitoneally and 24-h urine samples were again collected for 5 d. The percentage of this parenteral dose excreted in the urine was used to correct the values of the topical experiments for incomplete urinary excretion. The parenteral correction values for nicotinic acid and cortisone were 50.0 t 2.0 and 21.0 k 2.6%, respectively. Values are the mean t SEM of five determinations. In both the in vivo and in vitro experiments, the site of application was washed with soap and water at 24 h to remove unabsorbed material. In vitro studies were continued for an additional 48-72 h to collect the remaining absorbed compound. Damaged Skin-Skin was abraded by drawing the point of a 19gauge hypodermic needle across the surface. In rat studies, prior removal of hair by a 10-min treatment with a cream depilatory (Neet; Whitehall Laboratories, New York, NY) was required because the dense hair interfered with the abrasion process. The pressure applied with the needle was such that the stratum corneum was pierced, but only the epidermal layer was damaged, as evidenced by lack of bleeding in the in vivo rat experiments. Because the area of exposed skin in the diffusion cells (0.32 cm2) was smaller than the test area in the in vivo experiments (1.25 cm’), a smaller number of marks were made across the surface in the in vitro studies. Six marks were made in the in vivo studies: three in one direction and three perpendicular to the original marks. With the width of the mark at 0.01 cm, it was estimated that approximately 5.5%of the surface area was damaged. In the in vitro studies, a corresponding degree of damage was produced by making four marks (two in one direction and two perpendicular to the original ones), which caused abrasion to a n estimated 6.4% of the surface area. The stratum corneum was removed by repeated stripping with cellophane tape (Scotch Brand; 3M Co., Minneapolis, MN). Pressure was applied to the tape by hand before it was pulled from the surface of the skin. Rat skin was again pretreated with a depilatory to facilitate the tape-stripping process. The greatest amount of stratum corneum was observed on the tape from the first several strippings. After six strippings with either rat or human skin, the surface had a glistening appearance, indicating that essentially all of the stratum corneum had been removed and the aqueous “viable” epidermal layer now made up the surface layer. In some rat (Table I) and all human (Table 11) experiments, the skin was stripped 12 times to ensure that all of the stratum corneum had been removed. The backs of the rats were irradiated with a Kratos LH153 lamp (Kratos Analytical Instruments, Ramsey, NJ) that delivered 55% UVA and 45%UVB irradiation. The intensity of irradiation was 0.37 (UVA) and 0.30 (UVB) watt/cm2/min. The rats were anesthetized with pentobarbital, their backs were shaved with an electric clipper, and the animals were placed underneath the lamp for dosing. Each animal was covered with aluminum foil that contained an opening for irradiation of the back skin. Erythema was not observed until 2 d after irradiation, even at high doses. Erythema was greatest on d 3 and 4 after irradiation, with the maximum response observed on d 4. Percutaneous absorption studies were therefore initiated 3 d following the irradiation process. In the in vitro studies, the animals were sacrificed a t this time (3 d), and the skin was prepared as described above. The minimal erythmea dose (at 3 d) was 0.5 min (0.17 J/cm2). Permeation experiments were performed after 1.5 and 6.0 min of irradiation, which resulted in mild and intense erythema, respectively.
through tape-stripped skin was similar with six and 12 strippings and was comparable to the maximum degree of absorption caused by abrasion of the skin. An insignificant effect of the depilatory on water permeation through human skin was observed (Table 11, Fig. 1) Permeation through Damaged Rat Skin In Vltro’
Table I-Water
Treatment of Skin
Dose Absorbed, % (24h)
Dose/h, YO (Maximum)
None (8) Depilatory (4) Depilatory + abrasion 1 time (4) 3 times (4) 4 times (4) 6 times (4) Tape-stripping 6 times (3) 12 times (5)
4.1 +- 0.6 9.3 3.7
*
0.21 f 0.03 0.43 2 0.2
29.6 +- 7.8 70.0 f 2.6 78.5 f 2.7 76.4 3.2
*
1.4 f 0.4 4.6 f 0.4 6.2 0.7 5.9 2 0.5
81.7 2 1.3 80.6 3.8
8.3 2 0.4 7.5 2 1.0
*
*
~
‘Values are the mean t SEM of the number of determinations (in parentheses). Treatment with the depilatory was made for 10 min and was followed by a water rinse. Abrasion lines across the surface of the skin were made the indicated number of times with a 19-gauge hypodermic needle. Table 11-Water Permeation through Damaged Human Skin In Vltro’ Dose Absorbed, Doselh, Treatment of Skin (Maximum) (24h) O/O
None (7)
O/O
2.7 5 0.1
0.12 k 0.01
Abrasion 1 time (10) 3 times (7) 4 times (8)
10.7 t 1.6 30.2 ? 9.4 33.8 ? 7.1
0.52 t 0.09 2.4 5 1.0 2.4 5 0.9
Tape-stripping 12 times (7)
56.9
3.6
Depilatory (3)
?
7.2
3.2 ? 0.3
Depilatory + abrasion 4 times (7)
* 0.7
0.16 2 0.02
32.9 ? 7.1
2.0
2
0.6
~
*Values are the mean t SEM of the number of determinations (in parentheses). Treatment with depilatory was made for 10 min and was followed by a water rinse. Abrasion lines across the surface of the skin were made the indicated number of times with a 19-gaugehypodermic needle.
4r
t
Results The effect of skin damage caused by abrasion and tapestripping on water permeation through rat skin was determined (Table I). Treatment with the depilatory before the skin was damaged resulted in an increase in water absorption of approximately twofold. Making one line across the surface of the skin enhanced absorption additionally by threefold. Maximum absorption was obtained by abrading three, four, or six times. The degree of water absorption
0
1
0
5
-
10
I
15
-
0
I
20
0
25
Time. h Figure 1-Time course of the absorption of water through excised human skin. Key: 0,normal skin; 0 , one abrasion line; 0 , three abrasion lines; A,four abrasion lines; V, tape-stripped skin.
