BASIC INVESTIGATION
Self-Assembling Colloidal System for the Ocular Administration of Cyclosporine A Christoph Luschmann, PhD,* Joerg Tessmar, PhD,† Simon Schoeberl,‡ Olaf Strauß, PhD,‡§ Karl Luschmann, PhD,¶ and Achim Goepferich, PhD*
Purpose: In this study, we developed a self-assembling micellar system to deliver cyclosporine A (CsA) in an aqueous solution to the cornea.
Methods: Two nonionic surfactants of the poly(ethylene glycol)fatty alcohol ether type (Sympatens AS and Sympatens ACS) were characterized in terms of micelle size, shape, and charge, and their encapsulation efficiency for CsA. In an in situ single dose bioavailability study, the corneal CsA levels were determined in an enucleated porcine eye model. A commercial formulation and a 2% CsA olive oil solution served as references. Results: Both surfactants formed spherical micelles with a size of 9 to 12 nm in water. A concentration as low as 0.3% (wt/vol) Sympatens AS was sufficient to entrap therapeutic levels of at least 0.1% (wt/vol) CsA. In the porcine in situ model, exceptionally high drug levels in the cornea were obtained for the micellar CsA solution (1557 6 407 ngCsA/gcornea). They were significantly higher than those of Restasis (545 6 137 ngCsA/gcornea) or the olive oil solution (452 6 142 ngCsA/gcornea). Conclusions: In conclusion, we have shown a promising simple and efficient approach for the application of CsA in an aqueous solution to the cornea to treat inflammatory corneal diseases. Key Words: surfactant micelles, cyclosporin A, poor solubility, dry eye disease, bioavailability (Cornea 2014;33:77–81)
L
ocal therapy for the anterior eye and, therefore, the delivery of drugs to the cornea for the treatment of inflammatory
Received for publication June 5, 2013; revision received July 29, 2013; accepted August 2, 2013. Published online ahead of print October 24, 2013. From the *Department of Pharmaceutical Technology, University of Regensburg, Regensburg, Germany; †Department for Functional Materials in Medicine and Dentistry, University of Wuerzburg, Wuerzburg, Germany; ‡Department of Experimental Ophthalmology, University Hospital Regensburg, Regensburg, Germany; §Experimental Ophthalmology, Department Ophthalmology, Charité University Medicine Berlin, Berlin, Germany; and ¶Pharma Stulln GmbH, Stulln, Germany. Supported by Bavarian Research Foundation (BFS AZ 870-90). K. Luschmann is the director of Pharma Stulln GmbH. The other authors have no other funding or conflicts of interest to disclose. Reprints: Achim Goepferich, Department of Pharmaceutical Technology, University of Regensburg, Regensburg 93040, Germany (e-mail: achim. [email protected]). Copyright © 2013 by Lippincott Williams & Wilkins
Cornea Volume 33, Number 1, January 2014
corneal diseases is still a highly unmet need. Contemporary research focuses on increasing the bioavailability and the therapeutic efficacy of drugs and on avoiding their systemic side effects.1–4 An example of a highly potent antiinflammatory drug is cyclosporine A (CsA). CsA is successfully used in the treatment of uveitis,6 necrotizing scleritis,7 and thyroid ophthalmopathy8 and is one of the most promising substances for the treatment of dry eye disease.9 Unfortunately, it has a poor solubility in aqueous media (CsA, 6.6 mg/mL in water10) and a high octanol–water coefficient of log P = 3.11 Many different options, with a main focus on colloidal systems, such as micelles,12,13 liposomes,14 or various nanoparticular formulations,15–17 have been investigated to solubilize CsA. A cationic nanoemulsion is currently in phase 3 clinical trials and Restasis (Allergan), a 0.05% CsA microemulsion, is the only approved product, but it is available only on the US market.18 Hence, the dry eye syndrome is typically treated with oily 2% CsA eye drops that use vegetable oils as solvents.19,20 Unfortunately, all systems containing an oily phase usually cause side effects such as a burning, stinging sensation and blurred vision. Therefore, patient compliance is very low.21 Thus, there is still a tremendous need for a simple, yet efficient aqueous colloidal system, which does not need an oil phase. Ideally, it should be made of inexpensive surfactants, should self-assemble in the presence of water and drug, and should solubilize large amounts of the drug. Such a formulation would lead to a tremendous increase in patient compliance and would be a major option in the causal treatment of various diseases of the cornea. The aim of our study was to develop such a selfassembling micellar system. We were particularly interested in the CsA uptake into corneal tissue in a porcine in situ model.
