Original Paper Ophthalmic Res 1992;24:351-356
Division of Ophthalmology, University of Alicante Medical School, Alicante, Spain
Keywords Cyclosporin-A Cornea Topical treatment Ocular inflammation Albino rabbit
Corneal Concentration and Systemic Absorption of Cyclosporin-A Following its Topical Application in the Rabbit Eye Abstract To ascertain the corneal storage and the possible systemic absorption of 2% cyclosporin-A (CsA) eyedrops, the authors studied, by RIA with monoclonal specific antibodies, the cor neal and blood levels of the drug following its topical adminis tration on healthy albino rabbit eyes. When topical CsA was administered following a low-dose application schedule [such as a single dose of 100 pi every 12 h for 5 days (group 1)], CsA corneal levels of 260.8 ± 59.8 ng/ml and blood levels of 55.67 ± 24.4 ng/ml (expressed as means ± SD) were achieved. These levels significantly increased to 1,111.27 ± 449.4 ng/ml (p > 0.0001) and 88.66 ± 59.7 ng/ml (p > 0.05), respectively, when a higher dosage was used [100 pi every 6 h for 10 days (group 2)]. It is shown that a dose-dependent corneal concentration of CsA, due to a cumulative effect, is achieved in the intact rabbit cornea following topical application of the drug. Systemic absorption of CsA, although irregular, does exist. Therefore, when using topical 2% CsA as in this study, a systemic effect of the drug (especially in low weight animals) should be consid ered when analizing the results of topical application.
Indroduction Cyclosporin-A (CsA) is an immunosup pressive peptide which is extracted from the fungi Tolypocladium injlatum. Its molecular weight is 1,202.6 D, and it is highly lipophilic
This work has been supported in part by a grant from Sandoz laboratories Ltd. (Basel. Switzerland) and a fellowship from the Center to Prevent Blindness of the University of Alicante (J.L.B.)
Received: November 13. 1991 Accepted: June 25. 1992
[ 1]. CsA inhibits the activation and differenti ation of T lymphocytes, blocks the synthesis and the release of lymphokines [2], and it has been shown to be effective in some immunoinflammatory eye disorders [3],
Juan L. Bcllot. MI) Division of Ophthalmology University of Alicante Campus San Vicente del Raspeig E-03690 Alicante (Spain)
©1992 S. Karger AG. Basel 0030-3747/92/ 0246-0351$2.75/0
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:56:00 PM
Juan L. Bellot Jorge L. Alió José M. Ruiz Moreno Alberto Artola
Material and Methods Twenty-three healthy New' Zealand albino rabbits of 2-2.5 kg of weight were used in this study following the European Community standards for experimental animal manipulation.
352
Two groups of 10 rabbits were topically treated with 2% CsA eyedrops (20 mg/ml) in olive oil. follow ing two different dosage protocols: The 10 animals (20 eyes) of group 1 were treated, using a micropipctte. with 100 pi every 12 h for 5 days (in both eyes), receiving a theoretical dose of 4 mg/kg/ day, hence, a theoretical total dose of 20 mg/kg at the end of the study. The 10 animals (20 eyes) included in group 2 received 100 pi every 6 h for 10 days in both eyes. The theoretical administered dose was 8 mg/kg/ day. and a theoretical total dose of 80 mg/kg was achieved at the end of the study Five hours after the last topical application, the animals were anesthetized w'ith 50 mg/kg of intramus cular ketamine and 25 mg/kg of intraperitoneal thiopenthal sodium. A 5-m! sample of whole blood was immediately obtained by an intracardiac puncture, the animals were sacrificed with a lethal intracardiac dose of sodium thiopental, and their eyes were then enu cleated. The blood levels of the drug were investigated fol lowing the protocol of the Sandimmun® radioimmu noassay (RIA) kit (Sandoz, Basel, Switzerland) with monoclonal specific antibodies. The blood clotting was prevented with EDTA (Neotube. Nipro. Japan). Also, bacterial growth was inhibited by adding sodium azide in a final concentration of 0.01 % (w/v) in each blood tube. Following enucleation, the eyes were carefully washed with saline. A 9-mm central button was tre phined from each rabbit’s cornea, transferred into a tube containing 1 ml of a saline solution of 0.25% Tri ton X-100 (Sigma) and was shaken for 6 h using mag netic bars and stirrer. 100 pi were taken from this solution, treated with 900 pi of methanol, and centrifuged at 1,600 g, 50 pi of the supernatant obtained from such centrifugation were used as sample for CsA RIA. Previously, it was proved that Triton X-100 did not interfere w'ith the antigen-antibody reaction when samples were treated using the above procedure. Two different standard curves were plotted, using the same sample matrix (whole blood and cornea, both removed from normal non-CsA-treatcd rabbits) and processed in the same manner as the unknowm sam ples. RIA quality control tests of each standard curve were done: blood and cornea samples removed from 3 healthy nontreated rabbits were measured and used as normal controls. The results were statistically studied by Student’s t test after determining the normal distribution of data by the y- test. The Mann-Whitney U test was used