Journal of Pharmaceutical Sciences / 1063 Vol. 74, No. 10, October 1985
(use of the depilatory was only for comparison with rat skin; a depilatory was not used before damage of the relatively hairless abdominal human skin). The smallest increase in absorption was caused by an abrasion of one line. After abrasion of either three or four times, a similar increase in absorption occurred; water permeation was 12.5 times greater than the percentage absorbed through untreated skin after 24 h. The increase in absorption of water through tapestripped skin was greater than that through abraded skin. Absorption of cortisone through rat skin from a petrolatum vehicle was measured in vitro and in vivo (Table 111). No significant difference between the in vivo and in vitro results was obtained when normal and abraded skin were compared. A greater amount of cortisone was absorbed after the excised skin was tape-stripped but this increase was not significant. Permeation of nicotinic acid in rats was also compared in vivo and in vitro (Table IV). Again, no significant difference between in vitro and in vivo procedures was seen when normal and abraded skin were compared. The degree of absorption of nicotinic acid through tape-stripped skin was greater (but not significantly greater) than that through abraded skin. Pretreatment with UV irradiation 3 d before in vivo skin permeation studies were carried out resulted in a damaged barrier that correlated in severity with the length of irradiation. Mild erythema from the 1.5-min exposure resulted in a 3.2-fold increase in absorption. The severe erythema caused by 6 min of irradiation resulted in absorption of 51% of the applied dose. Rats were irradiated for 1.5 min and then sacrificed at 3 and 4 d for removal of skin for in vitro studies. Maximum in vitro absorption was obtained with application 4 d after irradiation. The percutaneous absorption of seven compounds, applied in lotion (5 mg/cm2), by normal and abraded skin was determined in humans and rats. The solubility properties of these chemicals are given in Table V. Permeation values with human skin (Table VI) were expressed as the percentage of the applied dose that was absorbed and as the Table Ill-Cortisone Absorption through Rat Skin Condition of Skin Normal Abraded Tape-stripped
-
Applied Dose Absorbed, %' In Vivo
In Vitro
19.9 t 1.3 (4) 55.4 t 6.1 (4)
20.1 4 1.1 (6) 41.7 4 4.0 (6) 49.5 2 3.3 (6)
aValues are the mean t SEM of the number of determinations (in parentheses).Cortisone was applied in a petrolatum vehicle at 25 mg/ cm2.No significant difference between the in vivo and in vitro results with the normal and abraded skin was obtained (t test, p < 0.05). Table IV-Nicotinic Condition of Skin Normal Abraded Tape-stripped UV irradiated 1.5 rnin 6.0 min
Acid Absorption through Rat Skin Applied Dose Absorbed, % a In Vivo
In Vitro
6.8 t 0.8 (5) 47.4 t 5.3 (5)
5.3 -c 1.1 (4) 50.9 2 6.4 (4) 57.5 t 2.9 (6)
21.8 2 3.6 (5) 51.1 2 5.1 (5)
6.5 2 0.8 (12) 13.0 1.3b(11)
*
____
~____
'Values are the mean 2 SEM of the number of determinations (in parentheses). Nicotinic acid was applied in a common cosmetic lotion (Vaseline Intensive Care) at 5 mg/cm2. No significant difference between the in vivo and in vitro results with the normal and abraded skin was obtained (ttest;p < 0.05). Compound was applied to skin 3 d after UV irradiation except as indicated. bCompound was applied 4 d after irradiation. 1064 / Journal of Pharmaceutical Sciences Vol. 74, No. 10, October 1985
Table V-Solubility Properties of Test Compounds* Compound Benzoic acid Caffeine Cortisone Nicotinic acid Phenol Propylene glycol Urea
Water Solubility,
Octanol Solubility,
glLb
g/L
3.4 21.7 0.28 16.7 66.7 1000 1000
251.9 21.7 12.3 10.5 2014 18 2.0
OctanolNVater Partition CoefficientC 74.1 1.o 44.0 0.63 30.2 0.018
0.002
aValues for the octanol solubility of each compound were calculated from the water solubility and partition data. *See ref. 10. 'See ref. 11. maximum rate (percent dose per h) absorbed. Permeation of the compounds through rat skin (Table VII) was expressed as the percentage of the applied dose that was absorbed. With human skin, the increase in absorption ranged from only 1.6fold with caffeine to 17.1-fold with urea.
Discussion Water permeation has been used as an indicator of the integrity of the skin barrier in both in vivo studies (measurements of transepidermal water loss) and diffusion cell experiments. The relatively slow permeation of water through skin allows good sensitivity for detecting damage to the membrane. We have used this method routinely to verify the integrity of excised human cadaver skin before using i t to study the permeation of other compounds. We examined the effect of a damaged skin barrier on water permeation in rats (Table I) and humans (Table 11). Permeation values were expressed in terms of the total percentage of the dose that was absorbed and as the maximum rate (percentage dose per h) measured. Similar trends in the effects on absorption were seen when the data were expressed in these ways. The rat skin was damaged by pretreatment for 10 min with the cream depilatory used to facilitate the abrasion and tape-stripping procedures. This result is in agreement with those in previous studies that have shown damage to the barrier properties of animal skin by depilatories.12J3 Absorption was further increased by abrasion; the greatest degree of absorption (obtained with three to six abrasion lines) was similar in magnitude to that observed through skin with the stratum corneum removed by tapestripping. Human skin (Table 11) was not damaged by the short treatment with the depilatory. Although there was a substantial increase in water absorption through abraded skin, it was significantly less than that which occurred after tapestripping (in contrast to results with rat skin). The 30-fold increase in the rate of water absorption by skin after tapestripping agrees with that observed by Blank5 in human skin. That more extensive damage was done by abrasion of rat skin than of human skin might be due to differences in the structures of the two barriers. It might also be due, however, at least partly to the pretreatment of rat skin with the depilatory, which results in a condition that might predispose the rat skin to enhanced damage by abrasion. Pretreatment of human skin with the depilatory, however, did not increase the damage produced by abrasion. Only a small fraction of the skin was damaged by the abrasion process (6.4% with four lines). In contrast, the stratum corneum was essentially completely removed by the tape-stripping process. Despite this large difference in the area of exposed skin that was damaged, little or no difference in the effect of water absorption in rats was seen, and with
Table VI-Effect
of Abrasion on Percutaneous Absorption through Human Skin’
Dose Absorbed, % (Total) Compound Benzoic acid Caffeine Cortisone Nicotinic acid Phenol Propylene glycol Urea
Normal Skin
Abraded Skin
27.5 t 4.1 (3) 40.6 5 4.8 (4) 6.7 t 1.7 (7) 2.1 ? 0.9 (7) 25.5 2 2.2 (7) 13.8 -t 2.2 (6) 9.5 2 2.3 (4)
57.7 63.2 26.3 24.2 42.3 73.2 67.9
Dose Absorbed per h , % (Maximum) Increase, Fold
2 2.1 (4) t 6.0 (3) 2 3.7 (6)
3.3 (6) t 4.5 (6) f 11.6 (6) 2 5.6 (4) 2
2.1 1.6 3.9 11.5 1.7 5.3 7.1
Normal Skin 1.3 1.7 0.2 0.07 4.1 0.93 0.27
t 0.2 t 0.2 2 0.04
t 0.04 t 0.4 2 0.2 t 0.1
Abraded Skin
Increase, Fold
5.6 t 0.9 3.9 t 1.1 1.8 t 0.4 3.4 t 1.0 6.8 t 0.7 10.9 2 2.0 9.2 2 1.1
4.2 2.3 9.0 48.6 1.7 11.7 34.1