MATERIALS AND METHODS Materials CsA was supplied by the Pharma Stulln GmbH (Stulln, Germany). Cyclosporin D was a generous gift from Prof. Dr F. Kees (University of Regensburg, Germany). Sympatens AS/200 G (AS) and Sympatens ACS/200 G (ACS) were provided by KOLB (Hedingen, Switzerland). Solutol HS 15 was a generous gift of BASF (Ludwigshafen, Germany). Mannitol was purchased from Caesar & Loretz GmbH (Hilden, Germany). Deionized water was obtained from a Milli-Q water purification system from Millipore (Schwalbach, Germany). www.corneajrnl.com |
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Methanol (MeOH), acetonitrile (MeCN), isopropanol [all high-performance liquid chromatography (HPLC) grade] and chloroform p.a. were purchased from Merck (Darmstadt, Germany). Hydrochloric acid (0.1 N) and sodium hydroxide (0.1 N) solutions were obtained from Carl Roth (Karlsruhe, Germany).
Entrapment Efficiency Four different surfactant concentrations were prepared to determine their entrapment efficiency of 0.1% drug. To solubilize CsA, aqueous solutions of 1%, 0.7%, 0.3%, and 0.1% (wt/vol) AS and ACS were prepared by gentle stirring. One milliliter of each surfactant concentration was spiked with 1 mg of CsA, and stirred for 24 hours at room temperature (n = 3). The dispersions were centrifuged at 16,000g for 10 minutes in Eppendorf tubes. One hundred microliters of the supernatant was diluted with 900 mL of MeOH in HPLC vials (2-mL Screw top vial; BGB Analytik AG, Rheinfelden, Germany). The samples were stored at 280°C (HERAfreeze; Thermo Fisher Scientific GmbH, Ulm, Germany) until further analysis was carried out. For the CsA analysis, a Shimadzu chromatographic setup consisting of an SCL-10AVP controller, an LC-10ATVP pump at a flow rate of 1 mL/min, an SIL-10ADVP autoinjector with a sample volume of 15 mL, a CTO-10ASVP oven with a temperature of 75°C and an SPD-10AVP ultraviolet detector at a wavelength of 210 nm were used (all from Shimadzu Deutschland GmbH, Duisburg, Germany). A reversed-phase octadecyl column Luna 3 mm C18(2) 100A with 100 · 4.6 mm served as the stationary phase. The mobile phase consisted of 75% MeCN and 25% H2O and was linearly changed to 90% MeCN and 10% H2O over 5 minutes. These concentrations were kept for 12.5 minutes, and then, the column was reequilibrated to the initial conditions until the end of the run at 15 minutes. The entrapment efficiency E was calculated as follows: cn1 cn2 cn3 þ þ co1 co2 co3 · 100; E½% ¼ 3 where cn was the concentration determined in the sample, which was divided by co, the initial concentration of 0.1% drug for each of the 3 measurements. The average was calculated by dividing by n = 3 and finally, the relative entrapment efficacy E was obtained by multiplication by 100.
Drug-Loaded Micelle Preparation One hundred milliliters of an isotonic 5.2% (wt/vol) aqueous mannitol solution was prepared by gentle stirring. Thirty milliliters of surfactant stock solutions (SSSs) with concentrations of 5% (wt/vol) AS, 5% (wt/vol) ACS, and 10% (wt/vol) Solutol, respectively, were prepared by dissolving the surfactants in aqueous mannitol solution under gentle stirring. One hundred milligrams of CsA was dissolved in 10 mL of each SSS by gentle stirring. The pH was adjusted to 7.4, and the SSSs were stored at 4°C until use.
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Micelle Size and Zeta-Potential Measurements The particle size and the polydispersity index, a measure of the size distribution of the micelles, were measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern, Herrenberg, Germany). The sample volume in the polystyrene semimicrocuvettes was 400 mL. The zeta potential was measured in folded capillary cells (Malvern, Herrenberg, Germany) with a sample volume of about 1 mL by laser Doppler electrophoresis using a Zetasizer Nano ZS.
