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An Unexpected Finding in Percutaneous Absorption Observed between Haired and Hairless Guinea Pig Skin
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An Unexpected Finding in Percutaneous Absorption Observed between Haired and Hairless Guinea Pig Skin To the Editor: Animal skin models are routinely used in place of human skin in in vitro percutaneous absorption studies due to their availability, viability, and reproducibility. Hairless animal skin has the added advantage of not requiring any shaving prior to testing. The purpose of this work was to evaluate the percutaneous absorption and possible targeted follicular delivery of retinoic acid (RA), in formulation with polyolprepolymer-2 (PP-2), a unique delivery compound. Haired and hairless guinea pig (GP) skins were used as models to represent skins of different follicular densities. The hair follicle is an invagination of the epidermis extending deep into the dermis. We hypothesized that if a follicular pathway is utilized, dermal drug deposition and penetration will be greater in skin with the higher follicular density. Follicular delivery has been observed in the skin from animals of high and low follicular density1 including hairless animals, which although called “hairless” possess underdeveloped hair follicles. Results from in vivo and in vitro studies suggest that cutaneous drug deposition is related to the follicular density of the skin. Illel and Schaefer2 used a totally follicle-free rat skin model which they adapted from Behl et al.3 and concluded that the in vitro steady state flux of [3H]hydrocortisone and total diffusion of hydrocortisone through and deposition in the hairless rat skin was significantly greater than the follicle-free skin, suggesting the importance of hair follicles in hydrocortisone delivery. Unfortunately, the procedure for developing a follicle-free skin involves immersion of the dorsal skin of the animal in 60 °C water, removal of the epidermis, and waiting several weeks for the formation of new follicle-free skin. Aside from the pain and suffering inflicted on the animal and the time involved in developing a follicle-free skin, there is the question of the structural composition and permeability behavior of the newly regrown tissue compared to the normal epidermis of hairless rat skin and whether a direct comparison of the two tissue types is completely valid.4 This experiment evaluated the percutaneous absorption of RA in haired and hairless GPs with the expectation that drug penetration will be lower in hairless GPs because they have
Figure 1sCumulative penetration profile of labeled retinoic acid from formulations: (0) RA + 10% PP-2 in haired GP skin, (]) RA + 0% PP-2 in haired GP skin, (O) RA + 10% PP-2 in hairless GP skin, (4) RA + 0% PP-2 in hairless GP skin.
398 / Journal of Pharmaceutical Sciences Vol. 86, No. 3, March 1997
Figure 2sDeposition of RA in haired and hairless GP skin from a formulation containing 0.025% RA and 10% PP-2.
Figure 3sDeposition of RA in normal haired and hairless GP skin from a formulation containing 0.025% RA and 0% PP-2.
fewer hair follicles than haired GPs.5 The variety of hairless GP used was the Crl:IAF/HA(hr/hr)BR strain, a stock of hardy and fertile hairless GPs. The normal haired GP was of the Hartly strain. Three animals of each strain were obtained from Charles River Laboratories, Wilmington, MA. Retinoic acid, a drug used for treating acne, a disease of follicular involvement, was formulated at 0.025% in an ethanolic gel formulation together with and without 10% PP-2, a TopiCare Delivery Compound. PP-2 consists of a mixture of high molecular weight oligomers, ranging in molecular weight from 1700 to 10 000 Da with an average of 4000, that can modify drug delivery by retaining drug on and in the upper layers of the skin.6 Tritiated RA at 2 µCi/dose was obtained from DuPont NEN Research Products, Boston, MA. Haired GP skin was clipped and shaved 1 day prior to sacrifice. No shaving was required for hairless GP skin. Full thickness dorsal skins from three haired and three hairless guinea pigs were obtained fresh. The skins were cut and mounted on flow-through diffusion cells. Each formulation was tested on skin from all three animals in each strain. Under yellow light conditions, a 3.2-mg dose of formulation was applied over a 0.64-cm2 test area (5 mg/cm2) on the epidermal surface of the skin. The dermal side of the skin was perfused with receptor fluid, phosphate-buffered saline
S0022-3549(96)00309-7 CCC: $14.00
© 1997, American Chemical Society and American Pharmaceutical Association
Figure 4s(a, top left) Control hairless guinea pig skin, (b, top right) control haired guinea pig skin, (c, bottom left) tape-stripped hairless guinea pig skin, (d, bottom right) tape-stripped haired guinea pig skin (all images 20× magnification).