‘Values for absorption by either method of calculation are the mean 5 SEM of the number of determinations (in parentheses). Compounds were applied to skin in a common Cosmetic lotion (Vaseline Intensive Care) (5 mg/cm*),and the surface of the skin was washed at 24 h. Table VII-Effect of Abrasion on Percutaneous Absorption through Rat Skina
Compound Benzoic acid Caffeine Cortisone Nicotinic acid Phenol Propylene glycol Urea
Applied Dose Absorbed, % Normal Skin Abraded Skin 19.9 ? 2.4 (4) 56.3 2 3.8 (5) 16.3 t 3.3 (4) 5.3 1.1 (4) 16.4 2 1.8 (6) 13.3 t 1.9 (6) 11.5 2 3.1 (6)
*
40.4 -C 89.8 _t 69.6 t 50.9 +60.0 ? 53.5 t 58.7 2
2.4 (4) 6.0 (5) 10.5 (4) 6.4 (4) 3.8 (6) 8.1 (5) 7.2 (6)
Increase, Fold 2.0 1.6 4.3 9.6 3.7 4.0 5.1
‘Values for absorption are the mean -C SEM of the number of determinations (in parentheses).Compounds were applied to skin in a common cosmetic lotion (Vaseline Intensive Care) (5 mg/cm2),and the surface of the skin was washed at 24 h. human skin a difference in water permeation of less than twofold was obtained. This lack of a large difference in water penetration is due to the fact that water absorption is enhanced so greatly by abrasion that there can be little further increase by tape-stripping the skin. Only when one abrasion line was made was a mild form of skin damage produced. The effect of in vitro skin damage on absorption was compared to the increased skin permeation seen after rat skin was abraded in vivo. Cortisone permeation in diffusion cell studies was lower than, but not significantly different from, corresponding results of studies on abraded skin in vivo (Table 111). The results of in vivo and in vitro absorption through abraded skin agreed even more closely in the study with nicotinic acid (Table IV). When cortisone and nicotinic acid were used with normal skin, good agreement between absorption in in vivo and absorption in vitro was obtained, a n observation that has been reported in other human and animal investigations.l.l”l6 Also, with both compounds, tape-stripping slightly but not significantly increased diffusion cell absorption values compared to those obtained with abraded skin. The effect of skin damage by UV irradiation on absorption was examined with nicotinic acid. In these studies, in which skin damage was caused by a biological response, the in vivo-in vitro comparison was of particular interest. After 6 min of irradiation to produce intense erythema, the skin became visibly damaged during the experiments, with cracks in the stratum corneum and subsequent eschar formation. That the degree of absorption through this damaged skin was similar to that observed through abraded skin is probably due to the broken skin barrier in each case. When mild erythema was produced, damage to the skin surface was not visible. The degree of nicotinic acid absorption in vivo was intermediate between those observed with normal skin and
intensely irradiated skin. When animals irradiated for 1.5 min were sacrificed a t 3 d and the back skin was used for in vitro permeability studies, the degree of absorption observed was significantly less than that seen with skin in vivo. The maximal erythema response was observed on d 4, and if 4 d elapsed between irradiation and sacrifice, the in vitro absorption values increased significantly from those obtained after the usual 3-d waiting period. These results indicate that the higher degree of absorption obtained in vivo after mild irradiation is due to changes i n the skin barrier during the percutaneous absorption study. Unlike damage to the barrier from abrasion or tape-stripping, which occurs instantaneously, damage from UV irradiation takes place over a period of days and is a function of the intensity of irradiation. Progressive damage to skin that occurs from a physiological response, of course, cannot be simulated in a diffusion cell experiment. In a n in vitro system, damage to the skin can be adjusted to simulate mild or severe effects on its barrier properties by varying the amount of physical damage to the skin, such as the number of abrasion lines. The magnitude of the increase in absorption through damaged skin is dependent on the ease with which a molecule penetrates the skin barrier. This was demonstrated with the compounds listed in Table V by determining the effect of damage caused by abrasion on absorption. Test compounds were selected with various solubility properties to provide a range in skin permeation values. The rapid rate of absorption expected of phenol and benzoic acid was limited to some extent by the volatility of these compounds. Finite dosing in a common cosmetic lotion was used to obtain values relevant to those obtained from actual skin exposure. Absorption of all compounds through human skin (Table VI) was increased by abrasion. The maximum percentage of dose absorbed per h seemed to be a more sensitive indicator of changes in absorption, since increases in permeation from absorption spanned a wider range (from 1.7-fold for phenol to 34.1-fold for urea). The rank order of increases caused by abrasion, however, was essentially the same as when the data were expressed as the percentage of the dose that was absorbed (phenol and caffeine were reversed as the two compounds affected least by skin damage). The absorption of the fastest-penetrating compounds (caffeine, benzoic acid, and phenol) was increased by abrasion by approximately twofold when the data were expressed as the percentage of the dose that was absorbed. The least well-absorbed compound, nicotinic acid, was affected to the greatest extent, an 11.5-fold increase in penetration. Rat skin (Table VII) served as a reasonable model for normal human skin. Most (four of seven) absorption values were slightly higher with rat skin, but other values were similar or slightly lower. Abrasion of rat skin resulted in a n enhancement of absorption similar to that of human skin. The fastest-penetrating compounds were caffeine and benzoic acid, for which a twofold increase in Journal of Pharmaceutical Sciences / 1065 Vol. 74, No. 10, October 1985
permeation was obtained. The penetration of nicotinic acid was increased to the greatest extent. When a finite dose of a compound is applied to skin, the effect of damage on absorption may be less than that obtained with infinite dosing and the resulting steady-state rate of absorption through the skin. For example, when 40% of a fast-penetrating compound like caffeine penetrates intact skin during the course of an experiment, the maximum possible observable effect of damage to skin would be a further increase of only 2.5-fold (100%). If the steady-state absorption rates were measured, it is likely that a greater increase in the absorption of caffeine through damaged skin would be seen. In summary, the increase in absorption through damaged skin in vivo can be measured with in vitro diffusion cell techniques. The rat is a convenient animal model that gives results comparable to those obtained with human skin. A sparsely haired animal might be advantageous in that pretreatment with a depilatory could be avoided. Increases in permeation through damaged skin are a function of the barrier property of skin for the test compound; as permeation increases, there is a diminished increase in absorption through damaged skin. In the application of chemicals to damaged skin, typical increases in permeation may range from <2- to >lOO-fold. Mild damage to skin resulted from abrasion with one line with a hypodermic needle, but as the number of abrasion lines increased, the increase in absorp-
1066 / Journal of Pharmaceutical Sciences Vol. 74, No. 10, October 1985
tion approached that caused by removal of the stratum corneum by tape-stripping the surface of the skin.