Bellot/Alió/Ruiz Moreno/Artola
Corneal Accumulation and Systemic Absorption of CsA
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:56:00 PM
Several authors have proposed the topical use of CsA 2% eyedrops for the treatment of some ocular diseases, such as corneal grafting [4. 5] and vernal conjunctivitis [6-8], How ever, the dosage and results of such topical treatment are still controversial, speculative and are now under study. Previous experimental studies have shown that intraocular penetration of topically ap plied CsA is poor in the intact eye. Due to its lipophilic properties, CsA accumulates in the cornea and conjunctiva following its topical application [9, 10]. These findings are rele vant to the expected efficacy of CsA on ocu lar corneal disease when it is topically ap plied. Although the cornea does not allow intrao cular penetration of CsA. its systemic absorp tion through the conjunctiva is possible. Such systemic absorption could influence the ef fects if topically applied CsA through the induction of systemic immune effects, espe cially in experimental animals of low weight. Conversely, if a significant systemic level of the drug could be achieved from the topical application, it could indicate the possibility of systemic toxicity of topically applied CsA. The purposes of this study were to ascer tain whether the topical instillation of CsA 2% leads to corneal accumulation of the drug in the intact eye of the rabbit and whether the corneal levels of CsA depend on the frequency of instillations. We have also investigated whether a systemic absorption occurs when CsA 2% eyedrops are applied on both eyes of low weight experimental animals.
when a nonnormal distribution was found. Linear cor relation studies were done by plotting the CsA levels in blood and cornea against the dosage of topical CsA. For these studies we have used the statistical computer program Microstat® (Ecosoft. 1984).
Results Coweal Levels Previously, it has been proved that there are no differences (p > 0.05, Student’s t test) between the standard curve plotted using a buffered solution of 10% rabbit plasma and the curve obtained by adding Triton X-100 to corneal samples without CsA and undergoing the same procedure as the unknown samples (stirred for 6 h and extracted with methanol). We obtained 813.23 ng/ml in buffer-plasma solution and 817.8 ng/ml in the tubes with Triton X-100, for a theoretical CsA value of 800 ng/ml and 411.2 and 431.5 ng/ml, respec tively for a theoretical point of 400 ng/ml. Six normal control corneal samples from 3 untreated rabbits gave CsA levels lower than the sensibility limit of the RIA method (25 ng/ml), as expected.
Dose, mg/kg
Group 1 (20 eyes) showed mean CsA levels of 260.8 ± 59.8 ng/ml (mean ± SD; SEM = 13.72), ranging from 113.35 to 359.7 ng/ml, while group 2 (20 eyes) showed 1,111.27 ± 449.4 ng/ml (mean ± SD; SEM = 100.48), ranging from 524 to 1,600 ng/ml (fig. 1). The differences between both groups were found to be highly significant (p < 0.0001, Mann-Whitney U test). Also, a signif icant linear correlation (r = 0.83) was found between the theoretical topical dose adminis tered and the corneal levels achieved by the CsA 2% treatment. Systemic Absorption Blood levels were 55.67 ± 24.4 ng/ml (mean ± SD; SEM = 8.15), ranging from 31.72 to 118.24 ng/ml, in group 1(10 sam ples). and 88.66 ± 59.7 ng/ml (SEM = 17.85), ranging from 35.41 to 181.82 ng/ml, in group 2(10 samples; fig. 1), with statistically signifi cant differences (p < 0.05, Student’s t test). However, we could not find a significant linear correlation between the increase in the theoretical topical dose and the increase in the blood levels of the drug (r = 0.36).
353
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:56:00 PM
Fig. 1. CsA levels in cornea and blood samples from healthy rabbits treated with topical eyedrops fol lowing two different theoretical doses. Each concentration point is represented as mean ± 95% confi dence limits.