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Journal of Pharmaceutical Sciences / 399 Vol. 86, No. 3, March 1997
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(PBS) containing 0.01% sodium azide and 1.5% Oleth-20 at pH 7.4, and a flow rate of 1.0 mL/h was maintained at 37 °C. Receptor fluid was collected at half-hour intervals for the first hour, and then at hourly intervals for the next 5 h. Thereafter, receptor fluid was collected at 6 h intervals for a total of 24 h. At the end of the exposure period, the surface material was removed from the skin with two dry wipes. This was followed by tape-stripping five times using Scotch 3M Magic tape which successively removed layers of the stratum corneum (SC). The residual SC and epidermis were then separated from the dermis by gently pulling them apart with tweezers prior to analysis by liquid scintillation counting (LSC). Similar to the in vitro percutaneous absorption study, dorsal skins from both GP strains were obtained fresh for histological analysis of species differences and effect of tape stripping. A 1-cm2 area on untreated skin was marked off and wiped twice consecutively with a dry cotton swab to remove moisture. Scotch 3M Magic Tape was applied to the skin with light pressure and peeled back to remove the SC. After five tape strips, the marked area was cut out and placed in a jar containing 10% neutral buffered formalin. The formalin-fixed tissue was trimmed and embedded into paraffin blocks that were sectioned, stained with Hematoxylin and Eosin, mounted on slides for evaluation, and subsequently photographed at 20× magnification. The penetration profile of RA from the formulations through normal haired and hairless skin is depicted in Figure 1. Contrary to expectation, the penetration profile indicates that hairless GP skin was much more permeable to RA than haired GP skin despite the lower follicular density in the hairless animal. Figures 2 and 3 show the effect of 10% PP-2 addition to the formulation on RA deposition on the skin surface and in the SC, residual epidermis, dermis, and receptor fluid for both haired and hairless skins. As previously observed in in vitro studies7 using human skin, the addition of PP-2 to the formulation significantly decreased RA penetration. Further comparison of Figures 2 and 3 suggests a significant depot for RA in the portion of the fully developed hair follicle residing in the dermis of the haired GP, as shown by the higher dermal levels yet correspondingly lower penetration levels relative to the hairless GP. PP-2 appeared to have no significant effect on the dermal depot of RA in haired GP skin, while its presence significantly reduced dermal levels in hairless GP skin. This suggests that, in the haired skin, RA dermal deposition is likely controlled by follicular deposition as well as intercellular diffusion through the epidermis and into the dermal tissue surrounding the pilosebaceous units. The observation that PP-2 significantly impeded the penetration of RA through hairless GP skin implies the ability of PP-2 to retain drug on and in the upper layers of the skin. This was demonstrated by the greater recovery of RA from the surface wipes following application of the formulation containing 10% PP-2. The drug-retaining property of PP-2 may be beneficial in decreasing potential side effects resulting from topical RA administration.7 RA skin penetration differences indicate dissimilarities in the barrier properties between these two strains of guinea pig.
400 / Journal of Pharmaceutical Sciences Vol. 86, No. 3, March 1997
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Histological data shown in Figure 4a confirmed findings from Charles River Laboratories that SC of hairless GP was thicker and the cells in the upper layer were more loosely associated than that of haired GP (Figure 4b). Figure 4c shows that after five tape strips, SC of the hairless GP was reduced in thickness, but a uniform thin layer remained. The same number of tape strips applied to the haired GP remove almost all SC with only focal areas generally associated with the infundibulum of hair follicles remaining (Figure 4d). The viable epidermis of the hairless GP was thicker (four to five cells thick) compared to the haired GP (two to three cells thick), as shown in Figure 4. Hyperkeratinization of the upper stratum corneum8 and epidermal hyperproliferation5,9 may have impaired the barrier function of hairless skin. Lauer et al. observed greater drug (mannitol and progesterone) permeation through hairless relative to haired rat skin in vivo, as well as gross histological differences in skin structure between the two rat strains.10 It remains to be determined whether histopathologic differences between haired and hairless skin are associated with alterations in lipid11 and protein composition of the stratum corneum, a general loss of integrity of the horny tissue, or some other physicochemical factors that affect percutaneous absorption. Greater RA penetration through hairless than haired GP skin suggests that absorption through GP skin was influenced more by the structure and composition of the stratum corneum than follicular density.
