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Skin development and permeability
Journal of Controlled Release, 6 (1987) 51-58 Elsevier Science Publishers B.V., Amsterdam -
SKIN DEVELOPMENT
51 Printed in The Netherlands
AND PERMEABILITY”
Tamie Kurihara-Bergstrom’”
and William
R. Good
Pharmaceuticals Division, Ciba-Geigy Corp., Ardsley, New York 10502 (U.S. A .)
The barrier properties of skin in terms of the physiochemical characteristics of the stratum corneum were examined by studying fetal pig skin penetration with emphasis on the influence of gestational age. Fetal pig gestational age has been shown to markedly effect in vitro skin permeability of various kinds of molecular species. Maturation of fetal pig skin undergoes striking changes in its epidermal structure, especially the stratum corneum, thus affecting its properties as a barrier to drug diffusion. Furthermore, and very significant, is the finding that permeation of fetal pig skin occurs not only for neutral species, but also for ionized species. These findings implicate an aqueous pore pathway as being the dominant route for transport of molecules across fetal skin, particularly at early gestational age. Skin of this gestational period can be likened to a swollen hydrogel matrix. However, as the epidermis matures, keratinization increases. These experimental results clearly demonstrate the significance of the lipid pathway in the stratum corneum permeability as a function of epidermal keratinization.
INTRODUCTION
It is well accepted that the stratum corneum is the major rate limiting barrier to molecular diffusion through mammalian epidermis [l-3]. This epidermal barrier limits the penetration of a wide variety of substances, the permeation of which most importantly depends upon their physico-chemical properties as well as the normal and abnormal state of the skin. The ease with which most substances penetrate the skin after injury to [ 4,5] or removal of [6,7] the stratum corneum clearly indicates that this layer has a significantly critical role in determining cutaneous permeability. Intact adult skin functions well as a barrier, but preterm infant skin is a less effective bar‘Paper presented at the Third International Symposium on Recent Advances in Drug Delivery Systems, February 24-27,1987, Salt Lake City, UT, U.S.A. “To whom correspondence should be addressed.
01683659/87/$03.50
rier. The permeability characteristics of the preterm neonate skin to drugs was first noted by Nachman and Esterly [8] in their original description of the blanching response to topical application of phenylephrine. They demonstrated that blanching occurred in preterm infants but not in full term infants, and it disappeared in preterm infants by three weeks of age. Difficulties in fluid balance [9,10] and temperature control [ 11,12] from high insensible water loss, percutaneous absorption of toxic agents resulting in illness and death [ 13-251, and the ease with which the skin is damaged by mild trauma indicate that the skin of the preterm infant is an imperfect barrier. These observations have been interpreted as indicating that the premature neonate has an increased permeability compared to the adult. However, none of these earlier studies have linked permeability phenomena directly with developing epidermal differentiation.
0 1987 Elsevier Science Publishers B.V.
52
The purpose of the present study is to understand the nature and origin of the barrier properties of skin in terms of physicochemical characteristics of the stratum corneum development. In this paper, we have examined the barrier characteristics of skin by studying fetal pig skin penetration with emphasis on the influence of gestational age. As a result, we have demonstrated experimentally the ability to correlate in vitro skin permeation properties with maturation of epidermal keratinization.
MATERIALS
AND METHODS
methods
Both arecoline and caffeine were assayed by liquid chromatography high performance (HPLC) , Using a C,, Millipore column with a flow rate of 1 ml/min of a mobile phase consisting of 30/70 water/acetonitrile adjusted to a pH of 6.8 for arecoline, and consisting of 28172 methanol/sodium acetate adjusted to a pH of 3.6 with acetic acid for caffeine. The retention time of arecoline was 4 minutes, and that of caffeine 2.7 minutes. Sample detection was accomplished by UV absorption at 214 nm for arecoline, and 272 nm for caffeine. In vitro fetal pig skin permeation
Materials
Fetal pigs, cross bred of Landrace/Large White, were obtained from pregnant SPF swine supplied by the Pig Improvement Company (Franklin, Kentucky). Arecoline hydrobromide and caffeine were purchased from ICN Pharmaceuticals (Plainview, New York) and Sigma Chemical Company (St. Louis, Missouri), respectively. Preparation
Analytical
of fetal pig skin
Swine fetuses at 55, 75 and 96 day old gestational age and full term (115 day old) were obtained from pregnant SPF swine by cesarean delivery and immediately placed in a germ free incubator. The fetuses were sacrificed by a cervical dislocation, and the dorsal and abdominal skin were then excised. These individual skin samples were placed in Eagle’s medium supplemented with 0.01% gentamicin for 30 minutes at room temperature, and incubated in 10% glycerol solution supplemented with 0.01% gentamicin for a minimum of 1 hour at 4” C. The skin samples were wrapped in gauze and aluminum foil, and sealed in heat-sealable polyester pouches. These sealed skin samples were stored at -20°C for 24 hours, and then they were stored at - 90’ C until further use.