Micelle Characterization by Transmission Electron Microscopy The micelle size and shape of a drug-free 5% ACS aqueous solution were visualized by transmission electron microscopy (TEM) using a negative staining technique. Two microliters of the sample solution was dried on the surface of carbon-coated copper grids (400 mesh; Plano GmbH, Wetzlar, Germany). The sample was then stained using a 2% (wt/vol) uranyl acetate aqueous solution. Images were recorded using a Philips CM12 transmission electron microscope (FEI Electron Optics, Eindhoven, Netherlands) equipped with a slow scan charge-coupled device (CCD) camera TEM1000 (TVIPS, Tietz GmbH, Gauting, Germany). Average particle sizes were determined by measuring 100 micelles using ImageJ software (National Institutes of Health).
In Situ Model for Corneal Drug Resorption The experiments were conducted as described by Luschmann et al.21a Briefly, enucleated pig eyes were supplied from a local abattoir and stored in a cooled (2–8°C) isotonic, sterile sodium chloride solution until use. All eyes were used at the same timepoint within 2 hours after enucleation to avoid differences in cell hydration. An eye was placed in the custom-made resorption chamber and fixed. Two hundred microliters of the solutions was applied on the cornea. After incubation for 30 minutes at room temperature, each eye was washed 4 times with 1 mL of aqueous 5.2% (wt/vol) mannitol solution. The corneas were excised using a surgical knife and homogenized in a tissue pulverizer (University workshop; analog to a Bessman Tissue Pulverizer) and liquid nitrogen. To extract CsA, the tissue pieces were incubated for 2 hours under gentle stirring with 1 mL MeOH, and spiked with 100 ng/ mL of cyclosporin D as an internal standard. After the extraction was performed, the suspension was centrifuged at 16,000g for 15 minutes. Five hundred microliters of the supernatant was transferred to HPLC vials and stored at 280°C until further use. All the samples were prepared and measured in triplicate.
Chromatographic Method and Equipment The ultra-HPLC–mass spectroscopy (UHPLC–MS) analyses were performed as described by Luschmann et al.21a Briefly, an Agilent Technologies UHPLC system equipped with a 6540 quadrupole time of flight LC/MS 2013 Lippincott Williams & Wilkins
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A Self-Assembling Colloidal System
system (Agilent Technologies Deutschland GmbH, Böblingen, Germany) was used. Separation was performed on a Waters Acquity BEH C18 column (1.7 mm, 2.1 · 50 mm). The mobile phase consisted of 40% (A) H2O with 0.1% formic acid and 60% (B) MeOH with 0.1% formic acid. A gradient was applied for 3 minutes to concentrations of 0% (A) and 100% (B). Afterward, a column reequilibration was performed for 3 minutes. The obtained data were analyzed using the t test at a significance level of P , 0.05 using SigmaPlot 11.0.
RESULTS AND DISCUSSION Entrapment Efficiency at Therapeutic Dosing
We first evaluated the efficiency by which CsA could be accumulated in aqueous solutions by means of encapsulation efficiency. The surfactant concentrations for CsA solutions ranged from 0.1% to 1.0% (wt/vol; Fig. 1) and the initial drug amount was set to 0.1% (wt/vol) to be comparable with Restasis (0.05% CsA).22 With at least 0.3% (wt/vol) and 0.5% (wt/vol) of AS and ACS, respectively, the encapsulation efficiency was 100% of the initial amount of the drug, but even at 0.3% (wt/vol) of ACS, it was still 91% 6 7.5%. Therefore, to prepare a formulation with 0.05% (wt/vol) CsA, the amount of surfactant could be reduced to 0.3% (wt/vol). In contrast, to prepare a 0.066% CsA solution, Di Tommaso et al12 needed 3.0% methoxy poly(ethylene glycol)-poly(hexyl-lactide) copolymer.