References and Notes 1. Wahlberg, J. E. Acta Dermatol. Venereol. 1968, 48, 336-344. 2. Illel, B.; Schaefer, H. Acta Dermatol. Venerol. 1988, 68, 427430. 3. Behl, C.; Wittkowsky, A.; Barrett, M.; Pierson, C.; Flynn, G. J. Pharm. Sci. 1981, 70, 835-837. 4. Cullender, C. Advance Drug Delivery Reviews; Elsevier Science Publishers: New York, 1992; Vol. 9, pp 119-135. 5. Unpublished reference from Charles River Laboratories, Inc., Wilmington, MA 10887. 6. Polyolprepolymers, Properties and Uses in Cosmetic Products; Penederm Technical Manual; Penederm Incorporated: Foster City, CA, 1994. 7. Bucks, D.; Quigley, J.; Lang, M.; Clark, E.; Jivani, N.; Blanock, K. J. Invest. Dermatol. 1992, 98 (4), 649 (SID Abstracts [587]). 8. Haigh, J.; Smith, E. Euro. J. Pharm. Sci. 1994, 311-330. 9. Reed, C.; O’Donoghue, J. L. Lab. Anim. Sci. 1979, 29 (6), 744748. 10. Lauer, A. C.; Elder, J. T.; Weiner, N. D. J. Pharm. Sci. 1997, in press. 11. Elias, P. M.; Maibach, H. I.; Lowe, N. J. (Eds.) Models in Dermatology; Karger: Basel, 1985; Vol. I, pp 272-285.
GRACE HISOIRE DANIEL BUCKSX
AND
Penederm Incorporated Drug Transport Department 320 Lakeside Drive Foster City, CA 94404 Received July 25, 1996 Final revised manuscript received October 15, 1996 Accepted for publication November 19, 1996 JS960309+
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An Unexpected Finding in Percutaneous Absorption Observed between Haired and Hairless Guinea Pig Skin To the Editor: Animal skin models are routinely used in place of human skin in in vitro percutaneous absorption studies due to their availability, viability, and reproducibility. Hairless animal skin has the added advantage of not requiring any shaving prior to testing. The purpose of this work was to evaluate the percutaneous absorption and possible targeted follicular delivery of retinoic acid (RA), in formulation with polyolprepolymer-2 (PP-2), a unique delivery compound. Haired and hairless guinea pig (GP) skins were used as models to represent skins of different follicular densities. The hair follicle is an invagination of the epidermis extending deep into the dermis. We hypothesized that if a follicular pathway is utilized, dermal drug deposition and penetration will be greater in skin with the higher follicular density. Follicular delivery has been observed in the skin from animals of high and low follicular density1 including hairless animals, which although called “hairless” possess underdeveloped hair follicles. Results from in vivo and in vitro studies suggest that cutaneous drug deposition is related to the follicular density of the skin. Illel and Schaefer2 used a totally follicle-free rat skin model which they adapted from Behl et al.3 and concluded that the in vitro steady state flux of [3H]hydrocortisone and total diffusion of hydrocortisone through and deposition in the hairless rat skin was significantly greater than the follicle-free skin, suggesting the importance of hair follicles in hydrocortisone delivery. Unfortunately, the procedure for developing a follicle-free skin involves immersion of the dorsal skin of the animal in 60 °C water, removal of the epidermis, and waiting several weeks for the formation of new follicle-free skin. Aside from the pain and suffering inflicted on the animal and the time involved in developing a follicle-free skin, there is the question of the structural composition and permeability behavior of the newly regrown tissue compared to the normal epidermis of hairless rat skin and whether a direct comparison of the two tissue types is completely valid.4 This experiment evaluated the percutaneous absorption of RA in haired and hairless GPs with the expectation that drug penetration will be lower in hairless GPs because they have
Figure 1sCumulative penetration profile of labeled retinoic acid from formulations: (0) RA + 10% PP-2 in haired GP skin, (]) RA + 0% PP-2 in haired GP skin, (O) RA + 10% PP-2 in hairless GP skin, (4) RA + 0% PP-2 in hairless GP skin.