Discussion We can conclude from the results of this study that a dose-dependent corneal storage of CsA can be achieved in the healthy rabbit cornea following topical application of this drug. Systemic absorption of CsA does exist at the doses here employed, but it is not directly dependent on the topical dose. Topical CsA does not easily penetrate the intact eye [9, 10]. The highest concentration was found in corneal tissue, where the drug is retained in the stroma due to a different polarity between the lipophilic CsA molecule and the hydrophilic corneal entothelium [11, 12]. Our results correspond with those reported by Mosteller et al. [ 11 ], who first reported the lack of intraocular penetration of the drug when topically applied, as well as the reten tion of CsA in the corneal stroma, findings which were confirmed by Wiederholt et al. [12], BenEzra and Maftzir [9, 10] studied the intraocular penetration of CsA 2% eyedrops, and definitely concluded that CsA does not penetrate the healthy intact eye of either the
354
rabbit or the rat when topically applied. In the human eye, similar findings were also con firmed [13. 14], However, to our knowledge, the possible relationship between the corneal levels of CsA and the frequency of topical applications of CsA has not been previously studied. In our opinion, such a study might be relevant to ascertain the optimal topical dose of CsA to be used in other experimental and clinical experiences. Our results show that a relatively low dose of topical 2% CsA in an olive oil vehicle, such as 100 |tl every 12 h for 5 days, leads to cor neal levels of the drug higher than the 100 ng/ml threshold level. Such levels are enough to inhibit in vitro T-cell functions, such as lymphokine release and cytotoxicity [15, 16], Thus, a low dose of topical CsA might be suf ficient to treat some corneal conditions (e.g. corneal graft rejection), if a local effect of the drug could be demonstrated. We have also shown that an increase in the frequency and duration of the treatment leads to a dose-dependent increase in the corneal levels of the drug, with a high correlation (r = 0.83). Thus, when a higher dose of CsA is needed, it can be achieved by increasing the frequency of topical applications. To the best of our knowledge, such findings, although ex pected. had not been reported previously. CsA has been shown to be toxic in vitro for concentrations higher than 1,000 ng/ml, espe cially for the human corneal endothelium [ 17] and corneal stromal cells [ 18]. Such data arc of interest, as higher concentrations have been found in the present study. However, important corneal side effects have not been reported with the clinical topical use of CsA in humans [7,8] except for a superficial punctate keratitis in some cases [3]. This relative lack of toxicity can be explained by the fact that only 8% of the drug absorbed by the cornea reaches the endothelium [ 12],
Bellot/Alió/Ruiz Moreno/Artola
Corneal Accumulation and Systemic Absorption of CsA
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:56:00 PM
Three separated quality controls per formed in the blood samples showed mean levels of 104.7 ng/ml for the theoretical value of 100 ng/ml (Q 1) and 427 ng/ml for Q2 (400 ng/ml). Three blood samples from the rabbits that did not receive CsA were also below the low est limit of the RIA technique ( < 25 ng/ml). To ascertain if any relation does exist be tween corneal and blood CsA levels, we have calculated a theoretical bioavailability factor by dividing the corneal by the blood concen trations in each animal. In group 1, the cor neal levels are 5.38 ± 2.02 (mean ± SD) times the blood levels, increasing to 17.5 ± 11.86 in group 2.
Regarding the systemic absorption of topi cal CsA, we have tried to study the blood lev els reached after bilateral topical application in our experimental model. This is necessary in some of the evolving ocular indications of topical CsA, such as vernal conjunctivitis [68,19]. Systemic toxicity, especially nephrotoxic ity [15], is the main disadvantage of CsA. Hence, it is necessary to establish the riskbenefit ratio that exists when the drug is administered, even topically, to treat some ocular immune conditions which are not life threatening. Topical treatment is the preferred route for drug administration in ophthalmology. Sys temic levels of the drug when topically ap plied are especially important in experimental studies with animals of low weight, as a sys temic effect on T-cell function might in fluence the results of local ocular experiences. In the present study, the blood levels were low enough to guarantee the lack of systemic tox icity of the drug, but not a systemic immuno logical effect in the rabbit, as the therapeutic window of CsA has been established in 50250 ng/ml [15]. However, such an effect is practically impossible in the human as the expected blood levels in a 70-kg human
should be 0.74 ng/ml, according to the results of our study, if it is assumed that CsA follows a similar absorption and metabolism in the human as well as the rabbit, as it has been pre viously reported [ 13, 14], We have not found a significant correla tion between dose and blood levels, although a significant but not parallel increase does exist in rabbits when a storage schedule is top ically used. The fact that topical CsA can achieve sys temic therapeutic levels in the rabbit is con tradictory to the data previously reported by BenEzra and Maftzir [9], who found blood levels lower than 25-30 ng/ml in healthy rab bits. Moreover, we have measured CsA levels by using the monoclonal specific RIA which shows no significant cross-reaction with CsA metabolites and, therefore, determines lower values of CsA than the polyclonal method [20], which was the one used by those authors [9]. The finding of therapeutic blood levels of CsA in the rabbit might be important, because a systemic immune effect in combination with the local effect of 2% CsA in animals less than 2 kg of weight has to be considered when some therapeutic action of CsA is being inves tigated.