studies
All experiments performed in these studies involved use of fullthickness fetal pig dorsal skin. The fetal pig skin was thawed in phosphate buffered saline solution for 15 minutes at room temperature. It was then incubated in phosphate buffered saline solution for 20 minutes at 32 ‘C to remove glycerol from the skin. Cells used for skin permeation experiments had an effective diffusional area of approximately 1 cm2 and a downstream volume of 4 ml. A stirrer, made of Teflon, was used only in the receiver compartment and was driven by constant speed motor. Fetal pig skin specimens were placed on the mouth of the horizontally mounted cells, epidermis side up, and the reservoir cap was clamped in place. The receiver side half-cell was filled with pH 7.4 buffered saline solution and the donor side half-cell with either pH 4.0, 7.4 or 9.0 buffered saline solution depending upon the specific study protocol. Before the experimental run the receiver compartment was filled with pH 7.4 phosphate buffered saline. At zero time the donor compartment was charged with arecoline or caffeine in the appropriate buffered solution. One hundred microliter samples were withdrawn from the receiver solution periodically and analyzed using the HPLC method described earlier.
53
The steady-state flux across a skin membrane, whose surfaces, X=0, X= h are maintained to constant solution concentration C, and CZ, respectively, is given by:
J,=K~D.(C,--C,)/h=KD
AC/h
(1) 10; ,_
where
P,=KD/H
(2)
is the permeability coefficient. When the permeation process attained a steady-state, as determined graphically, the flux for the test was calculated from:
(3) where V, is the receiver volume, dc/dt is the steady-state rate of change in concentration in the receiver compartment, and A is the diffusional area. RESULTS
The permeation of arecoline and caffeine through fetal pig skin was characterized as a function of the gestational age. Arecoline skin permeability measurements were conducted using arecoline hydrobromide in aqueous solutions below its pK, (pH 4.0) and above its pKa (pH 9.0). Arecoline pEc, was determined to be 7.9. Steady-state flux values from 0.1 M arecoline solutions were always compared to ensure an equal driving force at both pHs. Flux values observed through skin of 55,75,96 and 115 (full term) day old gestational age are calculated using eqn. (3). Caffeine skin permeability measurements were also conducted using caffeine in aqueous solution at pH 4,7.4 and 9, and its steady-state flux values from saturated solutions for all pHs were determined using eqn. (3). Permeability coefficients were then calculated by eqns. (I) and ( 2 ) . The permeability coefficient of arecoline from pH 4 aqueous solutions decreased gradually from 55 to 75 days gestational age, then sharply from 75 to 115 days gestational age (Fig. 1) . The average permeability coefficient of full term
10
1 /Y& 60
-70
‘8d
so ---
&
110.
120
Fig. 1. Permeability coefficients of arecoline through fetal pig skin from pH 4 aqueous solutions as a function of gestational age. The error bars represent rt CT.
(115 days) skin was determined to be 2.5~ lo-’ cm/s. The permeation of 96,75 and 55 day old gestational age skin represents some 17, 557 and 678 fold increase over the permeability coefficient from the full term skin (115 days gestational age), respectively. In contrast, the average permeability coefficient of arecoline through full term skin from pH 9 aqueous solutions was determined to be 40.2x lOwa cm/s (Fig. 2). This represents a 16 fold increase over that from pH 4 aqueous solution. As has been observed for the skin permeation of pH 4 aqueous solutions, the permeability coefficient of arecoline from pH 9 aqueous solutions also decreased greatly in going from 96 day old gestational age to full term skin. This skin permeation behavior shows nearly the same permeability coefficient for both 75 and 55 day old gestational ages, Gestational age profile of skin permeation was also studied using a neutral compound, caffeine. The permeability of caffeine through fetal pig skin from pH 7.4 aqueous solutions decreased sharply as a function of gestational age; from 75 days old to 115 days (full term) (Fig. 3 ) . The average permeability coefficient of full term skin was determined to be 3.1 x lo--’ cm/s. The permeation of 75 and 96 day old ges-
Permeability Coeftlcient
1600
x IO;1 (cm:sec) 1400.
Fig. 2. Permeability coefficients of arecoline through fetal pig skin from pH 9 aqueous solutions as a function of gestational age. The error bars represent I 6.
tation age skin represents some 346 and 11 fold increase over the permeability coefficient obtained from the full term skin, respectively (Fig. 3). This behavior is in good agreement with the skin permeation-gestational age profile of arecoline solution.
Fig. 4. Permeability coefficientsof caffeine through 75 dayold gestational age fetal pig skin from pH 4, 7.4 and 9 aqueous solutions. The error bars represent t o.
6
5: I
/
pH4
/-
li
pHi
4
pH90
Fig. 5. Permeability coefficients of caffeine through 115 dayold gestational age (full term) fetal pig skin from pH 4,7.4 and 9 aqueous solutions. The error bars represent + o.
102 _ t
/i,rr_-d.__
I 70
1
80
i 90 100 Gestational
1
Ild‘i20 Age (days)
Fig. 3. Permeability coefficients of caffeine through fetal pig skin from pH 7.4 aqueous solutions as a function of gestational age. The error bars represent + CJ.