References and Notes 1. Bronaugh, R. L.; Stewart, R. F.; Congdon, E. R.; Giles, A. L., J r . Toxicol. Appl. Pharmacol. 1982, 62, 474. 2. Bronaugh, R. L.; Stewart, R. F.; Congdon, E. R. Toxicol. ADp1. Pharmacol. 1982, 62, 481. 3. Bronaugh, R. L.; Stewart, R. F. J . Pharm. Sci. 1984, 73, 1255. 4. Bronau h, R. L.; Stewart, R. F. J . Pharm. Sci. 1985, 74, 64. 5. Blank, H. J . Invest. Dermatol. 1953,21, 259. 6. Feldmann, R. J.; Maibach, H. I. Arch. Dermatol. 1965,91,661. 7. Flynn, G. L.; Durrheim, H.; Higuchi, W. I. J . Phurm. Sci.1981, 70, 52. 8. Felsher, Z.; Rothman, S. J . Invest. Dermatol. 1945, 6, 271. 9. Moore, M. R.; Meredith, P. A.; Watson, W. S.; Summer, D. J.; Ta lor, M. K . ; Goldberg, A. Food Cosmet. Toxicol. 1980,18,399. 10. “Tie Merck Index”, 9th ed.; Windholz, M.; Budavari, S.; Blumett, R. F.; Otterbein, E. S., Eds.; Merck and Co.: Rahway, NJ, 1976. 11. Hansch, C.; Leo, A. “Substituent Constants for Correlation Analysis in Chemistry and Biology”; Wiley: New York, 1979. 12. Wolejsza, N. F.; Usdin, V. J . Soc. Cosmet. Chem. 1979,30, 375. 13. Ando, H. Y.; Escobar, A.; Schnaare, R. L.; Sugita, E. T. J. SOC. Cosmet. Chem. 1983,34, 159. 14. Bronaugh, R. L.; Maibach, H. I. J . Invest. Dermato2. 1985, 84, 180. 15. Bronaugh, R. L.; Stewart, R. F.; Wester, R. C.; Bucks, D.; Maibach, H. I.; Anderson, J. Food Chem. Toxicol. 1985,23, 111. 16. Bronaugh, R. L.; Franz, T. J. Br. J . Dermatol. (submitted for publication). I
f
__
Abstract 0 The permeation of compounds through skin damaged by
different methods was compared because agents that are absorbed through skin are sometimes applied to a damaged barrier. The removal of the stratum corneum by stripping the skin with cellophane tape was the most effective method for enhancing absorption. A minimal increase in water permeation was obtained when one abrasion line was made with a hypodermic needle, but the absorption increased substantially when three to six lines were made across the site of application. Similar values were obtained with in vivo and in vitro techniques for penetration of cortisone and nicotinic acid through normal and abraded rat skin. Severe damage by UV irradiation to rats in vivo resulted in nicotinic acid absorption similar to that obtained in vitro through abraded or tapestripped skin. Damage from mild irradiation could not be accurately duplicated by in vitro methods. The magnitude of the increase in absorption of seven chemicals through abraded human and rat skin was related to the extent to which the molecules were absorbed by the skin. The greatest increases in penetration were obtained with the compounds that were most poorly absorbed.
Reliable in vitro techniques have been developed to measure permeation through excised skin in diffusion cells.'" Topical exposure to chemicals, however, sometimes occurs on skin that is damaged or diseased and therefore is a potentially deficient barrier. In fact, drugs are often topically applied to skin which is damaged in some way. Ingredients in cosmetics may be absorbed more readily by skin that is damaged by dryness, irritation, allergic reactions, and abrasions such as those from shaving. Little information exists on the effect of a damaged barrier on the percutaneous absorption of chemicals. In a few studies, the stratum corneum has been removed by repeated stripping with cellophane tape and the effect on absorption observed. Blank5 reported that, with excised human skin, tape-stripping of the membrane resulted in increased permeation of water approaching two orders of magnitude. In the clinical study by Feldmann and Maibach,6 however, an increase of only twofold in the skin permeation of hydrocortisone was obtained through tape-stripped skin. In the most comprehensive tape-stripping study, Flynn and co-workers7 removed the stratum corneum of hairless mouse skin and found that the amount of increased absorption of each of a homologous series of alcohols depended on the chemical's solubility properties. Felsher and Rothman8 found an increase of three- to tenfold in transepidermal water loss in patients with psoriasis and exfoliative dermatitis. Skin was abraded with a hypodermic needle to measure the increased absorption of lead through damaged skin in human volunt e e r ~When . ~ a single line was made a t the site of application, an increase of slightly more than twofold in total body lead was obtained. To facilitate accurate determination of the potential for increased absorption through damaged skin, we compared the effect on absorption of different procedures for damaging the skin barrier: (a)abrasion with a hypodermic needle, (b) UV irradiation (erythema and inflammation), and (c) remov1062 /Journal of Pharmaceutical Sciences Vol. 74, No. lo,October 1985
Accepted for publication July
al of the stratum corneum by repeated tape-stripping. To determine the value of in vitro studies, we compared in vivo measurements through damaged skin with those made with skin in a diffusion cell. We measured the skin permeation of a series of compounds (with different rates of absorption and solubility properties) through excised normal and abraded skin of the human and rat.
