British Journal of Dermatology (1990) 123, 631-640.
Enhanced percutaneous absorption of a novel topical cyclosporin A formulation and assessment of its immunosuppressive activity J.I.DUNCAN, S.N.L.PAYNE, A.J.WINFIELD,* A.D.ORMERODf AND A.W.THOMSON Department of Pathology, University of Aberdeen, Foresterhill, Aberdeen, fDepartment of Dermatology, Aberdeen Royal Infirmary, Aberdeen and 'School of Pharmacy, Robert Gordon's Institute of Tecbnology, Aberdeen, Scotland, U.K. Accepted for publication 7 June 1990
SUMMARY
No clinically successful topical cyclosporin A (CyA) formulation has been produced, mainly due to the apparent lack of drug penetration. This study has produced the first in vitro kinetic data on CyA penetration across human cadavar stratum corneum and has shown that addition of the penetration enhancers (PE) Azone and propylene glycol to the CyA vehicle significantly enhanced drug permeation across the skin barrier. Using flow-through permeability cells with 5"o w/v CyA (Sandimmun') alone (CyA) or with PE (CyA + PE) in olive oil in the donor chamber, the penetration rate (mean + SD ^g/cm^/h) into receptor fluid vi'as 53 ± 43 (« ^ 13) for CyA and 660 ± 175 ( n - 7 ) for CyA + PE. The in vivo efficacy of this formulation was assessed in guinea-pigs undergoing delayed-type hypersensitivity (DTH) reactions to dinitrofluorobenzene (DNFB). CyA was applied topically at the time of challenge and twice daily thereafter. At 24 h, skin reactions revealed that compared with appropriate drug vehicles, concentrations of 0-25, 0 5 and 5*',, CyA ± PE had a significant inhibitory effect upon the erythema response and this corresponded with significant reductions in T-cell infiltrates (05 and 5",, CyA). No statistically significant reductions in erythema were demonstrated with 005",, CyA ± PE, but there was a reduction in the number of infiltrating lymphocytes in sites receiving o O5'^o CyA-|- PE compared with vehicle-treated sites (P < 001). This suggests that PE permitted some penetration of an otherwise non-immunosuppressive concentration of CyA through the skin.
Cyclosporin A (CyA), which is a fungal metabolite, is a potent inhibitor of T-lymphocytedependent cell-mediated immune processes. The systemic administration of this drug in low Correspondence: Dr J.I.Duncan, Departmenl of Patbology, University of Aberdeen, Foresterbill, Aberdeen AB9 2ZD, Scotland, U.K. 631
632
J.I.Duncan et al.
doses has been shown to be extremely beneficial in clearing chronic plaque psoriasis,' '^ a disease of unknown aetiology, but which appears to have a T-cell involvement.'^ Despite its clinical success, dose-dependent, drug-induced hypertension and nephrotoxicity have been found in psoriatic patients on long-term CyA therapy for up to 3 years.'^'* This has precluded use ofthe drug in less severe psoriasis and other immunodermatological disorders. These side-effects could be minimized or even abolished if CyA was administered topically. To date no effective formulation has been produced, primarily because little is known about the pharmacokinetics of percutaneous CyA absorption. Altbough CyA has been applied topically to humans, at concentrations of 01-10",, in oil or unguentum Merck vehicles, it was found to be largely ineffective in suppressing allergic contact sensitivity rcactions,"^'^ in inducing hair regrowth in alopecia areata**" "* or in reducing lesions in chronic plaque psoriasis.^'''" The failure to produce a therapeutic response resulted from inadequate penetration of CyA through tbe skin, especially as no drug was detected in the blood.^'^"'^ Despite these failures, it has been shown that intralesional injection of CyA on 6 alternate days, produced a significant resolution of psoriatic plaques within 2 weeks.'^^ This suggests that adequate amounts of locally applied CyA could be as effective as when the drug was administered systemically. The principal barrier to percutaneous absorption is the stratum corneum, which functions as a rate-limiting factor in the penetration process, and which many drugs, including CyA, cannot cross,'* Therefore solvents are usually incorporated into drug excipients to reversibly reduce the diffusional resistance of the stratum corneum. Water, alcohol, alkyl methyl sulphoxides, pyrrolidones, propylene glycol and Azonc arc all known penetration enhancers (PE) which collectively increase the hydration state and lipid fluidity ofthe stratum corneum.'^"' The purpose of tbis work was to formulate a topical CyA preparation incorporating suitable PE and to compare percutaneous absorption of CyA using this formulation witb that of CyA without PE, An in vitro technique involving isolated stratum corneum placed in open flowthrough permeability cells'^ was adopted. This method, which can be used to establish the steady-state passage of molecules, relies upon excess test solution being present on the donor (upper) side of the skin membrane, in order that the quantity of drug passing through the stratum corneum is negligible in proportion. Thereafter, the eflicacy ofthe formulation was evaluated in guinea-pigs undergoing cutaneous delayed-type hypersensitivity (DTH) reactions. Unlike humans, where there has been littic, or no, clinical response with topical CyA, guinea-pig skin appears to be relatively permeable to topically-applied CyA, since it effectively suppresses D T H responses' ** and reduces T-cell infiltrates.' ** The aim of this in vivo work was to ascertain whether the addition of PH, to an otherwise non-immunosupprcssive concentration of CyA, could permit the penetration of suflicient drug through the skin to prevent elicitation of the D T H response.