Micelle Characterization—Micelle Size and Charge Because of their great importance for the planned tissue intrusion, micelle size and zeta potential were determined. All micelles showed very small and uniform particle sizes (Fig. 2). Sympatens AS and ACS formed micelles of around 10 nm, and Solutol formed micelles of about 12 nm. It has been reported in the literature for other drug-loaded polymeric micelles that micelle size was nearly unaltered after drug loading occurred.23,24 CsA-loaded micelles, however, irrespective of the surfactant, were smaller than the drug-free
FIGURE 2. Micelle characterization: size, size distribution [polydispersity index (PdI) ( )] and Zeta potential of drugloaded and drug-free micellar systems of Sympatens AS ( ), Sympatens ACS ( ) and Solutol ( ); the drug loading was 1% (wt/vol) CsA.
control. All preparations had a narrow size distribution, with a polydispersity index (PdI) ,0.16 and a nearly neutral zeta potential with no significant differences between the drug-free and drug-loaded micelles. Although this indicates that the particles would not be able to stick to the negatively charged tissue surface via electrostatic interactions,25 this may be a major advantage over positively charged colloidal particles in terms of biocompatibility.14,26
Micelle Characterization—Micelle Shape and Distribution To visualize the micellar structures, and to confirm the results determined by dynamic light-scattering measurements, TEM photographs were taken of a 5% Sympatens AS aqueous solution (Figs. 3A, B). A homogeneous distribution of the colloidal structures with a size of 9.2 6 1.2 nm could be observed. The micelles were nearly spherical. These results agree well with the sizes determined by dynamic light scattering and the data presented by Di Tommaso et al.27
Drug Resorption In Situ
FIGURE 1. Encapsulation efficacy: encapsulation of an initial amount of 0.1% (wt/vol) CsA by micellar solutions of Sympatens AS ( ) and ACS ( ) at different concentrations. 2013 Lippincott Williams & Wilkins
To explore the potential of self-assembling colloidal systems to serve as a therapeutic formulation for the cornea, we investigated the CsA uptake from a 0.05% CsA micellar solution in porcine corneal tissue. We compared the micellar CsA solution with Restasis and with a 2% CsA olive oil solution (Fig. 4). The average www.corneajrnl.com |
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FIGURE 3. Micelle characterization: (A) + (B) TEM images for a 5% Sympatens ACS solution in water; (A) overview of the homogeneous preparation; (B) single micellar structures.
drug concentration in the cornea for the micellar CsA solution was 1557 6 407 ngCsA/gcornea. It was significantly higher than that reached with Restasis (545 6 137 ngCsA/gcornea; n = 3; P , 0.01) or the 2% CsA oily solution (452 6 142 ngCsA/gcornea; n = 3; P , 0.01). These values agree well with literature data. Daull et al25 observed comparable levels 20 minutes after a single administration of a cationic 0.05% CsA nanoemulsion and Restasis in rabbits. Even though the volume of 200 mL, which was needed in our study to ensure a complete wetting of the porcine cornea, was well above the volume of a typical eye drop (25–50 mL), the in situ model seemed to be quite suitable to compare the drug uptake from different types of formulations. The 2% CsA oily solution did not show any significant differences compared with that of Restasis. Despite its high drug loading, which was 40-fold higher than our micellar CsA solution and Restasis, the oily
FIGURE 4. In situ resorption of CsA by porcine cornea from 200 mL of sample solution over a period of 30 minutes. A micellar solution, Restasis and an olive oil solution were compared. Measurements were performed in triplicate, and the results are shown as average 6 SD [**P , 0.01 (t test); n.s, not significant].
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solution showed the lowest tissue levels. This seems to be caused by the expected partition of CsA, which is known to have a higher affinity for lipophilic vehicles than to the hydrophilic milieu of the cornea.11 In accordance with the therapeutic tissue levels of CsA reported by Kaswan et al (50–300 ng/gtissue)16 and the results of this in vitro investigation, the 0.05% micellar CsA solution developed by our group should easily be able to generate therapeutic levels in the cornea, which are significantly higher than those of Restasis. In comparison with the tissue levels reported by Daull et al25 and Di Tommaso et al,21,22 it would be possible to lower the number of applications per day compared with that of Restasis, which should be administered at least twice a day. Thus, the 0.05% micellar CsA solution would further enhance patient compliance. In summary, our self-assembling micellar solution is a highly promising approach to deliver high CsA concentrations to the corneal tissue. Only small amounts of the nonionic surfactants were necessary to create a drug loading at therapeutic dosage. The tremendous advantages of the small sized and neutrally charged micelles were their selfassembly and the drug loading directly in the aqueous medium by the use of only gentle stirring. In conclusion, we have shown a promising, simple, and effective approach for the application of an aqueous dispersion of cyclosporine A to treat inflammatory corneal diseases.