Experimental Section Percutaneous absorption was quantitated by measuring the permeation of radiolabeled compounds. The following compounds were obtained from New England Nuclear Corp., Boston, MA: tritiated water (specific activity, 1.0 mCilg); [1,2-3Hlcortisone (43.0 Ci/mmol); [7-'4C]benzoic acid (29.4 mCi/mmol); [l4C1urea (57.0 mCi/mmol); and [l-rnethyZ-14C]caffeine(49.3 mciimmol). [Carboxyl-'4Clnicotinic acid (61.0 mCi/mmol) was obtained from Amersham Corp., Arlington Heights, IL. Pathfinder Laboratories, St. Louis, MO, supplied [U-14C]phenol (11.6 mCilmmol), and ICN Pharmaceuticals, Irvine, CA, was the source of [2-3H]propylene glycol (400 mCi/mmol). All compounds were determined to have a radiochemical purity of 297%. Experimental samples were counted in a Beckman LS9000 scintillation counter with Beckman Ready-Solv MP scintillation fluid. Vehicle Preparation-Tritiated water was added to distilled water to give a final concentration of 3.4 pCi/cm2 of skin a t application. In the preparation of the petrolatum and cosmetic lotion (Vaseline Intensive Care) solutions, small portions of each radiolabeled compound in methanol were added to vials and dried. The vehicle was then added with mixing (on a hot plate to liquify petrolatum solutions). An amount of radioisotope was added to give a concentration of approximately 4 pg/cm2 (a typical level of exposure to many chemicals) a t application. For tritiated compounds, the addition of unlabeled material in methanol was also required. In Vitro Studies-Skin permeation was determined by the flowthrough cell techniques previously de ~c ribe d.~ Human abdominal skin was obtained at autopsy within 24 h of death. To be usable, water permeation measurements through the skin were required to yield a permeability constant (steady state ratelapplied concentrac d h . If the skin sample had been frozen for tion) of (2.5 x storage, it was rechecked for the rate of water permeation before use. Skin was not stored for longer than 2 months. Rat skin was freshly obtained from the backs of 3- to 6-month-old female Osborne-Mendel animals. Split-thickness 350-pm sections of both human and rat skin were prepared with a dermatome (Padgett, Kansas City, MO). Circles of skin were placed in the diffusion cells, and the normal saline receptor fluid was pumped underneath the skin a t a rate of approximately 1.5 mL/h. The effluent containing the absorbed radioactivity was collected in scintillation vials in a fraction collector. Skin surface temperature was maintained a t 32°C by heating the aluminum holding block to 35°C. In experiments in which I3H1water permeation was measured, 300 pL of the compound in water was applied to the surface of the skin and the cell tops were covered with Parafilm to prevent evaporation. When compounds were applied in a thin layer of petrolatum or cosmetic lotion, the surface of the skin was washed at 24 h as described in the next section. In Vivo Studies-Values for cortisone and nicotinic acid absorption were obtained by the method previously described.' Rats were partially restrained with rubber tubing tied around the body behind 0022-3549/85/10001062$0 1 .OO/O 0 1985,American Pharmaceutical Association
the front legs and in front of the rear legs. The back of each rat was lightly shaved with electric clippers. A nylon ring (area = 1.25 cm2) was attached to the back with a cyanoacrylate adhesive. The vehicle containing the test compound was applied in a thin layer to the back skin, and the site of application was covered with a screen. Urine samples were collected at 24-h intervals for 5 d, at which time background levels of radioactivity were approached. In separate experiments, approximately 1 pCi of each test compound was injected intraperitoneally and 24-h urine samples were again collected for 5 d. The percentage of this parenteral dose excreted in the urine was used to correct the values of the topical experiments for incomplete urinary excretion. The parenteral correction values for nicotinic acid and cortisone were 50.0 t 2.0 and 21.0 k 2.6%, respectively. Values are the mean t SEM of five determinations. In both the in vivo and in vitro experiments, the site of application was washed with soap and water at 24 h to remove unabsorbed material. In vitro studies were continued for an additional 48-72 h to collect the remaining absorbed compound. Damaged Skin-Skin was abraded by drawing the point of a 19gauge hypodermic needle across the surface. In rat studies, prior removal of hair by a 10-min treatment with a cream depilatory (Neet; Whitehall Laboratories, New York, NY) was required because the dense hair interfered with the abrasion process. The pressure applied with the needle was such that the stratum corneum was pierced, but only the epidermal layer was damaged, as evidenced by lack of bleeding in the in vivo rat experiments. Because the area of exposed skin in the diffusion cells (0.32 cm2) was smaller than the test area in the in vivo experiments (1.25 cm’), a smaller number of marks were made across the surface in the in vitro studies. Six marks were made in the in vivo studies: three in one direction and three perpendicular to the original marks. With the width of the mark at 0.01 cm, it was estimated that approximately 5.5%of the surface area was damaged. In the in vitro studies, a corresponding degree of damage was produced by making four marks (two in one direction and two perpendicular to the original ones), which caused abrasion to a n estimated 6.4% of the surface area. The stratum corneum was removed by repeated stripping with cellophane tape (Scotch Brand; 3M Co., Minneapolis, MN). Pressure was applied to the tape by hand before it was pulled from the surface of the skin. Rat skin was again pretreated with a depilatory to facilitate the tape-stripping process. The greatest amount of stratum corneum was observed on the tape from the first several strippings. After six strippings with either rat or human skin, the surface had a glistening appearance, indicating that essentially all of the stratum corneum had been removed and the aqueous “viable” epidermal layer now made up the surface layer. In some rat (Table I) and all human (Table 11) experiments, the skin was stripped 12 times to ensure that all of the stratum corneum had been removed. The backs of the rats were irradiated with a Kratos LH153 lamp (Kratos Analytical Instruments, Ramsey, NJ) that delivered 55% UVA and 45%UVB irradiation. The intensity of irradiation was 0.37 (UVA) and 0.30 (UVB) watt/cm2/min. The rats were anesthetized with pentobarbital, their backs were shaved with an electric clipper, and the animals were placed underneath the lamp for dosing. Each animal was covered with aluminum foil that contained an opening for irradiation of the back skin. Erythema was not observed until 2 d after irradiation, even at high doses. Erythema was greatest on d 3 and 4 after irradiation, with the maximum response observed on d 4. Percutaneous absorption studies were therefore initiated 3 d following the irradiation process. In the in vitro studies, the animals were sacrificed a t this time (3 d), and the skin was prepared as described above. The minimal erythmea dose (at 3 d) was 0.5 min (0.17 J/cm2). Permeation experiments were performed after 1.5 and 6.0 min of irradiation, which resulted in mild and intense erythema, respectively.