METHODS
Drugs CyA solutions were made from the 10",, w/v oral preparation of CyA (Sandimmun, Sandoz Ltd, Basle, Switzerland) which was diluted with olive oil to the required concentrations ofo 05-5"o. The CyA plus PE (CyA -f PE) formulations contained in addition to CyA, 2"o v/v Azone (AZ, Nelson Research, Irvine, CA, U.S.A.) and 18",) v/v propylene glycol (PG, Sigma, Poole, Dorset, U.K.).
Absorption of a novel topical cyclosporin A
633
In vitro studies Percutaneous absorption measurements were made using modified open flow-through permeability cells^" containing stratum corneum prepared from human cadaver abdominal skin.^' Upper donor chambers held 0 5 ml of 5",, CyA + PE containing 30 ^1 -^H-CyA (Amersham International PLC) of 370 kBq/ml activity. The total area of exposed stratum corneum in the cell was 0 2 cm^. A non-ionic surfactant receptor solution of 6",, w/v Volpo 20 in distilled water (Croda Chemicals Ltd., Goole, U.K.) was driven through the lower chamber (volume 80 /d) of the cell at 5 ml/h, thereby replacing the receptor fluid 63 times/h and maintaining sink conditions. The apparatus and solutions were maintained at 32 ± i C. The run-through receptor solution (25 ml) was collected every 30 min during a 5-hr period and the amount of 'H-CyA penetrating the stratum corneum assessed by adding 10 ml PICO FLUOR 40 (Canberra Packard, Pangbourne, U.K,) scintillation fluid to these samples and counting them in a Packard Tri-Carb 4530 scintillation counter. In vivo studies Animals. Male Dunkin-Hartley guinea-pigs purchased from David Hall, Newchurch, Staffs, U.K. and weighing 700-900 g were used. They received a pelleted diet, supplemented with vegetables and hay. Sensitization. Animals were sensitized to i-fluoro-2,4-dinitrobenzene (DNFB) by epicutaneous application of 50 ^\ 10",, (w/v) DNFB solution in acetone: olive oil ( i : r) to the dorsum of the right ear. Skin testing. Skin tests were performed 8 days after sensitization, by application of a nonirritant dose of 20/J1 DNFB solution (o-5'\, w/v) in acetone:olive oil (4:1) to the shaved flank. The erythematous reactions were assessed 24 h later using the following scale: 4, red and elevated; 3, red but not elevated; 2, confluent pink; i, patchy pink spots; 0 5 , feeble reaction; o, no change. Topical regimen. AmmahiAndomXy received 20/il topical applications of either 0 0 5 , o 25,0 5 or 5",, CyA + PE, olive oil or olive oil -|- PE to three test sites on each flank, immediately after drying ofthe challenge DNFB (o h). The drug was re-applied after 5 h and the animals were killed at 24 h when biopsies were taken for immunohistochemical analysis. Immunohistochemistry. Following excision, a portion of each skin biopsy was embedded in OCT (Raymond A Lamb, London U.K.) and snap frozen in liquid Arcton, previously cooled to freezing point by immersion in liquid nitrogen. Cryostat sections (6-8 /(m), cut in such an orientation as to include both the reaction 'epicentre' and outer margin ofthe reaction site, were mounted on alum-gelatin-coated slides and allowed to dry overnight at room temperature. Slides were stored wrapped in aluminium foil at - 20 C then brought to room temperature before fixation for 20 min in acetone prior to staining. The three-stage alkaline phosphatase-anti-alkaline phosphatase (APAAP) method was employed to demonstrate leucocyte phenotypes, using primary mouse monoclonal antibodies to guinea-pig mononuclear cells (Table i). Secondary antibody, rabbit anti-mouse Igs (Dako, Copenhagen, Denmark) was diluted 1:20 in io"o normal guinea-pig serum.