The skin permeation of caffeine through 75 and 115 (full term) day old gestational age skin was characterized as a function of donor solution pH. The permeability coefficients of caffeine were found to be near the same value at all pHs; 4, 7.4 and 9, in the same gestational period (Figs. 4 and 5). The average permeability coefficient of 75 day old gestat.ional age skin
55 was calculated to be 1.08 X 10V5 cm/s, and that of full term skin 3.0 x 100~ cm/s.
20 Ratlo P(pHS)/P(pH4) 18
r~-
----
-~
DISCUSSION The results of these investigations of skin development and permeability have demonstrated the signi~cance of the lipophilic pathway in the stratum corneum as a function of epidermal keratinization, Arecoline and caffeine have been chosen for the skin diffusion study since they possess widely different lipophilic properties. In studies of the effect of epidermal keratinization on transdermal penetration, they make a useful set of prototype compounds because of their varying physiochemical properties. The skin permeability measurements were conducted using charged and uncharged permeants, arecoline and caffeine, respectively. The permeability coefficients of both arecoline and caffeine displayed in Figs. 1, 2 and 3 are clearly affected by the gestational age of fetal pig skin. In the profiles the pattern of behavior for both compounds is similar. Fetal pig gestational age has been shown to markedly affect in vitro skin permeability of both charged and uncharged permeants. The stratum corneum which represents the major barrier to drug absorption appears to develop with increasing fetal pig gestational age. Maturation of fetal pig skin undergoes striking changes in its epidermal structure, especially the stratum corneum, thus affecting its properties as a barrier to drug diffusion. These functional changes of the epidermal barrier are also closely mirrored by histological changes [ 26 ] . Furthermore, and very significant, is the finding that permeation of fetal pig skin occurs not only for neutral species, but also for charged species. The permeability of arecoline significantly increased with pH of the aqueous solution going from pH 4 to pH 9 in both fuil term and 96 day old fetal skin, but not in either 75 or 55 day old fetal skin (Figs. 1 and 2 ) . In contrast, the permeability of caffeine is indepen-
Gestakmal
Age (days)
Fig. 6. Ratio of arecoline permeability coefficient [p(pH 9) /p (pH 4) ] through fetal pig skin as a function of gestational age.
dent of the solution pH in both full term and preterm skin (Figs. 4 and 5). As has been known for adult human skin, the epidermal penetration is highly dependent upon the pH of the donor solution, which determines the activity of the transportable species, the uncharged form. Arecoline exists mostly in a protonated form at pH 4, and as a free base at pH 9 since its pK, is 7.9. Caffeine however exists as an unionized form at all pHs. Indeed, in our experiment, the influence of skin development and gestational age on fetal skin properties is more clearly seen by examining the ratio of arecoline delivery rate at pH 9 and pH 4 (Fig. 6 1. These ratios are 15 for both full term and 96 day old fetuses, and 1.1 for both 75 and 55 day old fetuses. These findings implicate an aqueous pore pathway as being the dominant route for transport of molecules across the fetal skin, particularly at early gestational age. This is the first time that experimental results clearly demonstrate the significance of the lipid pathway in the stratum corneum as a function of epidermal keratinization. Transdermal absorption usually means the transport of a substance through the stratum
56 55 Days (Gestational
age) r---l
75 Days (Gestational
96 Days (Gestational
age)
age)
115 Days (Full Term)
Fig. 7. Idealized model of the fetal pig skin transport. “L” and “I”’ represent lipid and aqueous pores pathways for passive diffusion of drugs in the stratum corneum.
corneum into the blood stream. The fundamental physical properties of the drug that influence its passive absorption are diffusion, partitioning and ionic equilibria in the case of ionizable solutes. Despite the histological, anatomical, functional and compositional complexity of the stratum corneum, the anatomical barrier can be conceptualized simply as the biomembrane. It is compartmentalized conceptually into the lipid and aqueous pore pathways. Although aqueous pores have not been observed microscopically, pores are meant to be highly polar regions of the membrane which are more aqueous than the other lipoidal regions. Figure 7 depicts a general way to categorize the pathways for transport in pig skin as a function of gestational ages. This schematic diagram provides parallel lipoidal and aqueous pore pathways for the simple passive diffusion of drugs in the outer layer of the pig skin, and is not made to assign a pathway to a particular
morphological structure. In the case of full term and 96 day old fetal pig skin, the outer layer is keratinized, namely the stratum corneum. The permeability coefficient, p8 for steady state permeation of full term pig stratum corneum is described by eqn. ( 4 ) : Ps=qJ&+
Cl-qh,
(4)
where cy, is the area fraction of the aqueous pore pathway; p,, andpL are the permeability coefficient across the aqueous pore and the lipid pathway, respectively. All molecular drug species, ionic and nondisassociated, are able to diffuse across the aqueous boundary layer and permeate through the skin. Arecoline flux through full term pig skin obtained from the pH 4 aqueous solution indicates that there exists a continuous aqueous pore pathway which ionized species pass through. The observation by Cooper [ 271 of the human skin barrier in which surfactants act primarily to increase the transport of polar molecules is certainly consistent in this regard. The data for arecoline flux from pH 9 aqueous solution indicates that a lipid continuous pathway exists; the principal transport region as the compounds become more hydrophobic. A similar permeability behavior of arecoline transport was also observed in mature human and miniature pig skin [ 291. For the transport of very lipophilic molecules the skin transport becomes aqueous boundary layer-controlled. The significance of this aqueous boundary layer to skin transport in both human and animals for in vitro experiments has been reported elsewhere [ 27,281. The aqueous pores are available to all small molecular species while lipoidal pathway is accessible only to uncharged drug species that partition into the lipoidal pathway. What drug species will be dominant at the stratum corneum surface is governed by the pK, relative to the surface pH. Hence, it is clearly demonstrated by our experimental findings such that molecules possessing high solubility in both oil and water (i.e., arecoline ) can produce large skin permeabilities.