ACKNOWLEDGMENTS The authors thank the Bavarian Research Foundation (BFS) for financial support (BFS AZ 870-90). The authors are grateful to Elfriede Eckert (University Hospital Regensburg) for her help in the cellular tolerability studies, to Prof. Dr Rainhard Rachel (University of Regensburg) for his assistance with TEM and to Angelika Berié (University of Regensburg) for her help with the sample preparation for the TEM investigations. The authors also thank Mr Josef Kiermaier (University of Regensburg) for his support with UHPLC–MS analysis and Prof. Dr Frieder Kees (University of Regensburg) for his advice on tissue homogenization. 2013 Lippincott Williams & Wilkins
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REFERENCES 1. Avunduk AM, Avunduk MC, Varnell ED, et al. The comparison of efficacies of topical corticosteroids and nonsteroidal anti-inflammatory drops on dry eye patients: a clinical and immunocytochemical study. Am J Ophthalmol. 2003;136:593–602. 2. Kassem MA, Abdel Rahman AA, Ghorab MM, et al. Nanosuspension as an ophthalmic delivery system for certain glucocorticoid drugs. Int J Pharm. 2007;340:126–133. 3. Lee HK, Ryu IH, Seo KY, et al. Topical 0.1% prednisolone lowers nerve growth factor expression in keratoconjunctivitis sicca patients. Ophthalmology. 2006;113:198–205. 4. Waldrep J, Arppe J, Jansa K, et al. High dose cyclosporin A and budesonide–liposome aerosols. Int J Pharm. 1997;152:27–36. 5. Deleted in proof. 6. Nussenblatt RB, Palestine AG. Cyclosporine: immunology, pharmacology and therapeutic uses. Surv Ophthalmol. 1986;31:159–169. 7. Hoffmann F, Wiederholt M. Local treatment of necrotizing scleritis with cyclosporin A. Cornea. 1985–1986;4:3–7. 8. Witte A, Landgraf R, Markl A, et al. Treatment of Graves’ ophthalmopathy with cyclosporin A. Klin Wochenschr. 1985;63:1000–1004. 9. Lallemand F, Felt-Baeyens O, Besseghir K, et al. Cyclosporine A delivery to the eye: a pharmaceutical challenge. Eur J Pharm Biopharm. 2003;56: 307–318. 10. Mithani SD, Bakatselou V, TenHoor CN, et al. Estimation of the increase in solubility of drugs as a function of bile salt concentration. Pharm Res. 1996;13:163–167. 11. el Tayar N, Mark AE, Vallat P, et al. Solvent-dependent conformation and hydrogen-bonding capacity of cyclosporin A: evidence from partition coefficients and molecular dynamics simulations. J Med Chem. 1993;36:3757–3764. 12. Di Tommaso C, Bourges JL, Valamanesh F, et al. Novel micelle carriers for cyclosporin A topical ocular delivery: in vivo cornea penetration, ocular distribution and efficacy studies. Eur J Pharm Biopharm. 2012; 81:257–264. 13. Kuwano M, Ibuki H, Morikawa N, et al. Cyclosporine A formulation affects its ocular distribution in rabbits. Pharm Res. 2002;19:108–111. 14. Milani JK, Pleyer U, Dukes A, et al. Prolongation of corneal allograft survival with liposome-encapsulated cyclosporine in the rat eye. Ophthalmology. 1993;100:890–896.