634
y.I.Duncan et al. TABLE I. Monoclonal antibodies used in the immunocytochemical analysis of guinea-pig mononuclear cells Antibody isotype CT7 IgGl CT6 IgGl
Specificity
Source
pan T cells Dr R,J.Scheper* T cytotoxic/suppressor cells Dr R,J.Scheper
Dilution 1:2000 1:5000
*A gift from Dr R,J.Schcper, Parhological Institute, Free University Hospital, Amsterdam, The Netherlands,
Quantitative assessment of cellular infiltration. Cell counts in the epidermis and upper papillary dermis were performed blind on coded sections. Positive cells (in five consecutive areas encompassed by an eyepiece grid occupying c. 50",, ofthe field of view) were counted using a X 10 eyepiece and x 25 objective. Statistics Results are expressed as means ± i standard deviation. The significance of differences between means was determined using Student's t-test (for cellular infiltrates) or Mann-Whitney nonparametric test (for erythema scores).
RESULTS
In-vitro penetration of CyA The addition of Azone (AZ) and propylene glycol (PG) to 5",, CyA enhanced the accumulation of 'H-CyA in the receptor fiuid during 5 h (Fig. ia). The amount of CyA penetrating the stratum corneum in the absence of PE was 53 ± 43 /xg/cm^/h, whereas the addition of PE significantly (P < 0001) increased CyA absorption tenfold to 660 ± 175 |tg CyA/cm^/h. After 5 h, 2-6"o CyA from the PE-containing formulation had penetrated the stratum corneum compared with only o 2"n from the CyA-only solution (Fig. ib). Delayed-type hypersensitivity reactions in the guinea-pig Skin reactions. The influence of topical CyA, at different concentrations, either alone or in combination with PE, on the intensity of skin reactions to DNFB at 24 h is shown in Table 2. Examination ofthe guinea-pigs revealed that CyA concentrations between o 25 and s'\, were significantly immunosuppressive and that almost complete suppression of the DTH reaction occurred with 5% CyA. When CyA was applied at a concentration of 005% there was no inhibitory effect without PE, although a 30",, reduction (which did not quite reach significance at P
Enhanced percutaneous absorption of a novel topical cyclosporin A formulation and assessment of its immunosuppressive activity J.I.DUNCAN, S.N.L.PAYNE, A.J.WINFIELD,* A.D.ORMERODf AND A.W.THOMSON Department of Pathology, University of Aberdeen, Foresterhill, Aberdeen, fDepartment of Dermatology, Aberdeen Royal Infirmary, Aberdeen and 'School of Pharmacy, Robert Gordon's Institute of Tecbnology, Aberdeen, Scotland, U.K. Accepted for publication 7 June 1990
SUMMARY
No clinically successful topical cyclosporin A (CyA) formulation has been produced, mainly due to the apparent lack of drug penetration. This study has produced the first in vitro kinetic data on CyA penetration across human cadavar stratum corneum and has shown that addition of the penetration enhancers (PE) Azone and propylene glycol to the CyA vehicle significantly enhanced drug permeation across the skin barrier. Using flow-through permeability cells with 5"o w/v CyA (Sandimmun') alone (CyA) or with PE (CyA + PE) in olive oil in the donor chamber, the penetration rate (mean + SD ^g/cm^/h) into receptor fluid vi'as 53 ± 43 (« ^ 13) for CyA and 660 ± 175 ( n - 7 ) for CyA + PE. The in vivo efficacy of this formulation was assessed in guinea-pigs undergoing delayed-type hypersensitivity (DTH) reactions to dinitrofluorobenzene (DNFB). CyA was applied topically at the time of challenge and twice daily thereafter. At 24 h, skin reactions revealed that compared with appropriate drug vehicles, concentrations of 0-25, 0 5 and 5*',, CyA ± PE had a significant inhibitory effect upon the erythema response and this corresponded with significant reductions in T-cell infiltrates (05 and 5",, CyA). No statistically significant reductions in erythema were demonstrated with 005",, CyA ± PE, but there was a reduction in the number of infiltrating lymphocytes in sites receiving o O5'^o CyA-|- PE compared with vehicle-treated sites (P < 001). This suggests that PE permitted some penetration of an otherwise non-immunosuppressive concentration of CyA through the skin.