57 2
Fetal pig skin of early gestational period can be likened to a swollen hydrogen matrix. However, as the epidermis matures, keratinization increases. At 96 day gestational age skin, arecoline permeability coefficients of both ionized and unionized species increase although the permeability ratio (ppHS/ppH4) remains the same (Fig. 6). This indicates that the area fraction of both aqueous pore and lipid pathway remains the same as those of the full term pig skin; however, the properties of both pathways appear to be altered. It is also observed histologically that an epidermal keratinization has been started by this gestational period [ 261. In 75 day old or younger fetuses, there does not exist a parallel pathway in skin transport as pictured in the model for fetal pig skin transport (Fig. 7). The skin offers little more than the resistance of a gelled aqueous phase to mass transfer. It resembles stripped skin when the stratum corneum is removed by stripping, as is suggested by Scheuplein [ 61. Consequently, a critical gestational period of the fetal pig skin at which the stratum corneum starts developing appears to be between 75 and 96 day old gestational age. The results presented here clearly demonstrate the significance of the lipid pathway in the stratum corneum as a function of epidermal keratinization. This investigation can apply not only to premature infant skin, but also to understanding the barrier properties of the stratum corneum to drug transport in general.
12
ACKNOWLEDGEMENT
13
The authors would like to acknowledge Ms. Carol Signor for conducting the skin transport experiments presented in this paper. REFERENCES 1
R.J. Scheuplein and I.H. Blank, Permeability skin, Physiol. Rev. 51 (1971) 702.
3
4
5
6
7
8
9
10
11
14
15
16 of the
T. Kurihara-Bergstrom, M. Woodworth, S. Feisullin and P. Beall, Characterization of the Yucatan miniature pig skin and small intestine for pharmaceutical applications, Lab. Anim. Sci., 36 (4) (1986) 396. M. Bartek, J. LaBudde, H. Maibach, Skin permeability in uitro: Cornprison in rat, rabbit, pig and man, J. Invest. Dermatol., 58 (1972) 114. R.K. Loeffler, J.W. Herron and V. Thomas, A quantitative study of percutaneous absorption of quantities of radiostrontium chloride through burned skin, U.S. Atomic Energy Comm., Nucl. Sci. Abstract 5 (1951) 4959. C. Behl, G. Flynn, T. Kurihara, W. Smith, 0. Gatmaitan, W. Higuchi, N. Ho and C. Pierson, Permeability of thermally damaged skin: I. Immediate Influence of 60°C scalding on hairless mouse skin, J. Invest. Dermatol., 75 (4) (1980) 340. R.J. Scheuplein, Mechanism of percutaneous absorption: I. Routes of penetration and the influence of sol$ility, J. Invest. Dermatol., 45 (1965) 334. CD. Yu, WI. Higuchi, N.F.H. Ho, J.L. Fox and G.L. Flynn, Physical model evaluation of topical prodrug delivery: Simultaneous transport and bioconversion of vidarabine-5’ -valerate, J. Pharm. Sci., 69 (7) (1980) 770. R.L. Nachman and N.B. Estedy, Increased skin permeability in preterm infants, J. Pediatrics, 79 (4) (1971) 628. A.A. Fanaroff, M. Wald, H.S. Gruber and M.H. Klaus, Insensible water loss in low-bi~h-weight infants, Pediatrics, 50 (1972) 236. R.W.A. Jones, M.J. Rochefort and J.D. Baum, Increased insensible water loss in newborn infants nursed under radiant heaters, Br. Med. J., 2 (1976) 1347. N. Rutter and D. Hull, Water loss from the skin of term and preterm babies, Arch. Dis. Child., 54 (1979) 858. T.K. Belgaumkar and K.E. Scott, Effect of low humidity on small premature infants in servocontrol incubators: I. Decrease in rectal temperature, Biol. Neonate, 26 (1975) 377. E.P. Scott, G.E. Prince and C.C. Rotondo, Dye poisoning in infancy, J. Pediatrics, 28 (1946) 713. B.M. Kagan, B. Mirman, J. Calvin and E. Lundeen, Cyanosis in premature infants due to aniline dye intoxication, J. Pediatrics, 34 (1949) 574. R.O. Fisch, E.G. Berglund, A.G. Bridge, P.R. Finley, P.G. Quie and R. Raille, Methemoglobinemia in a hospital nursery, J. Am. Med. Assoc. 185 (1963) 760. A.M. Robson, J.M. Kissan, H.H. Elvick and L. Pundavela, Pentachlorphenol poisoning in a nursery for newborn infants I: Clinical features and treatments, J. Pediatrics, 75 (1969) 309.