References 4 Hoffman F, Wiederholt M: Lokale Behandlung des Hornhauttrans plantates beim Menschen mit Cy closporin A. Klin Monatsbl Augenheilkd 1985;187:92-96. 5 Pouliquen YJM, Goichot-Bonnat EL: Ciclosporine A collyre dans les greffes de cornée à haut risque. Oph talmologie 1987;2:311-313.
6 BenEzra D. Cohen E: Cyclosporin A eyedrops in severe vernal keratocon junctivitis (abstract A -14). 1st Sym posium on Ocular Inflammation, Alicante, May 1990. 7 BenEzra D, Peer J. Brodsky M, Co hen E: Cyclosporin A for the treat ment of severe vernal keratocon junctivitis. Am J Ophthalmol 1986; 101:278.
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1 Wenger R.: Chemistry of cyclospo rin; in White DJG (ed): Cyclospo rine A. Amsterdam. Elsevier Bio medical Press. 1982, pp 19-34. 2 Hess AD. Tutschka PJ. Santos GW, Pu Z: Effect of cyclosporin A on human lymphocyte responses in vi tro. J Immunol 1982;128:355-367. 3 Nussenblatt RB: The use of cyclo sporine in ocular inflammatory dis orders. Transplant Proc 1988; 20(suppl 4): 114-121.
12 Wiederholt M, Kossendrup D. Hoff man F: Pharmacokinetic of topical cyclosporin A in the rabbit eye. In vest Ophthalmol Vis Sei 1986:27: 519-524. 13 BenEzra D. Maftzir G, de Courten Ch. Timonen P: Ocular penetration of cyclosporin A: The human eye. Br J Ophthalmol 1990:74:350. 14 Díaz M. Planclls C, Claramunt T, et al: Penetración en humor acuoso hu mano del colirio de ciclosporina al 2%. Arch Soc Esp Oftalmol 1990; 58:89-106. 15 Keown PA, Stiller CR: Cyclospo rine: A double-edged sword. Hosp Pract 1987:22:207-215,219-220. 16 BenEzra D: Cyclosporin A in Behçet’s disease - An overview: in Barnes L (ed): Recent Advances in Behçet’s Disease. London, Royal Society of Medicine Services. 1986, pp 319-325.
17 Singh G. Lindstrom RL, Doughman DJ: Cyclosporin A on human cor neal endothelium. Cornea 1984/85: 3:272-277. 18 BenEzra D. Antebe I. Maftzir G: Differential effect o f cyclosporin A on lymphocyte and keratoevte pro liferation. ARVO 1987:28:42. 19 Secchi AG. Tognon MS, Leonardi A: T opical use of cyclosporine in the treatment of verncl keratoconjunc tivitis. Am J Ophthalmol 1990:110: 641-645. 20 Holt DW: Sandimmun® (cyclospo rin) Monitoring. A Practical Guide. Sandoz, Basle. 1987.
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8 BenEzra D. Matamoros N. Cohen E: Treatment of severe keratocon junctivitis with cyclosporin A. Transplant Proc 1988;20(suppl): 136. 9 BenEzra D. Maftzir G: Ocular pene tration o f cyclosporine A: The rabbit eye. Invest Ophthalmol Vis Sei 1990;31:1362-1366. 10 BenEzra D. Maftzir G: Ocular pene tration of cyclosporin A in the rat eye. Arch Ophthalmol 1990:108: 584-587. 11 Mosteller MW. Gebhardt BM. Hamilton AM. Kaufman E: Pene tration of topical cyclosporine into the rabbit cornea, aqueous humor and serum. Arch Ophthalmol 1985: 103:101-102.