through tape-stripped skin was similar with six and 12 strippings and was comparable to the maximum degree of absorption caused by abrasion of the skin. An insignificant effect of the depilatory on water permeation through human skin was observed (Table 11, Fig. 1) Permeation through Damaged Rat Skin In Vltro’
Table I-Water
Treatment of Skin
Dose Absorbed, % (24h)
Dose/h, YO (Maximum)
None (8) Depilatory (4) Depilatory + abrasion 1 time (4) 3 times (4) 4 times (4) 6 times (4) Tape-stripping 6 times (3) 12 times (5)
4.1 +- 0.6 9.3 3.7
*
0.21 f 0.03 0.43 2 0.2
29.6 +- 7.8 70.0 f 2.6 78.5 f 2.7 76.4 3.2
*
1.4 f 0.4 4.6 f 0.4 6.2 0.7 5.9 2 0.5
81.7 2 1.3 80.6 3.8
8.3 2 0.4 7.5 2 1.0
*
*
~
‘Values are the mean t SEM of the number of determinations (in parentheses). Treatment with the depilatory was made for 10 min and was followed by a water rinse. Abrasion lines across the surface of the skin were made the indicated number of times with a 19-gauge hypodermic needle. Table 11-Water Permeation through Damaged Human Skin In Vltro’ Dose Absorbed, Doselh, Treatment of Skin (Maximum) (24h) O/O
None (7)
O/O
2.7 5 0.1
0.12 k 0.01
Abrasion 1 time (10) 3 times (7) 4 times (8)
10.7 t 1.6 30.2 ? 9.4 33.8 ? 7.1
0.52 t 0.09 2.4 5 1.0 2.4 5 0.9
Tape-stripping 12 times (7)
56.9
3.6
Depilatory (3)
?
7.2
3.2 ? 0.3
Depilatory + abrasion 4 times (7)
* 0.7
0.16 2 0.02
32.9 ? 7.1
2.0
2
0.6
~
*Values are the mean t SEM of the number of determinations (in parentheses). Treatment with depilatory was made for 10 min and was followed by a water rinse. Abrasion lines across the surface of the skin were made the indicated number of times with a 19-gaugehypodermic needle.
4r
t
Results The effect of skin damage caused by abrasion and tapestripping on water permeation through rat skin was determined (Table I). Treatment with the depilatory before the skin was damaged resulted in an increase in water absorption of approximately twofold. Making one line across the surface of the skin enhanced absorption additionally by threefold. Maximum absorption was obtained by abrading three, four, or six times. The degree of water absorption
0
1
0
5
-
10
I
15
-
0
I
20
0
25
Time. h Figure 1-Time course of the absorption of water through excised human skin. Key: 0,normal skin; 0 , one abrasion line; 0 , three abrasion lines; A,four abrasion lines; V, tape-stripped skin.
Journal of Pharmaceutical Sciences / 1063 Vol. 74, No. 10, October 1985
(use of the depilatory was only for comparison with rat skin; a depilatory was not used before damage of the relatively hairless abdominal human skin). The smallest increase in absorption was caused by an abrasion of one line. After abrasion of either three or four times, a similar increase in absorption occurred; water permeation was 12.5 times greater than the percentage absorbed through untreated skin after 24 h. The increase in absorption of water through tapestripped skin was greater than that through abraded skin. Absorption of cortisone through rat skin from a petrolatum vehicle was measured in vitro and in vivo (Table 111). No significant difference between the in vivo and in vitro results was obtained when normal and abraded skin were compared. A greater amount of cortisone was absorbed after the excised skin was tape-stripped but this increase was not significant. Permeation of nicotinic acid in rats was also compared in vivo and in vitro (Table IV). Again, no significant difference between in vitro and in vivo procedures was seen when normal and abraded skin were compared. The degree of absorption of nicotinic acid through tape-stripped skin was greater (but not significantly greater) than that through abraded skin. Pretreatment with UV irradiation 3 d before in vivo skin permeation studies were carried out resulted in a damaged barrier that correlated in severity with the length of irradiation. Mild erythema from the 1.5-min exposure resulted in a 3.2-fold increase in absorption. The severe erythema caused by 6 min of irradiation resulted in absorption of 51% of the applied dose. Rats were irradiated for 1.5 min and then sacrificed at 3 and 4 d for removal of skin for in vitro studies. Maximum in vitro absorption was obtained with application 4 d after irradiation. The percutaneous absorption of seven compounds, applied in lotion (5 mg/cm2), by normal and abraded skin was determined in humans and rats. The solubility properties of these chemicals are given in Table V. Permeation values with human skin (Table VI) were expressed as the percentage of the applied dose that was absorbed and as the Table Ill-Cortisone Absorption through Rat Skin Condition of Skin Normal Abraded Tape-stripped
-
Applied Dose Absorbed, %' In Vivo
In Vitro
19.9 t 1.3 (4) 55.4 t 6.1 (4)
20.1 4 1.1 (6) 41.7 4 4.0 (6) 49.5 2 3.3 (6)
aValues are the mean t SEM of the number of determinations (in parentheses).Cortisone was applied in a petrolatum vehicle at 25 mg/ cm2.No significant difference between the in vivo and in vitro results with the normal and abraded skin was obtained (t test, p < 0.05). Table IV-Nicotinic Condition of Skin Normal Abraded Tape-stripped UV irradiated 1.5 rnin 6.0 min
Acid Absorption through Rat Skin Applied Dose Absorbed, % a In Vivo
In Vitro
6.8 t 0.8 (5) 47.4 t 5.3 (5)
5.3 -c 1.1 (4) 50.9 2 6.4 (4) 57.5 t 2.9 (6)
21.8 2 3.6 (5) 51.1 2 5.1 (5)
6.5 2 0.8 (12) 13.0 1.3b(11)
*
____
~____
'Values are the mean 2 SEM of the number of determinations (in parentheses). Nicotinic acid was applied in a common cosmetic lotion (Vaseline Intensive Care) at 5 mg/cm2. No significant difference between the in vivo and in vitro results with the normal and abraded skin was obtained (ttest;p < 0.05). Compound was applied to skin 3 d after UV irradiation except as indicated. bCompound was applied 4 d after irradiation. 1064 / Journal of Pharmaceutical Sciences Vol. 74, No. 10, October 1985
Table V-Solubility Properties of Test Compounds* Compound Benzoic acid Caffeine Cortisone Nicotinic acid Phenol Propylene glycol Urea
Water Solubility,
Octanol Solubility,
glLb
g/L
3.4 21.7 0.28 16.7 66.7 1000 1000
251.9 21.7 12.3 10.5 2014 18 2.0
OctanolNVater Partition CoefficientC 74.1 1.o 44.0 0.63 30.2 0.018
0.002
aValues for the octanol solubility of each compound were calculated from the water solubility and partition data. *See ref. 10. 'See ref. 11. maximum rate (percent dose per h) absorbed. Permeation of the compounds through rat skin (Table VII) was expressed as the percentage of the applied dose that was absorbed. With human skin, the increase in absorption ranged from only 1.6fold with caffeine to 17.1-fold with urea.