58 17
18
19
20
21
22
23
R.W. Armstrong, E.R. Eichner, D.E. Klein, et al., Pentachlorophenol poisoning in a nursery for newborn infants II: Epidemiologic and toxicologic studies, J. Pediatrics, 75 (1969) 317. B.I. Feinblatt, T. Aceto, G. Becham, et al., Percutaneous absorption of hydrocortisone in children, Am. J. Dis. Child., 112 (1966) 218. A. Curley, R.E. Hawk, R.D. Kimbrough, et al., Dermal absorption of hexachlorophene in infants, Lancet, 2 (1971) 296. H. Powell, 0. Swarner, L. Gluck, et al., Hexachlorophene myelinopathy in premature infants, J. Pediatrics, 82 (1973) 976. R.M. Shuman, R.W. Leech, E.C. Alvord, et al, Neurotoxicity of hexachlorophene in the human I: A clinicopathological study of 248 children, Pediatrics, 54 (1974) 689. G. Martin-Bouyer, R. Lebreton, M. Toga, et al., Outbreaks of accidental hexachlorophene poisoning in France, Lancet, 1 (1982) 91. J.P. Chabrolle and A. Rossier, Goiter and hypothy-
24 25
26
27 28
29
roidism in the newborn after cutaneous absorption of iodine, Arch. Dis. Child., 53 (1978) 495. J.B. Shick and J.M. Milstein, Burn hazard of isopropyl alcohol in the neonate, Pediatrics, 68 (1981) 587. V.A. Harpin and N. Rutter, Percutaneous alcohol absorption and skin necrosis in a premature infant, Arch. Dis. Child., 57 (1982) 477. T. Kurihara-Bergstrom, W.R. Good, C. Signor and M. Woodworth, Epidermal differentiation and permeability during skin development in pig fetuses, Submitted to Science, March 1987. E.R. Cooper, Increased skin permeability for lipophilic molecules, J. Pharm. Sci., 73 (8) (1984) 1153. G.L. Flynn, H. Durrheim and W.I. Higuchi, Permeation of hairless mouse skin II: Mebrane sectioning techniques and influence on alkanol permeabilities, J. Pharma. Sci. 70(l) (1981) 52. S. Feisullin, M. Woodworth, L. DeNoble and T. Kurihara-Bergstrom, Pharmaceutical applications of the miniature pig skin, abstract in the 1st meeting of American Association of Pharmaceutical Scientists, Pharm. Res., 3 (5) (1986) 585.
SKIN DEVELOPMENT
51 Printed in The Netherlands
AND PERMEABILITY”
Tamie Kurihara-Bergstrom’”
and William
R. Good
Pharmaceuticals Division, Ciba-Geigy Corp., Ardsley, New York 10502 (U.S. A .)
The barrier properties of skin in terms of the physiochemical characteristics of the stratum corneum were examined by studying fetal pig skin penetration with emphasis on the influence of gestational age. Fetal pig gestational age has been shown to markedly effect in vitro skin permeability of various kinds of molecular species. Maturation of fetal pig skin undergoes striking changes in its epidermal structure, especially the stratum corneum, thus affecting its properties as a barrier to drug diffusion. Furthermore, and very significant, is the finding that permeation of fetal pig skin occurs not only for neutral species, but also for ionized species. These findings implicate an aqueous pore pathway as being the dominant route for transport of molecules across fetal skin, particularly at early gestational age. Skin of this gestational period can be likened to a swollen hydrogel matrix. However, as the epidermis matures, keratinization increases. These experimental results clearly demonstrate the significance of the lipid pathway in the stratum corneum permeability as a function of epidermal keratinization.
INTRODUCTION
It is well accepted that the stratum corneum is the major rate limiting barrier to molecular diffusion through mammalian epidermis [l-3]. This epidermal barrier limits the penetration of a wide variety of substances, the permeation of which most importantly depends upon their physico-chemical properties as well as the normal and abnormal state of the skin. The ease with which most substances penetrate the skin after injury to [ 4,5] or removal of [6,7] the stratum corneum clearly indicates that this layer has a significantly critical role in determining cutaneous permeability. Intact adult skin functions well as a barrier, but preterm infant skin is a less effective bar‘Paper presented at the Third International Symposium on Recent Advances in Drug Delivery Systems, February 24-27,1987, Salt Lake City, UT, U.S.A. “To whom correspondence should be addressed.