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15. Calvo P, Sanchez A, Martinez J, et al. Polyester nanocapsules as new topical ocular delivery systems for cyclosporin A. Pharm Res. 1996;13:311–315. 16. Kaswan RL. Intraocular penetration of topically applied cyclosporine. Transplant Proc. 1988;20:650–655. 17. Le Bourlais CA, Chevanne F, Turlin B, et al. Effect of cyclosporine A formulations on bovine corneal absorption: ex-vivo study. J Microencapsul. 1997;14:457–467. 18. Di Tommaso C, Behar-Cohen F, Gurny R, et al. Colloidal systems for the delivery of cyclosporin A to the anterior segment of the eye. Ann Pharm Fr. 2011;69:116–123. 19. BenEzra D, Maftzir G. Ocular penetration of cyclosporin A. The rabbit eye. Invest Ophthalmol Vis Sci. 1990;31:1362–1366. 20. Holland EJ, Olsen TW, Ketcham JM, et al. Topical cyclosporin A in the treatment of anterior segment inflammatory disease. Cornea. 1993;12: 413–419. 21. Di Tommaso C, Valamanesh F, Miller F, et al. A novel cyclosporin a aqueous formulation for dry eye treatment: in vitro and in vivo evaluation. Invest Ophthalmol Vis Sci. 2012;53:2292–2299. 21a. Luschmann C, Tessmar J, Schoeberl S, et al. Developing an in situ nanosuspension: A novel approach towards the efficient administration of poorly soluble drugs at the anterior eye, Eur J Pharm Sci. 2013;50:385–392. 22. Restasis (cyclosporine ophthalmic emulsion) 0.05% [package insert]. Irvine, CA: Allergan, Inc; 2010. 23. Elsabahy M, Perron ME, Bertrand N, et al. Solubilization of docetaxel in poly(ethylene oxide)-block-poly(butylene/styrene oxide) micelles. Biomacromolecules. 2007;8:2250–2257. 24. Mondon K, Zeisser-Labouèbe M, Gurny R, et al. Novel cyclosporin A formulations using MPEG–hexyl-substituted polylactide micelles: a suitability study. Eur J Pharm Biopharm. 2011;77:56–65. 25. Daull P, Lallemand F, Philips B, et al. Distribution of cyclosporine A in ocular tissues after topical administration of cyclosporine A cationic emulsions to pigmented rabbits. Cornea. 2013;32:345–354. 26. Yavuz B, Bozdag Pehlivan S, Unlü N. An overview on dry eye treatment: approaches for cyclosporin a delivery. Scientific World Journal. 2012;2012:194848. 27. Di Tommaso C, Torriglia A, Furrer P, et al. Ocular biocompatibility of novel cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers. Int J Pharm. 2011;416:515–524.
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Self-Assembling Colloidal System for the Ocular Administration of Cyclosporine A Christoph Luschmann, PhD,* Joerg Tessmar, PhD,† Simon Schoeberl,‡ Olaf Strauß, PhD,‡§ Karl Luschmann, PhD,¶ and Achim Goepferich, PhD*
Purpose: In this study, we developed a self-assembling micellar system to deliver cyclosporine A (CsA) in an aqueous solution to the cornea.
Methods: Two nonionic surfactants of the poly(ethylene glycol)fatty alcohol ether type (Sympatens AS and Sympatens ACS) were characterized in terms of micelle size, shape, and charge, and their encapsulation efficiency for CsA. In an in situ single dose bioavailability study, the corneal CsA levels were determined in an enucleated porcine eye model. A commercial formulation and a 2% CsA olive oil solution served as references. Results: Both surfactants formed spherical micelles with a size of 9 to 12 nm in water. A concentration as low as 0.3% (wt/vol) Sympatens AS was sufficient to entrap therapeutic levels of at least 0.1% (wt/vol) CsA. In the porcine in situ model, exceptionally high drug levels in the cornea were obtained for the micellar CsA solution (1557 6 407 ngCsA/gcornea). They were significantly higher than those of Restasis (545 6 137 ngCsA/gcornea) or the olive oil solution (452 6 142 ngCsA/gcornea). Conclusions: In conclusion, we have shown a promising simple and efficient approach for the application of CsA in an aqueous solution to the cornea to treat inflammatory corneal diseases. Key Words: surfactant micelles, cyclosporin A, poor solubility, dry eye disease, bioavailability (Cornea 2014;33:77–81)
L
ocal therapy for the anterior eye and, therefore, the delivery of drugs to the cornea for the treatment of inflammatory
Received for publication June 5, 2013; revision received July 29, 2013; accepted August 2, 2013. Published online ahead of print October 24, 2013. From the *Department of Pharmaceutical Technology, University of Regensburg, Regensburg, Germany; †Department for Functional Materials in Medicine and Dentistry, University of Wuerzburg, Wuerzburg, Germany; ‡Department of Experimental Ophthalmology, University Hospital Regensburg, Regensburg, Germany; §Experimental Ophthalmology, Department Ophthalmology, Charité University Medicine Berlin, Berlin, Germany; and ¶Pharma Stulln GmbH, Stulln, Germany. Supported by Bavarian Research Foundation (BFS AZ 870-90). K. Luschmann is the director of Pharma Stulln GmbH. The other authors have no other funding or conflicts of interest to disclose. Reprints: Achim Goepferich, Department of Pharmaceutical Technology, University of Regensburg, Regensburg 93040, Germany (e-mail: achim. [email protected]). Copyright © 2013 by Lippincott Williams & Wilkins