Division of Ophthalmology, University of Alicante Medical School, Alicante, Spain
Keywords Cyclosporin-A Cornea Topical treatment Ocular inflammation Albino rabbit
Corneal Concentration and Systemic Absorption of Cyclosporin-A Following its Topical Application in the Rabbit Eye Abstract To ascertain the corneal storage and the possible systemic absorption of 2% cyclosporin-A (CsA) eyedrops, the authors studied, by RIA with monoclonal specific antibodies, the cor neal and blood levels of the drug following its topical adminis tration on healthy albino rabbit eyes. When topical CsA was administered following a low-dose application schedule [such as a single dose of 100 pi every 12 h for 5 days (group 1)], CsA corneal levels of 260.8 ± 59.8 ng/ml and blood levels of 55.67 ± 24.4 ng/ml (expressed as means ± SD) were achieved. These levels significantly increased to 1,111.27 ± 449.4 ng/ml (p > 0.0001) and 88.66 ± 59.7 ng/ml (p > 0.05), respectively, when a higher dosage was used [100 pi every 6 h for 10 days (group 2)]. It is shown that a dose-dependent corneal concentration of CsA, due to a cumulative effect, is achieved in the intact rabbit cornea following topical application of the drug. Systemic absorption of CsA, although irregular, does exist. Therefore, when using topical 2% CsA as in this study, a systemic effect of the drug (especially in low weight animals) should be consid ered when analizing the results of topical application.
Indroduction Cyclosporin-A (CsA) is an immunosup pressive peptide which is extracted from the fungi Tolypocladium injlatum. Its molecular weight is 1,202.6 D, and it is highly lipophilic
This work has been supported in part by a grant from Sandoz laboratories Ltd. (Basel. Switzerland) and a fellowship from the Center to Prevent Blindness of the University of Alicante (J.L.B.)
Received: November 13. 1991 Accepted: June 25. 1992
[ 1]. CsA inhibits the activation and differenti ation of T lymphocytes, blocks the synthesis and the release of lymphokines [2], and it has been shown to be effective in some immunoinflammatory eye disorders [3],
Juan L. Bcllot. MI) Division of Ophthalmology University of Alicante Campus San Vicente del Raspeig E-03690 Alicante (Spain)
©1992 S. Karger AG. Basel 0030-3747/92/ 0246-0351$2.75/0
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:56:00 PM
Juan L. Bellot Jorge L. Alió José M. Ruiz Moreno Alberto Artola
Material and Methods Twenty-three healthy New' Zealand albino rabbits of 2-2.5 kg of weight were used in this study following the European Community standards for experimental animal manipulation.
352
Two groups of 10 rabbits were topically treated with 2% CsA eyedrops (20 mg/ml) in olive oil. follow ing two different dosage protocols: The 10 animals (20 eyes) of group 1 were treated, using a micropipctte. with 100 pi every 12 h for 5 days (in both eyes), receiving a theoretical dose of 4 mg/kg/ day, hence, a theoretical total dose of 20 mg/kg at the end of the study. The 10 animals (20 eyes) included in group 2 received 100 pi every 6 h for 10 days in both eyes. The theoretical administered dose was 8 mg/kg/ day. and a theoretical total dose of 80 mg/kg was achieved at the end of the study Five hours after the last topical application, the animals were anesthetized w'ith 50 mg/kg of intramus cular ketamine and 25 mg/kg of intraperitoneal thiopenthal sodium. A 5-m! sample of whole blood was immediately obtained by an intracardiac puncture, the animals were sacrificed with a lethal intracardiac dose of sodium thiopental, and their eyes were then enu cleated. The blood levels of the drug were investigated fol lowing the protocol of the Sandimmun® radioimmu noassay (RIA) kit (Sandoz, Basel, Switzerland) with monoclonal specific antibodies. The blood clotting was prevented with EDTA (Neotube. Nipro. Japan). Also, bacterial growth was inhibited by adding sodium azide in a final concentration of 0.01 % (w/v) in each blood tube. Following enucleation, the eyes were carefully washed with saline. A 9-mm central button was tre phined from each rabbit’s cornea, transferred into a tube containing 1 ml of a saline solution of 0.25% Tri ton X-100 (Sigma) and was shaken for 6 h using mag netic bars and stirrer. 100 pi were taken from this solution, treated with 900 pi of methanol, and centrifuged at 1,600 g, 50 pi of the supernatant obtained from such centrifugation were used as sample for CsA RIA. Previously, it was proved that Triton X-100 did not interfere w'ith the antigen-antibody reaction when samples were treated using the above procedure. Two different standard curves were plotted, using the same sample matrix (whole blood and cornea, both removed from normal non-CsA-treatcd rabbits) and processed in the same manner as the unknowm sam ples. RIA quality control tests of each standard curve were done: blood and cornea samples removed from 3 healthy nontreated rabbits were measured and used as normal controls. The results were statistically studied by Student’s t test after determining the normal distribution of data by the y- test. The Mann-Whitney U test was used