01683659/87/$03.50
rier. The permeability characteristics of the preterm neonate skin to drugs was first noted by Nachman and Esterly [8] in their original description of the blanching response to topical application of phenylephrine. They demonstrated that blanching occurred in preterm infants but not in full term infants, and it disappeared in preterm infants by three weeks of age. Difficulties in fluid balance [9,10] and temperature control [ 11,12] from high insensible water loss, percutaneous absorption of toxic agents resulting in illness and death [ 13-251, and the ease with which the skin is damaged by mild trauma indicate that the skin of the preterm infant is an imperfect barrier. These observations have been interpreted as indicating that the premature neonate has an increased permeability compared to the adult. However, none of these earlier studies have linked permeability phenomena directly with developing epidermal differentiation.
0 1987 Elsevier Science Publishers B.V.
52
The purpose of the present study is to understand the nature and origin of the barrier properties of skin in terms of physicochemical characteristics of the stratum corneum development. In this paper, we have examined the barrier characteristics of skin by studying fetal pig skin penetration with emphasis on the influence of gestational age. As a result, we have demonstrated experimentally the ability to correlate in vitro skin permeation properties with maturation of epidermal keratinization.
MATERIALS
AND METHODS
methods
Both arecoline and caffeine were assayed by liquid chromatography high performance (HPLC) , Using a C,, Millipore column with a flow rate of 1 ml/min of a mobile phase consisting of 30/70 water/acetonitrile adjusted to a pH of 6.8 for arecoline, and consisting of 28172 methanol/sodium acetate adjusted to a pH of 3.6 with acetic acid for caffeine. The retention time of arecoline was 4 minutes, and that of caffeine 2.7 minutes. Sample detection was accomplished by UV absorption at 214 nm for arecoline, and 272 nm for caffeine. In vitro fetal pig skin permeation
Materials
Fetal pigs, cross bred of Landrace/Large White, were obtained from pregnant SPF swine supplied by the Pig Improvement Company (Franklin, Kentucky). Arecoline hydrobromide and caffeine were purchased from ICN Pharmaceuticals (Plainview, New York) and Sigma Chemical Company (St. Louis, Missouri), respectively. Preparation
Analytical
of fetal pig skin
Swine fetuses at 55, 75 and 96 day old gestational age and full term (115 day old) were obtained from pregnant SPF swine by cesarean delivery and immediately placed in a germ free incubator. The fetuses were sacrificed by a cervical dislocation, and the dorsal and abdominal skin were then excised. These individual skin samples were placed in Eagle’s medium supplemented with 0.01% gentamicin for 30 minutes at room temperature, and incubated in 10% glycerol solution supplemented with 0.01% gentamicin for a minimum of 1 hour at 4” C. The skin samples were wrapped in gauze and aluminum foil, and sealed in heat-sealable polyester pouches. These sealed skin samples were stored at -20°C for 24 hours, and then they were stored at - 90’ C until further use.
studies
All experiments performed in these studies involved use of fullthickness fetal pig dorsal skin. The fetal pig skin was thawed in phosphate buffered saline solution for 15 minutes at room temperature. It was then incubated in phosphate buffered saline solution for 20 minutes at 32 ‘C to remove glycerol from the skin. Cells used for skin permeation experiments had an effective diffusional area of approximately 1 cm2 and a downstream volume of 4 ml. A stirrer, made of Teflon, was used only in the receiver compartment and was driven by constant speed motor. Fetal pig skin specimens were placed on the mouth of the horizontally mounted cells, epidermis side up, and the reservoir cap was clamped in place. The receiver side half-cell was filled with pH 7.4 buffered saline solution and the donor side half-cell with either pH 4.0, 7.4 or 9.0 buffered saline solution depending upon the specific study protocol. Before the experimental run the receiver compartment was filled with pH 7.4 phosphate buffered saline. At zero time the donor compartment was charged with arecoline or caffeine in the appropriate buffered solution. One hundred microliter samples were withdrawn from the receiver solution periodically and analyzed using the HPLC method described earlier.
53
The steady-state flux across a skin membrane, whose surfaces, X=0, X= h are maintained to constant solution concentration C, and CZ, respectively, is given by:
J,=K~D.(C,--C,)/h=KD
AC/h
(1) 10; ,_
where
P,=KD/H
(2)
is the permeability coefficient. When the permeation process attained a steady-state, as determined graphically, the flux for the test was calculated from:
(3) where V, is the receiver volume, dc/dt is the steady-state rate of change in concentration in the receiver compartment, and A is the diffusional area. RESULTS
The permeation of arecoline and caffeine through fetal pig skin was characterized as a function of the gestational age. Arecoline skin permeability measurements were conducted using arecoline hydrobromide in aqueous solutions below its pK, (pH 4.0) and above its pKa (pH 9.0). Arecoline pEc, was determined to be 7.9. Steady-state flux values from 0.1 M arecoline solutions were always compared to ensure an equal driving force at both pHs. Flux values observed through skin of 55,75,96 and 115 (full term) day old gestational age are calculated using eqn. (3). Caffeine skin permeability measurements were also conducted using caffeine in aqueous solution at pH 4,7.4 and 9, and its steady-state flux values from saturated solutions for all pHs were determined using eqn. (3). Permeability coefficients were then calculated by eqns. (I) and ( 2 ) . The permeability coefficient of arecoline from pH 4 aqueous solutions decreased gradually from 55 to 75 days gestational age, then sharply from 75 to 115 days gestational age (Fig. 1) . The average permeability coefficient of full term
10
1 /Y& 60
-70
‘8d
so ---
&
110.