398 / Journal of Pharmaceutical Sciences Vol. 86, No. 3, March 1997
Figure 2sDeposition of RA in haired and hairless GP skin from a formulation containing 0.025% RA and 10% PP-2.
Figure 3sDeposition of RA in normal haired and hairless GP skin from a formulation containing 0.025% RA and 0% PP-2.
fewer hair follicles than haired GPs.5 The variety of hairless GP used was the Crl:IAF/HA(hr/hr)BR strain, a stock of hardy and fertile hairless GPs. The normal haired GP was of the Hartly strain. Three animals of each strain were obtained from Charles River Laboratories, Wilmington, MA. Retinoic acid, a drug used for treating acne, a disease of follicular involvement, was formulated at 0.025% in an ethanolic gel formulation together with and without 10% PP-2, a TopiCare Delivery Compound. PP-2 consists of a mixture of high molecular weight oligomers, ranging in molecular weight from 1700 to 10 000 Da with an average of 4000, that can modify drug delivery by retaining drug on and in the upper layers of the skin.6 Tritiated RA at 2 µCi/dose was obtained from DuPont NEN Research Products, Boston, MA. Haired GP skin was clipped and shaved 1 day prior to sacrifice. No shaving was required for hairless GP skin. Full thickness dorsal skins from three haired and three hairless guinea pigs were obtained fresh. The skins were cut and mounted on flow-through diffusion cells. Each formulation was tested on skin from all three animals in each strain. Under yellow light conditions, a 3.2-mg dose of formulation was applied over a 0.64-cm2 test area (5 mg/cm2) on the epidermal surface of the skin. The dermal side of the skin was perfused with receptor fluid, phosphate-buffered saline
S0022-3549(96)00309-7 CCC: $14.00
© 1997, American Chemical Society and American Pharmaceutical Association
Figure 4s(a, top left) Control hairless guinea pig skin, (b, top right) control haired guinea pig skin, (c, bottom left) tape-stripped hairless guinea pig skin, (d, bottom right) tape-stripped haired guinea pig skin (all images 20× magnification).
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Journal of Pharmaceutical Sciences / 399 Vol. 86, No. 3, March 1997
+
(PBS) containing 0.01% sodium azide and 1.5% Oleth-20 at pH 7.4, and a flow rate of 1.0 mL/h was maintained at 37 °C. Receptor fluid was collected at half-hour intervals for the first hour, and then at hourly intervals for the next 5 h. Thereafter, receptor fluid was collected at 6 h intervals for a total of 24 h. At the end of the exposure period, the surface material was removed from the skin with two dry wipes. This was followed by tape-stripping five times using Scotch 3M Magic tape which successively removed layers of the stratum corneum (SC). The residual SC and epidermis were then separated from the dermis by gently pulling them apart with tweezers prior to analysis by liquid scintillation counting (LSC). Similar to the in vitro percutaneous absorption study, dorsal skins from both GP strains were obtained fresh for histological analysis of species differences and effect of tape stripping. A 1-cm2 area on untreated skin was marked off and wiped twice consecutively with a dry cotton swab to remove moisture. Scotch 3M Magic Tape was applied to the skin with light pressure and peeled back to remove the SC. After five tape strips, the marked area was cut out and placed in a jar containing 10% neutral buffered formalin. The formalin-fixed tissue was trimmed and embedded into paraffin blocks that were sectioned, stained with Hematoxylin and Eosin, mounted on slides for evaluation, and subsequently photographed at 20× magnification. The penetration profile of RA from the formulations through normal haired and hairless skin is depicted in Figure 1. Contrary to expectation, the penetration profile indicates that hairless GP skin was much more permeable to RA than haired GP skin despite the lower follicular density in the hairless animal. Figures 2 and 3 show the effect of 10% PP-2 addition to the formulation on RA deposition on the skin surface and in the SC, residual epidermis, dermis, and receptor fluid for both haired and hairless skins. As previously observed in in vitro studies7 using human skin, the addition of PP-2 to the formulation significantly decreased RA penetration. Further comparison of Figures 2 and 3 suggests a significant depot for RA in the portion of the fully developed hair follicle residing in the dermis of the haired GP, as shown by the higher dermal levels yet correspondingly lower penetration levels relative to the hairless GP. PP-2 appeared to have no significant effect on the dermal depot of RA in haired GP skin, while its presence significantly reduced dermal levels in hairless GP skin. This suggests that, in the haired skin, RA dermal deposition is likely controlled by follicular deposition as well as intercellular diffusion through the epidermis and into the dermal tissue surrounding the pilosebaceous units. The observation that PP-2 significantly impeded the penetration of RA through hairless GP skin implies the ability of PP-2 to retain drug on and in the upper layers of the skin. This was demonstrated by the greater recovery of RA from the surface wipes following application of the formulation containing 10% PP-2. The drug-retaining property of PP-2 may be beneficial in decreasing potential side effects resulting from topical RA administration.7 RA skin penetration differences indicate dissimilarities in the barrier properties between these two strains of guinea pig.