Bellot/Alió/Ruiz Moreno/Artola
Corneal Accumulation and Systemic Absorption of CsA
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:56:00 PM
Several authors have proposed the topical use of CsA 2% eyedrops for the treatment of some ocular diseases, such as corneal grafting [4. 5] and vernal conjunctivitis [6-8], How ever, the dosage and results of such topical treatment are still controversial, speculative and are now under study. Previous experimental studies have shown that intraocular penetration of topically ap plied CsA is poor in the intact eye. Due to its lipophilic properties, CsA accumulates in the cornea and conjunctiva following its topical application [9, 10]. These findings are rele vant to the expected efficacy of CsA on ocu lar corneal disease when it is topically ap plied. Although the cornea does not allow intrao cular penetration of CsA. its systemic absorp tion through the conjunctiva is possible. Such systemic absorption could influence the ef fects if topically applied CsA through the induction of systemic immune effects, espe cially in experimental animals of low weight. Conversely, if a significant systemic level of the drug could be achieved from the topical application, it could indicate the possibility of systemic toxicity of topically applied CsA. The purposes of this study were to ascer tain whether the topical instillation of CsA 2% leads to corneal accumulation of the drug in the intact eye of the rabbit and whether the corneal levels of CsA depend on the frequency of instillations. We have also investigated whether a systemic absorption occurs when CsA 2% eyedrops are applied on both eyes of low weight experimental animals.
when a nonnormal distribution was found. Linear cor relation studies were done by plotting the CsA levels in blood and cornea against the dosage of topical CsA. For these studies we have used the statistical computer program Microstat® (Ecosoft. 1984).
Results Coweal Levels Previously, it has been proved that there are no differences (p > 0.05, Student’s t test) between the standard curve plotted using a buffered solution of 10% rabbit plasma and the curve obtained by adding Triton X-100 to corneal samples without CsA and undergoing the same procedure as the unknown samples (stirred for 6 h and extracted with methanol). We obtained 813.23 ng/ml in buffer-plasma solution and 817.8 ng/ml in the tubes with Triton X-100, for a theoretical CsA value of 800 ng/ml and 411.2 and 431.5 ng/ml, respec tively for a theoretical point of 400 ng/ml. Six normal control corneal samples from 3 untreated rabbits gave CsA levels lower than the sensibility limit of the RIA method (25 ng/ml), as expected.
Dose, mg/kg
Group 1 (20 eyes) showed mean CsA levels of 260.8 ± 59.8 ng/ml (mean ± SD; SEM = 13.72), ranging from 113.35 to 359.7 ng/ml, while group 2 (20 eyes) showed 1,111.27 ± 449.4 ng/ml (mean ± SD; SEM = 100.48), ranging from 524 to 1,600 ng/ml (fig. 1). The differences between both groups were found to be highly significant (p < 0.0001, Mann-Whitney U test). Also, a signif icant linear correlation (r = 0.83) was found between the theoretical topical dose adminis tered and the corneal levels achieved by the CsA 2% treatment. Systemic Absorption Blood levels were 55.67 ± 24.4 ng/ml (mean ± SD; SEM = 8.15), ranging from 31.72 to 118.24 ng/ml, in group 1(10 sam ples). and 88.66 ± 59.7 ng/ml (SEM = 17.85), ranging from 35.41 to 181.82 ng/ml, in group 2(10 samples; fig. 1), with statistically signifi cant differences (p < 0.05, Student’s t test). However, we could not find a significant linear correlation between the increase in the theoretical topical dose and the increase in the blood levels of the drug (r = 0.36).
353
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:56:00 PM
Fig. 1. CsA levels in cornea and blood samples from healthy rabbits treated with topical eyedrops fol lowing two different theoretical doses. Each concentration point is represented as mean ± 95% confi dence limits.