120
Fig. 1. Permeability coefficients of arecoline through fetal pig skin from pH 4 aqueous solutions as a function of gestational age. The error bars represent rt CT.
(115 days) skin was determined to be 2.5~ lo-’ cm/s. The permeation of 96,75 and 55 day old gestational age skin represents some 17, 557 and 678 fold increase over the permeability coefficient from the full term skin (115 days gestational age), respectively. In contrast, the average permeability coefficient of arecoline through full term skin from pH 9 aqueous solutions was determined to be 40.2x lOwa cm/s (Fig. 2). This represents a 16 fold increase over that from pH 4 aqueous solution. As has been observed for the skin permeation of pH 4 aqueous solutions, the permeability coefficient of arecoline from pH 9 aqueous solutions also decreased greatly in going from 96 day old gestational age to full term skin. This skin permeation behavior shows nearly the same permeability coefficient for both 75 and 55 day old gestational ages, Gestational age profile of skin permeation was also studied using a neutral compound, caffeine. The permeability of caffeine through fetal pig skin from pH 7.4 aqueous solutions decreased sharply as a function of gestational age; from 75 days old to 115 days (full term) (Fig. 3 ) . The average permeability coefficient of full term skin was determined to be 3.1 x lo--’ cm/s. The permeation of 75 and 96 day old ges-
Permeability Coeftlcient
1600
x IO;1 (cm:sec) 1400.
Fig. 2. Permeability coefficients of arecoline through fetal pig skin from pH 9 aqueous solutions as a function of gestational age. The error bars represent I 6.
tation age skin represents some 346 and 11 fold increase over the permeability coefficient obtained from the full term skin, respectively (Fig. 3). This behavior is in good agreement with the skin permeation-gestational age profile of arecoline solution.
Fig. 4. Permeability coefficientsof caffeine through 75 dayold gestational age fetal pig skin from pH 4, 7.4 and 9 aqueous solutions. The error bars represent t o.
6
5: I
/
pH4
/-
li
pHi
4
pH90
Fig. 5. Permeability coefficients of caffeine through 115 dayold gestational age (full term) fetal pig skin from pH 4,7.4 and 9 aqueous solutions. The error bars represent + o.
102 _ t
/i,rr_-d.__
I 70
1
80
i 90 100 Gestational
1
Ild‘i20 Age (days)
Fig. 3. Permeability coefficients of caffeine through fetal pig skin from pH 7.4 aqueous solutions as a function of gestational age. The error bars represent + CJ.
The skin permeation of caffeine through 75 and 115 (full term) day old gestational age skin was characterized as a function of donor solution pH. The permeability coefficients of caffeine were found to be near the same value at all pHs; 4, 7.4 and 9, in the same gestational period (Figs. 4 and 5). The average permeability coefficient of 75 day old gestat.ional age skin
55 was calculated to be 1.08 X 10V5 cm/s, and that of full term skin 3.0 x 100~ cm/s.
20 Ratlo P(pHS)/P(pH4) 18
r~-
----
-~
DISCUSSION The results of these investigations of skin development and permeability have demonstrated the signi~cance of the lipophilic pathway in the stratum corneum as a function of epidermal keratinization, Arecoline and caffeine have been chosen for the skin diffusion study since they possess widely different lipophilic properties. In studies of the effect of epidermal keratinization on transdermal penetration, they make a useful set of prototype compounds because of their varying physiochemical properties. The skin permeability measurements were conducted using charged and uncharged permeants, arecoline and caffeine, respectively. The permeability coefficients of both arecoline and caffeine displayed in Figs. 1, 2 and 3 are clearly affected by the gestational age of fetal pig skin. In the profiles the pattern of behavior for both compounds is similar. Fetal pig gestational age has been shown to markedly affect in vitro skin permeability of both charged and uncharged permeants. The stratum corneum which represents the major barrier to drug absorption appears to develop with increasing fetal pig gestational age. Maturation of fetal pig skin undergoes striking changes in its epidermal structure, especially the stratum corneum, thus affecting its properties as a barrier to drug diffusion. These functional changes of the epidermal barrier are also closely mirrored by histological changes [ 26 ] . Furthermore, and very significant, is the finding that permeation of fetal pig skin occurs not only for neutral species, but also for charged species. The permeability of arecoline significantly increased with pH of the aqueous solution going from pH 4 to pH 9 in both fuil term and 96 day old fetal skin, but not in either 75 or 55 day old fetal skin (Figs. 1 and 2 ) . In contrast, the permeability of caffeine is indepen-
Gestakmal
Age (days)
Fig. 6. Ratio of arecoline permeability coefficient [p(pH 9) /p (pH 4) ] through fetal pig skin as a function of gestational age.