Cyclosporin A (CyA), which is a fungal metabolite, is a potent inhibitor of T-lymphocytedependent cell-mediated immune processes. The systemic administration of this drug in low Correspondence: Dr J.I.Duncan, Departmenl of Patbology, University of Aberdeen, Foresterbill, Aberdeen AB9 2ZD, Scotland, U.K. 631
632
J.I.Duncan et al.
doses has been shown to be extremely beneficial in clearing chronic plaque psoriasis,' '^ a disease of unknown aetiology, but which appears to have a T-cell involvement.'^ Despite its clinical success, dose-dependent, drug-induced hypertension and nephrotoxicity have been found in psoriatic patients on long-term CyA therapy for up to 3 years.'^'* This has precluded use ofthe drug in less severe psoriasis and other immunodermatological disorders. These side-effects could be minimized or even abolished if CyA was administered topically. To date no effective formulation has been produced, primarily because little is known about the pharmacokinetics of percutaneous CyA absorption. Altbough CyA has been applied topically to humans, at concentrations of 01-10",, in oil or unguentum Merck vehicles, it was found to be largely ineffective in suppressing allergic contact sensitivity rcactions,"^'^ in inducing hair regrowth in alopecia areata**" "* or in reducing lesions in chronic plaque psoriasis.^'''" The failure to produce a therapeutic response resulted from inadequate penetration of CyA through tbe skin, especially as no drug was detected in the blood.^'^"'^ Despite these failures, it has been shown that intralesional injection of CyA on 6 alternate days, produced a significant resolution of psoriatic plaques within 2 weeks.'^^ This suggests that adequate amounts of locally applied CyA could be as effective as when the drug was administered systemically. The principal barrier to percutaneous absorption is the stratum corneum, which functions as a rate-limiting factor in the penetration process, and which many drugs, including CyA, cannot cross,'* Therefore solvents are usually incorporated into drug excipients to reversibly reduce the diffusional resistance of the stratum corneum. Water, alcohol, alkyl methyl sulphoxides, pyrrolidones, propylene glycol and Azonc arc all known penetration enhancers (PE) which collectively increase the hydration state and lipid fluidity ofthe stratum corneum.'^"' The purpose of tbis work was to formulate a topical CyA preparation incorporating suitable PE and to compare percutaneous absorption of CyA using this formulation witb that of CyA without PE, An in vitro technique involving isolated stratum corneum placed in open flowthrough permeability cells'^ was adopted. This method, which can be used to establish the steady-state passage of molecules, relies upon excess test solution being present on the donor (upper) side of the skin membrane, in order that the quantity of drug passing through the stratum corneum is negligible in proportion. Thereafter, the eflicacy ofthe formulation was evaluated in guinea-pigs undergoing cutaneous delayed-type hypersensitivity (DTH) reactions. Unlike humans, where there has been littic, or no, clinical response with topical CyA, guinea-pig skin appears to be relatively permeable to topically-applied CyA, since it effectively suppresses D T H responses' ** and reduces T-cell infiltrates.' ** The aim of this in vivo work was to ascertain whether the addition of PH, to an otherwise non-immunosupprcssive concentration of CyA, could permit the penetration of suflicient drug through the skin to prevent elicitation of the D T H response.
METHODS
Drugs CyA solutions were made from the 10",, w/v oral preparation of CyA (Sandimmun, Sandoz Ltd, Basle, Switzerland) which was diluted with olive oil to the required concentrations ofo 05-5"o. The CyA plus PE (CyA -f PE) formulations contained in addition to CyA, 2"o v/v Azone (AZ, Nelson Research, Irvine, CA, U.S.A.) and 18",) v/v propylene glycol (PG, Sigma, Poole, Dorset, U.K.).