Discussion Water permeation has been used as an indicator of the integrity of the skin barrier in both in vivo studies (measurements of transepidermal water loss) and diffusion cell experiments. The relatively slow permeation of water through skin allows good sensitivity for detecting damage to the membrane. We have used this method routinely to verify the integrity of excised human cadaver skin before using i t to study the permeation of other compounds. We examined the effect of a damaged skin barrier on water permeation in rats (Table I) and humans (Table 11). Permeation values were expressed in terms of the total percentage of the dose that was absorbed and as the maximum rate (percentage dose per h) measured. Similar trends in the effects on absorption were seen when the data were expressed in these ways. The rat skin was damaged by pretreatment for 10 min with the cream depilatory used to facilitate the abrasion and tape-stripping procedures. This result is in agreement with those in previous studies that have shown damage to the barrier properties of animal skin by depilatories.12J3 Absorption was further increased by abrasion; the greatest degree of absorption (obtained with three to six abrasion lines) was similar in magnitude to that observed through skin with the stratum corneum removed by tapestripping. Human skin (Table 11) was not damaged by the short treatment with the depilatory. Although there was a substantial increase in water absorption through abraded skin, it was significantly less than that which occurred after tapestripping (in contrast to results with rat skin). The 30-fold increase in the rate of water absorption by skin after tapestripping agrees with that observed by Blank5 in human skin. That more extensive damage was done by abrasion of rat skin than of human skin might be due to differences in the structures of the two barriers. It might also be due, however, at least partly to the pretreatment of rat skin with the depilatory, which results in a condition that might predispose the rat skin to enhanced damage by abrasion. Pretreatment of human skin with the depilatory, however, did not increase the damage produced by abrasion. Only a small fraction of the skin was damaged by the abrasion process (6.4% with four lines). In contrast, the stratum corneum was essentially completely removed by the tape-stripping process. Despite this large difference in the area of exposed skin that was damaged, little or no difference in the effect of water absorption in rats was seen, and with
Table VI-Effect
of Abrasion on Percutaneous Absorption through Human Skin’
Dose Absorbed, % (Total) Compound Benzoic acid Caffeine Cortisone Nicotinic acid Phenol Propylene glycol Urea
Normal Skin
Abraded Skin
27.5 t 4.1 (3) 40.6 5 4.8 (4) 6.7 t 1.7 (7) 2.1 ? 0.9 (7) 25.5 2 2.2 (7) 13.8 -t 2.2 (6) 9.5 2 2.3 (4)
57.7 63.2 26.3 24.2 42.3 73.2 67.9
Dose Absorbed per h , % (Maximum) Increase, Fold
2 2.1 (4) t 6.0 (3) 2 3.7 (6)
3.3 (6) t 4.5 (6) f 11.6 (6) 2 5.6 (4) 2
2.1 1.6 3.9 11.5 1.7 5.3 7.1
Normal Skin 1.3 1.7 0.2 0.07 4.1 0.93 0.27
t 0.2 t 0.2 2 0.04
t 0.04 t 0.4 2 0.2 t 0.1
Abraded Skin
Increase, Fold
5.6 t 0.9 3.9 t 1.1 1.8 t 0.4 3.4 t 1.0 6.8 t 0.7 10.9 2 2.0 9.2 2 1.1
4.2 2.3 9.0 48.6 1.7 11.7 34.1
‘Values for absorption by either method of calculation are the mean 5 SEM of the number of determinations (in parentheses). Compounds were applied to skin in a common Cosmetic lotion (Vaseline Intensive Care) (5 mg/cm*),and the surface of the skin was washed at 24 h. Table VII-Effect of Abrasion on Percutaneous Absorption through Rat Skina
Compound Benzoic acid Caffeine Cortisone Nicotinic acid Phenol Propylene glycol Urea
Applied Dose Absorbed, % Normal Skin Abraded Skin 19.9 ? 2.4 (4) 56.3 2 3.8 (5) 16.3 t 3.3 (4) 5.3 1.1 (4) 16.4 2 1.8 (6) 13.3 t 1.9 (6) 11.5 2 3.1 (6)
*
40.4 -C 89.8 _t 69.6 t 50.9 +60.0 ? 53.5 t 58.7 2
2.4 (4) 6.0 (5) 10.5 (4) 6.4 (4) 3.8 (6) 8.1 (5) 7.2 (6)
Increase, Fold 2.0 1.6 4.3 9.6 3.7 4.0 5.1
‘Values for absorption are the mean -C SEM of the number of determinations (in parentheses).Compounds were applied to skin in a common cosmetic lotion (Vaseline Intensive Care) (5 mg/cm2),and the surface of the skin was washed at 24 h. human skin a difference in water permeation of less than twofold was obtained. This lack of a large difference in water penetration is due to the fact that water absorption is enhanced so greatly by abrasion that there can be little further increase by tape-stripping the skin. Only when one abrasion line was made was a mild form of skin damage produced. The effect of in vitro skin damage on absorption was compared to the increased skin permeation seen after rat skin was abraded in vivo. Cortisone permeation in diffusion cell studies was lower than, but not significantly different from, corresponding results of studies on abraded skin in vivo (Table 111). The results of in vivo and in vitro absorption through abraded skin agreed even more closely in the study with nicotinic acid (Table IV). When cortisone and nicotinic acid were used with normal skin, good agreement between absorption in in vivo and absorption in vitro was obtained, a n observation that has been reported in other human and animal investigations.l.l”l6 Also, with both compounds, tape-stripping slightly but not significantly increased diffusion cell absorption values compared to those obtained with abraded skin. The effect of skin damage by UV irradiation on absorption was examined with nicotinic acid. In these studies, in which skin damage was caused by a biological response, the in vivo-in vitro comparison was of particular interest. After 6 min of irradiation to produce intense erythema, the skin became visibly damaged during the experiments, with cracks in the stratum corneum and subsequent eschar formation. That the degree of absorption through this damaged skin was similar to that observed through abraded skin is probably due to the broken skin barrier in each case. When mild erythema was produced, damage to the skin surface was not visible. The degree of nicotinic acid absorption in vivo was intermediate between those observed with normal skin and