400 / Journal of Pharmaceutical Sciences Vol. 86, No. 3, March 1997
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Histological data shown in Figure 4a confirmed findings from Charles River Laboratories that SC of hairless GP was thicker and the cells in the upper layer were more loosely associated than that of haired GP (Figure 4b). Figure 4c shows that after five tape strips, SC of the hairless GP was reduced in thickness, but a uniform thin layer remained. The same number of tape strips applied to the haired GP remove almost all SC with only focal areas generally associated with the infundibulum of hair follicles remaining (Figure 4d). The viable epidermis of the hairless GP was thicker (four to five cells thick) compared to the haired GP (two to three cells thick), as shown in Figure 4. Hyperkeratinization of the upper stratum corneum8 and epidermal hyperproliferation5,9 may have impaired the barrier function of hairless skin. Lauer et al. observed greater drug (mannitol and progesterone) permeation through hairless relative to haired rat skin in vivo, as well as gross histological differences in skin structure between the two rat strains.10 It remains to be determined whether histopathologic differences between haired and hairless skin are associated with alterations in lipid11 and protein composition of the stratum corneum, a general loss of integrity of the horny tissue, or some other physicochemical factors that affect percutaneous absorption. Greater RA penetration through hairless than haired GP skin suggests that absorption through GP skin was influenced more by the structure and composition of the stratum corneum than follicular density.
References and Notes 1. Wahlberg, J. E. Acta Dermatol. Venereol. 1968, 48, 336-344. 2. Illel, B.; Schaefer, H. Acta Dermatol. Venerol. 1988, 68, 427430. 3. Behl, C.; Wittkowsky, A.; Barrett, M.; Pierson, C.; Flynn, G. J. Pharm. Sci. 1981, 70, 835-837. 4. Cullender, C. Advance Drug Delivery Reviews; Elsevier Science Publishers: New York, 1992; Vol. 9, pp 119-135. 5. Unpublished reference from Charles River Laboratories, Inc., Wilmington, MA 10887. 6. Polyolprepolymers, Properties and Uses in Cosmetic Products; Penederm Technical Manual; Penederm Incorporated: Foster City, CA, 1994. 7. Bucks, D.; Quigley, J.; Lang, M.; Clark, E.; Jivani, N.; Blanock, K. J. Invest. Dermatol. 1992, 98 (4), 649 (SID Abstracts [587]). 8. Haigh, J.; Smith, E. Euro. J. Pharm. Sci. 1994, 311-330. 9. Reed, C.; O’Donoghue, J. L. Lab. Anim. Sci. 1979, 29 (6), 744748. 10. Lauer, A. C.; Elder, J. T.; Weiner, N. D. J. Pharm. Sci. 1997, in press. 11. Elias, P. M.; Maibach, H. I.; Lowe, N. J. (Eds.) Models in Dermatology; Karger: Basel, 1985; Vol. I, pp 272-285.
GRACE HISOIRE DANIEL BUCKSX
AND
Penederm Incorporated Drug Transport Department 320 Lakeside Drive Foster City, CA 94404 Received July 25, 1996 Final revised manuscript received October 15, 1996 Accepted for publication November 19, 1996 JS960309+