Cornea Volume 33, Number 1, January 2014
corneal diseases is still a highly unmet need. Contemporary research focuses on increasing the bioavailability and the therapeutic efficacy of drugs and on avoiding their systemic side effects.1–4 An example of a highly potent antiinflammatory drug is cyclosporine A (CsA). CsA is successfully used in the treatment of uveitis,6 necrotizing scleritis,7 and thyroid ophthalmopathy8 and is one of the most promising substances for the treatment of dry eye disease.9 Unfortunately, it has a poor solubility in aqueous media (CsA, 6.6 mg/mL in water10) and a high octanol–water coefficient of log P = 3.11 Many different options, with a main focus on colloidal systems, such as micelles,12,13 liposomes,14 or various nanoparticular formulations,15–17 have been investigated to solubilize CsA. A cationic nanoemulsion is currently in phase 3 clinical trials and Restasis (Allergan), a 0.05% CsA microemulsion, is the only approved product, but it is available only on the US market.18 Hence, the dry eye syndrome is typically treated with oily 2% CsA eye drops that use vegetable oils as solvents.19,20 Unfortunately, all systems containing an oily phase usually cause side effects such as a burning, stinging sensation and blurred vision. Therefore, patient compliance is very low.21 Thus, there is still a tremendous need for a simple, yet efficient aqueous colloidal system, which does not need an oil phase. Ideally, it should be made of inexpensive surfactants, should self-assemble in the presence of water and drug, and should solubilize large amounts of the drug. Such a formulation would lead to a tremendous increase in patient compliance and would be a major option in the causal treatment of various diseases of the cornea. The aim of our study was to develop such a selfassembling micellar system. We were particularly interested in the CsA uptake into corneal tissue in a porcine in situ model.
MATERIALS AND METHODS Materials CsA was supplied by the Pharma Stulln GmbH (Stulln, Germany). Cyclosporin D was a generous gift from Prof. Dr F. Kees (University of Regensburg, Germany). Sympatens AS/200 G (AS) and Sympatens ACS/200 G (ACS) were provided by KOLB (Hedingen, Switzerland). Solutol HS 15 was a generous gift of BASF (Ludwigshafen, Germany). Mannitol was purchased from Caesar & Loretz GmbH (Hilden, Germany). Deionized water was obtained from a Milli-Q water purification system from Millipore (Schwalbach, Germany). www.corneajrnl.com |
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Methanol (MeOH), acetonitrile (MeCN), isopropanol [all high-performance liquid chromatography (HPLC) grade] and chloroform p.a. were purchased from Merck (Darmstadt, Germany). Hydrochloric acid (0.1 N) and sodium hydroxide (0.1 N) solutions were obtained from Carl Roth (Karlsruhe, Germany).
Entrapment Efficiency Four different surfactant concentrations were prepared to determine their entrapment efficiency of 0.1% drug. To solubilize CsA, aqueous solutions of 1%, 0.7%, 0.3%, and 0.1% (wt/vol) AS and ACS were prepared by gentle stirring. One milliliter of each surfactant concentration was spiked with 1 mg of CsA, and stirred for 24 hours at room temperature (n = 3). The dispersions were centrifuged at 16,000g for 10 minutes in Eppendorf tubes. One hundred microliters of the supernatant was diluted with 900 mL of MeOH in HPLC vials (2-mL Screw top vial; BGB Analytik AG, Rheinfelden, Germany). The samples were stored at 280°C (HERAfreeze; Thermo Fisher Scientific GmbH, Ulm, Germany) until further analysis was carried out. For the CsA analysis, a Shimadzu chromatographic setup consisting of an SCL-10AVP controller, an LC-10ATVP pump at a flow rate of 1 mL/min, an SIL-10ADVP autoinjector with a sample volume of 15 mL, a CTO-10ASVP oven with a temperature of 75°C and an SPD-10AVP ultraviolet detector at a wavelength of 210 nm were used (all from Shimadzu Deutschland GmbH, Duisburg, Germany). A reversed-phase octadecyl column Luna 3 mm C18(2) 100A with 100 · 4.6 mm served as the stationary phase. The mobile phase consisted of 75% MeCN and 25% H2O and was linearly changed to 90% MeCN and 10% H2O over 5 minutes. These concentrations were kept for 12.5 minutes, and then, the column was reequilibrated to the initial conditions until the end of the run at 15 minutes. The entrapment efficiency E was calculated as follows: cn1 cn2 cn3 þ þ co1 co2 co3 · 100; E½% ¼ 3 where cn was the concentration determined in the sample, which was divided by co, the initial concentration of 0.1% drug for each of the 3 measurements. The average was calculated by dividing by n = 3 and finally, the relative entrapment efficacy E was obtained by multiplication by 100.