intensely irradiated skin. When animals irradiated for 1.5 min were sacrificed a t 3 d and the back skin was used for in vitro permeability studies, the degree of absorption observed was significantly less than that seen with skin in vivo. The maximal erythema response was observed on d 4, and if 4 d elapsed between irradiation and sacrifice, the in vitro absorption values increased significantly from those obtained after the usual 3-d waiting period. These results indicate that the higher degree of absorption obtained in vivo after mild irradiation is due to changes i n the skin barrier during the percutaneous absorption study. Unlike damage to the barrier from abrasion or tape-stripping, which occurs instantaneously, damage from UV irradiation takes place over a period of days and is a function of the intensity of irradiation. Progressive damage to skin that occurs from a physiological response, of course, cannot be simulated in a diffusion cell experiment. In a n in vitro system, damage to the skin can be adjusted to simulate mild or severe effects on its barrier properties by varying the amount of physical damage to the skin, such as the number of abrasion lines. The magnitude of the increase in absorption through damaged skin is dependent on the ease with which a molecule penetrates the skin barrier. This was demonstrated with the compounds listed in Table V by determining the effect of damage caused by abrasion on absorption. Test compounds were selected with various solubility properties to provide a range in skin permeation values. The rapid rate of absorption expected of phenol and benzoic acid was limited to some extent by the volatility of these compounds. Finite dosing in a common cosmetic lotion was used to obtain values relevant to those obtained from actual skin exposure. Absorption of all compounds through human skin (Table VI) was increased by abrasion. The maximum percentage of dose absorbed per h seemed to be a more sensitive indicator of changes in absorption, since increases in permeation from absorption spanned a wider range (from 1.7-fold for phenol to 34.1-fold for urea). The rank order of increases caused by abrasion, however, was essentially the same as when the data were expressed as the percentage of the dose that was absorbed (phenol and caffeine were reversed as the two compounds affected least by skin damage). The absorption of the fastest-penetrating compounds (caffeine, benzoic acid, and phenol) was increased by abrasion by approximately twofold when the data were expressed as the percentage of the dose that was absorbed. The least well-absorbed compound, nicotinic acid, was affected to the greatest extent, an 11.5-fold increase in penetration. Rat skin (Table VII) served as a reasonable model for normal human skin. Most (four of seven) absorption values were slightly higher with rat skin, but other values were similar or slightly lower. Abrasion of rat skin resulted in a n enhancement of absorption similar to that of human skin. The fastest-penetrating compounds were caffeine and benzoic acid, for which a twofold increase in Journal of Pharmaceutical Sciences / 1065 Vol. 74, No. 10, October 1985
permeation was obtained. The penetration of nicotinic acid was increased to the greatest extent. When a finite dose of a compound is applied to skin, the effect of damage on absorption may be less than that obtained with infinite dosing and the resulting steady-state rate of absorption through the skin. For example, when 40% of a fast-penetrating compound like caffeine penetrates intact skin during the course of an experiment, the maximum possible observable effect of damage to skin would be a further increase of only 2.5-fold (100%). If the steady-state absorption rates were measured, it is likely that a greater increase in the absorption of caffeine through damaged skin would be seen. In summary, the increase in absorption through damaged skin in vivo can be measured with in vitro diffusion cell techniques. The rat is a convenient animal model that gives results comparable to those obtained with human skin. A sparsely haired animal might be advantageous in that pretreatment with a depilatory could be avoided. Increases in permeation through damaged skin are a function of the barrier property of skin for the test compound; as permeation increases, there is a diminished increase in absorption through damaged skin. In the application of chemicals to damaged skin, typical increases in permeation may range from <2- to >lOO-fold. Mild damage to skin resulted from abrasion with one line with a hypodermic needle, but as the number of abrasion lines increased, the increase in absorp-
1066 / Journal of Pharmaceutical Sciences Vol. 74, No. 10, October 1985
tion approached that caused by removal of the stratum corneum by tape-stripping the surface of the skin.
References and Notes 1. Bronaugh, R. L.; Stewart, R. F.; Congdon, E. R.; Giles, A. L., J r . Toxicol. Appl. Pharmacol. 1982, 62, 474. 2. Bronaugh, R. L.; Stewart, R. F.; Congdon, E. R. Toxicol. ADp1. Pharmacol. 1982, 62, 481. 3. Bronaugh, R. L.; Stewart, R. F. J . Pharm. Sci. 1984, 73, 1255. 4. Bronau h, R. L.; Stewart, R. F. J . Pharm. Sci. 1985, 74, 64. 5. Blank, H. J . Invest. Dermatol. 1953,21, 259. 6. Feldmann, R. J.; Maibach, H. I. Arch. Dermatol. 1965,91,661. 7. Flynn, G. L.; Durrheim, H.; Higuchi, W. I. J . Phurm. Sci.1981, 70, 52. 8. Felsher, Z.; Rothman, S. J . Invest. Dermatol. 1945, 6, 271. 9. Moore, M. R.; Meredith, P. A.; Watson, W. S.; Summer, D. J.; Ta lor, M. K . ; Goldberg, A. Food Cosmet. Toxicol. 1980,18,399. 10. “Tie Merck Index”, 9th ed.; Windholz, M.; Budavari, S.; Blumett, R. F.; Otterbein, E. S., Eds.; Merck and Co.: Rahway, NJ, 1976. 11. Hansch, C.; Leo, A. “Substituent Constants for Correlation Analysis in Chemistry and Biology”; Wiley: New York, 1979. 12. Wolejsza, N. F.; Usdin, V. J . Soc. Cosmet. Chem. 1979,30, 375. 13. Ando, H. Y.; Escobar, A.; Schnaare, R. L.; Sugita, E. T. J. SOC. Cosmet. Chem. 1983,34, 159. 14. Bronaugh, R. L.; Maibach, H. I. J . Invest. Dermato2. 1985, 84, 180. 15. Bronaugh, R. L.; Stewart, R. F.; Wester, R. C.; Bucks, D.; Maibach, H. I.; Anderson, J. Food Chem. Toxicol. 1985,23, 111. 16. Bronaugh, R. L.; Franz, T. J. Br. J . Dermatol. (submitted for publication). I
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