Discussion We can conclude from the results of this study that a dose-dependent corneal storage of CsA can be achieved in the healthy rabbit cornea following topical application of this drug. Systemic absorption of CsA does exist at the doses here employed, but it is not directly dependent on the topical dose. Topical CsA does not easily penetrate the intact eye [9, 10]. The highest concentration was found in corneal tissue, where the drug is retained in the stroma due to a different polarity between the lipophilic CsA molecule and the hydrophilic corneal entothelium [11, 12]. Our results correspond with those reported by Mosteller et al. [ 11 ], who first reported the lack of intraocular penetration of the drug when topically applied, as well as the reten tion of CsA in the corneal stroma, findings which were confirmed by Wiederholt et al. [12], BenEzra and Maftzir [9, 10] studied the intraocular penetration of CsA 2% eyedrops, and definitely concluded that CsA does not penetrate the healthy intact eye of either the
354
rabbit or the rat when topically applied. In the human eye, similar findings were also con firmed [13. 14], However, to our knowledge, the possible relationship between the corneal levels of CsA and the frequency of topical applications of CsA has not been previously studied. In our opinion, such a study might be relevant to ascertain the optimal topical dose of CsA to be used in other experimental and clinical experiences. Our results show that a relatively low dose of topical 2% CsA in an olive oil vehicle, such as 100 |tl every 12 h for 5 days, leads to cor neal levels of the drug higher than the 100 ng/ml threshold level. Such levels are enough to inhibit in vitro T-cell functions, such as lymphokine release and cytotoxicity [15, 16], Thus, a low dose of topical CsA might be suf ficient to treat some corneal conditions (e.g. corneal graft rejection), if a local effect of the drug could be demonstrated. We have also shown that an increase in the frequency and duration of the treatment leads to a dose-dependent increase in the corneal levels of the drug, with a high correlation (r = 0.83). Thus, when a higher dose of CsA is needed, it can be achieved by increasing the frequency of topical applications. To the best of our knowledge, such findings, although ex pected. had not been reported previously. CsA has been shown to be toxic in vitro for concentrations higher than 1,000 ng/ml, espe cially for the human corneal endothelium [ 17] and corneal stromal cells [ 18]. Such data arc of interest, as higher concentrations have been found in the present study. However, important corneal side effects have not been reported with the clinical topical use of CsA in humans [7,8] except for a superficial punctate keratitis in some cases [3]. This relative lack of toxicity can be explained by the fact that only 8% of the drug absorbed by the cornea reaches the endothelium [ 12],
Bellot/Alió/Ruiz Moreno/Artola
Corneal Accumulation and Systemic Absorption of CsA
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:56:00 PM
Three separated quality controls per formed in the blood samples showed mean levels of 104.7 ng/ml for the theoretical value of 100 ng/ml (Q 1) and 427 ng/ml for Q2 (400 ng/ml). Three blood samples from the rabbits that did not receive CsA were also below the low est limit of the RIA technique ( < 25 ng/ml). To ascertain if any relation does exist be tween corneal and blood CsA levels, we have calculated a theoretical bioavailability factor by dividing the corneal by the blood concen trations in each animal. In group 1, the cor neal levels are 5.38 ± 2.02 (mean ± SD) times the blood levels, increasing to 17.5 ± 11.86 in group 2.
Regarding the systemic absorption of topi cal CsA, we have tried to study the blood lev els reached after bilateral topical application in our experimental model. This is necessary in some of the evolving ocular indications of topical CsA, such as vernal conjunctivitis [68,19]. Systemic toxicity, especially nephrotoxic ity [15], is the main disadvantage of CsA. Hence, it is necessary to establish the riskbenefit ratio that exists when the drug is administered, even topically, to treat some ocular immune conditions which are not life threatening. Topical treatment is the preferred route for drug administration in ophthalmology. Sys temic levels of the drug when topically ap plied are especially important in experimental studies with animals of low weight, as a sys temic effect on T-cell function might in fluence the results of local ocular experiences. In the present study, the blood levels were low enough to guarantee the lack of systemic tox icity of the drug, but not a systemic immuno logical effect in the rabbit, as the therapeutic window of CsA has been established in 50250 ng/ml [15]. However, such an effect is practically impossible in the human as the expected blood levels in a 70-kg human
should be 0.74 ng/ml, according to the results of our study, if it is assumed that CsA follows a similar absorption and metabolism in the human as well as the rabbit, as it has been pre viously reported [ 13, 14], We have not found a significant correla tion between dose and blood levels, although a significant but not parallel increase does exist in rabbits when a storage schedule is top ically used. The fact that topical CsA can achieve sys temic therapeutic levels in the rabbit is con tradictory to the data previously reported by BenEzra and Maftzir [9], who found blood levels lower than 25-30 ng/ml in healthy rab bits. Moreover, we have measured CsA levels by using the monoclonal specific RIA which shows no significant cross-reaction with CsA metabolites and, therefore, determines lower values of CsA than the polyclonal method [20], which was the one used by those authors [9]. The finding of therapeutic blood levels of CsA in the rabbit might be important, because a systemic immune effect in combination with the local effect of 2% CsA in animals less than 2 kg of weight has to be considered when some therapeutic action of CsA is being inves tigated.