dent of the solution pH in both full term and preterm skin (Figs. 4 and 5). As has been known for adult human skin, the epidermal penetration is highly dependent upon the pH of the donor solution, which determines the activity of the transportable species, the uncharged form. Arecoline exists mostly in a protonated form at pH 4, and as a free base at pH 9 since its pK, is 7.9. Caffeine however exists as an unionized form at all pHs. Indeed, in our experiment, the influence of skin development and gestational age on fetal skin properties is more clearly seen by examining the ratio of arecoline delivery rate at pH 9 and pH 4 (Fig. 6 1. These ratios are 15 for both full term and 96 day old fetuses, and 1.1 for both 75 and 55 day old fetuses. These findings implicate an aqueous pore pathway as being the dominant route for transport of molecules across the fetal skin, particularly at early gestational age. This is the first time that experimental results clearly demonstrate the significance of the lipid pathway in the stratum corneum as a function of epidermal keratinization. Transdermal absorption usually means the transport of a substance through the stratum
56 55 Days (Gestational
age) r---l
75 Days (Gestational
96 Days (Gestational
age)
age)
115 Days (Full Term)
Fig. 7. Idealized model of the fetal pig skin transport. “L” and “I”’ represent lipid and aqueous pores pathways for passive diffusion of drugs in the stratum corneum.
corneum into the blood stream. The fundamental physical properties of the drug that influence its passive absorption are diffusion, partitioning and ionic equilibria in the case of ionizable solutes. Despite the histological, anatomical, functional and compositional complexity of the stratum corneum, the anatomical barrier can be conceptualized simply as the biomembrane. It is compartmentalized conceptually into the lipid and aqueous pore pathways. Although aqueous pores have not been observed microscopically, pores are meant to be highly polar regions of the membrane which are more aqueous than the other lipoidal regions. Figure 7 depicts a general way to categorize the pathways for transport in pig skin as a function of gestational ages. This schematic diagram provides parallel lipoidal and aqueous pore pathways for the simple passive diffusion of drugs in the outer layer of the pig skin, and is not made to assign a pathway to a particular
morphological structure. In the case of full term and 96 day old fetal pig skin, the outer layer is keratinized, namely the stratum corneum. The permeability coefficient, p8 for steady state permeation of full term pig stratum corneum is described by eqn. ( 4 ) : Ps=qJ&+
Cl-qh,
(4)
where cy, is the area fraction of the aqueous pore pathway; p,, andpL are the permeability coefficient across the aqueous pore and the lipid pathway, respectively. All molecular drug species, ionic and nondisassociated, are able to diffuse across the aqueous boundary layer and permeate through the skin. Arecoline flux through full term pig skin obtained from the pH 4 aqueous solution indicates that there exists a continuous aqueous pore pathway which ionized species pass through. The observation by Cooper [ 271 of the human skin barrier in which surfactants act primarily to increase the transport of polar molecules is certainly consistent in this regard. The data for arecoline flux from pH 9 aqueous solution indicates that a lipid continuous pathway exists; the principal transport region as the compounds become more hydrophobic. A similar permeability behavior of arecoline transport was also observed in mature human and miniature pig skin [ 291. For the transport of very lipophilic molecules the skin transport becomes aqueous boundary layer-controlled. The significance of this aqueous boundary layer to skin transport in both human and animals for in vitro experiments has been reported elsewhere [ 27,281. The aqueous pores are available to all small molecular species while lipoidal pathway is accessible only to uncharged drug species that partition into the lipoidal pathway. What drug species will be dominant at the stratum corneum surface is governed by the pK, relative to the surface pH. Hence, it is clearly demonstrated by our experimental findings such that molecules possessing high solubility in both oil and water (i.e., arecoline ) can produce large skin permeabilities.
57 2
Fetal pig skin of early gestational period can be likened to a swollen hydrogen matrix. However, as the epidermis matures, keratinization increases. At 96 day gestational age skin, arecoline permeability coefficients of both ionized and unionized species increase although the permeability ratio (ppHS/ppH4) remains the same (Fig. 6). This indicates that the area fraction of both aqueous pore and lipid pathway remains the same as those of the full term pig skin; however, the properties of both pathways appear to be altered. It is also observed histologically that an epidermal keratinization has been started by this gestational period [ 261. In 75 day old or younger fetuses, there does not exist a parallel pathway in skin transport as pictured in the model for fetal pig skin transport (Fig. 7). The skin offers little more than the resistance of a gelled aqueous phase to mass transfer. It resembles stripped skin when the stratum corneum is removed by stripping, as is suggested by Scheuplein [ 61. Consequently, a critical gestational period of the fetal pig skin at which the stratum corneum starts developing appears to be between 75 and 96 day old gestational age. The results presented here clearly demonstrate the significance of the lipid pathway in the stratum corneum as a function of epidermal keratinization. This investigation can apply not only to premature infant skin, but also to understanding the barrier properties of the stratum corneum to drug transport in general.
12
ACKNOWLEDGEMENT
13
The authors would like to acknowledge Ms. Carol Signor for conducting the skin transport experiments presented in this paper. REFERENCES 1
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