Absorption of a novel topical cyclosporin A
633
In vitro studies Percutaneous absorption measurements were made using modified open flow-through permeability cells^" containing stratum corneum prepared from human cadaver abdominal skin.^' Upper donor chambers held 0 5 ml of 5",, CyA + PE containing 30 ^1 -^H-CyA (Amersham International PLC) of 370 kBq/ml activity. The total area of exposed stratum corneum in the cell was 0 2 cm^. A non-ionic surfactant receptor solution of 6",, w/v Volpo 20 in distilled water (Croda Chemicals Ltd., Goole, U.K.) was driven through the lower chamber (volume 80 /d) of the cell at 5 ml/h, thereby replacing the receptor fluid 63 times/h and maintaining sink conditions. The apparatus and solutions were maintained at 32 ± i C. The run-through receptor solution (25 ml) was collected every 30 min during a 5-hr period and the amount of 'H-CyA penetrating the stratum corneum assessed by adding 10 ml PICO FLUOR 40 (Canberra Packard, Pangbourne, U.K,) scintillation fluid to these samples and counting them in a Packard Tri-Carb 4530 scintillation counter. In vivo studies Animals. Male Dunkin-Hartley guinea-pigs purchased from David Hall, Newchurch, Staffs, U.K. and weighing 700-900 g were used. They received a pelleted diet, supplemented with vegetables and hay. Sensitization. Animals were sensitized to i-fluoro-2,4-dinitrobenzene (DNFB) by epicutaneous application of 50 ^\ 10",, (w/v) DNFB solution in acetone: olive oil ( i : r) to the dorsum of the right ear. Skin testing. Skin tests were performed 8 days after sensitization, by application of a nonirritant dose of 20/J1 DNFB solution (o-5'\, w/v) in acetone:olive oil (4:1) to the shaved flank. The erythematous reactions were assessed 24 h later using the following scale: 4, red and elevated; 3, red but not elevated; 2, confluent pink; i, patchy pink spots; 0 5 , feeble reaction; o, no change. Topical regimen. AmmahiAndomXy received 20/il topical applications of either 0 0 5 , o 25,0 5 or 5",, CyA + PE, olive oil or olive oil -|- PE to three test sites on each flank, immediately after drying ofthe challenge DNFB (o h). The drug was re-applied after 5 h and the animals were killed at 24 h when biopsies were taken for immunohistochemical analysis. Immunohistochemistry. Following excision, a portion of each skin biopsy was embedded in OCT (Raymond A Lamb, London U.K.) and snap frozen in liquid Arcton, previously cooled to freezing point by immersion in liquid nitrogen. Cryostat sections (6-8 /(m), cut in such an orientation as to include both the reaction 'epicentre' and outer margin ofthe reaction site, were mounted on alum-gelatin-coated slides and allowed to dry overnight at room temperature. Slides were stored wrapped in aluminium foil at - 20 C then brought to room temperature before fixation for 20 min in acetone prior to staining. The three-stage alkaline phosphatase-anti-alkaline phosphatase (APAAP) method was employed to demonstrate leucocyte phenotypes, using primary mouse monoclonal antibodies to guinea-pig mononuclear cells (Table i). Secondary antibody, rabbit anti-mouse Igs (Dako, Copenhagen, Denmark) was diluted 1:20 in io"o normal guinea-pig serum.
634
y.I.Duncan et al. TABLE I. Monoclonal antibodies used in the immunocytochemical analysis of guinea-pig mononuclear cells Antibody isotype CT7 IgGl CT6 IgGl
Specificity
Source
pan T cells Dr R,J.Scheper* T cytotoxic/suppressor cells Dr R,J.Scheper
Dilution 1:2000 1:5000
*A gift from Dr R,J.Schcper, Parhological Institute, Free University Hospital, Amsterdam, The Netherlands,
Quantitative assessment of cellular infiltration. Cell counts in the epidermis and upper papillary dermis were performed blind on coded sections. Positive cells (in five consecutive areas encompassed by an eyepiece grid occupying c. 50",, ofthe field of view) were counted using a X 10 eyepiece and x 25 objective. Statistics Results are expressed as means ± i standard deviation. The significance of differences between means was determined using Student's t-test (for cellular infiltrates) or Mann-Whitney nonparametric test (for erythema scores).
RESULTS
In-vitro penetration of CyA The addition of Azone (AZ) and propylene glycol (PG) to 5",, CyA enhanced the accumulation of 'H-CyA in the receptor fiuid during 5 h (Fig. ia). The amount of CyA penetrating the stratum corneum in the absence of PE was 53 ± 43 /xg/cm^/h, whereas the addition of PE significantly (P < 0001) increased CyA absorption tenfold to 660 ± 175 |tg CyA/cm^/h. After 5 h, 2-6"o CyA from the PE-containing formulation had penetrated the stratum corneum compared with only o 2"n from the CyA-only solution (Fig. ib). Delayed-type hypersensitivity reactions in the guinea-pig Skin reactions. The influence of topical CyA, at different concentrations, either alone or in combination with PE, on the intensity of skin reactions to DNFB at 24 h is shown in Table 2. Examination ofthe guinea-pigs revealed that CyA concentrations between o 25 and s'\, were significantly immunosuppressive and that almost complete suppression of the DTH reaction occurred with 5% CyA. When CyA was applied at a concentration of 005% there was no inhibitory effect without PE, although a 30",, reduction (which did not quite reach significance at P