References 4 Hoffman F, Wiederholt M: Lokale Behandlung des Hornhauttrans plantates beim Menschen mit Cy closporin A. Klin Monatsbl Augenheilkd 1985;187:92-96. 5 Pouliquen YJM, Goichot-Bonnat EL: Ciclosporine A collyre dans les greffes de cornée à haut risque. Oph talmologie 1987;2:311-313.
6 BenEzra D. Cohen E: Cyclosporin A eyedrops in severe vernal keratocon junctivitis (abstract A -14). 1st Sym posium on Ocular Inflammation, Alicante, May 1990. 7 BenEzra D, Peer J. Brodsky M, Co hen E: Cyclosporin A for the treat ment of severe vernal keratocon junctivitis. Am J Ophthalmol 1986; 101:278.
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1 Wenger R.: Chemistry of cyclospo rin; in White DJG (ed): Cyclospo rine A. Amsterdam. Elsevier Bio medical Press. 1982, pp 19-34. 2 Hess AD. Tutschka PJ. Santos GW, Pu Z: Effect of cyclosporin A on human lymphocyte responses in vi tro. J Immunol 1982;128:355-367. 3 Nussenblatt RB: The use of cyclo sporine in ocular inflammatory dis orders. Transplant Proc 1988; 20(suppl 4): 114-121.
12 Wiederholt M, Kossendrup D. Hoff man F: Pharmacokinetic of topical cyclosporin A in the rabbit eye. In vest Ophthalmol Vis Sei 1986:27: 519-524. 13 BenEzra D. Maftzir G, de Courten Ch. Timonen P: Ocular penetration of cyclosporin A: The human eye. Br J Ophthalmol 1990:74:350. 14 Díaz M. Planclls C, Claramunt T, et al: Penetración en humor acuoso hu mano del colirio de ciclosporina al 2%. Arch Soc Esp Oftalmol 1990; 58:89-106. 15 Keown PA, Stiller CR: Cyclospo rine: A double-edged sword. Hosp Pract 1987:22:207-215,219-220. 16 BenEzra D: Cyclosporin A in Behçet’s disease - An overview: in Barnes L (ed): Recent Advances in Behçet’s Disease. London, Royal Society of Medicine Services. 1986, pp 319-325.
17 Singh G. Lindstrom RL, Doughman DJ: Cyclosporin A on human cor neal endothelium. Cornea 1984/85: 3:272-277. 18 BenEzra D. Antebe I. Maftzir G: Differential effect o f cyclosporin A on lymphocyte and keratoevte pro liferation. ARVO 1987:28:42. 19 Secchi AG. Tognon MS, Leonardi A: T opical use of cyclosporine in the treatment of verncl keratoconjunc tivitis. Am J Ophthalmol 1990:110: 641-645. 20 Holt DW: Sandimmun® (cyclospo rin) Monitoring. A Practical Guide. Sandoz, Basle. 1987.
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8 BenEzra D. Matamoros N. Cohen E: Treatment of severe keratocon junctivitis with cyclosporin A. Transplant Proc 1988;20(suppl): 136. 9 BenEzra D. Maftzir G: Ocular pene tration o f cyclosporine A: The rabbit eye. Invest Ophthalmol Vis Sei 1990;31:1362-1366. 10 BenEzra D. Maftzir G: Ocular pene tration of cyclosporin A in the rat eye. Arch Ophthalmol 1990:108: 584-587. 11 Mosteller MW. Gebhardt BM. Hamilton AM. Kaufman E: Pene tration of topical cyclosporine into the rabbit cornea, aqueous humor and serum. Arch Ophthalmol 1985: 103:101-102.