Drug-Loaded Micelle Preparation One hundred milliliters of an isotonic 5.2% (wt/vol) aqueous mannitol solution was prepared by gentle stirring. Thirty milliliters of surfactant stock solutions (SSSs) with concentrations of 5% (wt/vol) AS, 5% (wt/vol) ACS, and 10% (wt/vol) Solutol, respectively, were prepared by dissolving the surfactants in aqueous mannitol solution under gentle stirring. One hundred milligrams of CsA was dissolved in 10 mL of each SSS by gentle stirring. The pH was adjusted to 7.4, and the SSSs were stored at 4°C until use.
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Micelle Size and Zeta-Potential Measurements The particle size and the polydispersity index, a measure of the size distribution of the micelles, were measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern, Herrenberg, Germany). The sample volume in the polystyrene semimicrocuvettes was 400 mL. The zeta potential was measured in folded capillary cells (Malvern, Herrenberg, Germany) with a sample volume of about 1 mL by laser Doppler electrophoresis using a Zetasizer Nano ZS.
Micelle Characterization by Transmission Electron Microscopy The micelle size and shape of a drug-free 5% ACS aqueous solution were visualized by transmission electron microscopy (TEM) using a negative staining technique. Two microliters of the sample solution was dried on the surface of carbon-coated copper grids (400 mesh; Plano GmbH, Wetzlar, Germany). The sample was then stained using a 2% (wt/vol) uranyl acetate aqueous solution. Images were recorded using a Philips CM12 transmission electron microscope (FEI Electron Optics, Eindhoven, Netherlands) equipped with a slow scan charge-coupled device (CCD) camera TEM1000 (TVIPS, Tietz GmbH, Gauting, Germany). Average particle sizes were determined by measuring 100 micelles using ImageJ software (National Institutes of Health).
In Situ Model for Corneal Drug Resorption The experiments were conducted as described by Luschmann et al.21a Briefly, enucleated pig eyes were supplied from a local abattoir and stored in a cooled (2–8°C) isotonic, sterile sodium chloride solution until use. All eyes were used at the same timepoint within 2 hours after enucleation to avoid differences in cell hydration. An eye was placed in the custom-made resorption chamber and fixed. Two hundred microliters of the solutions was applied on the cornea. After incubation for 30 minutes at room temperature, each eye was washed 4 times with 1 mL of aqueous 5.2% (wt/vol) mannitol solution. The corneas were excised using a surgical knife and homogenized in a tissue pulverizer (University workshop; analog to a Bessman Tissue Pulverizer) and liquid nitrogen. To extract CsA, the tissue pieces were incubated for 2 hours under gentle stirring with 1 mL MeOH, and spiked with 100 ng/ mL of cyclosporin D as an internal standard. After the extraction was performed, the suspension was centrifuged at 16,000g for 15 minutes. Five hundred microliters of the supernatant was transferred to HPLC vials and stored at 280°C until further use. All the samples were prepared and measured in triplicate.
Chromatographic Method and Equipment The ultra-HPLC–mass spectroscopy (UHPLC–MS) analyses were performed as described by Luschmann et al.21a Briefly, an Agilent Technologies UHPLC system equipped with a 6540 quadrupole time of flight LC/MS 2013 Lippincott Williams & Wilkins
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system (Agilent Technologies Deutschland GmbH, Böblingen, Germany) was used. Separation was performed on a Waters Acquity BEH C18 column (1.7 mm, 2.1 · 50 mm). The mobile phase consisted of 40% (A) H2O with 0.1% formic acid and 60% (B) MeOH with 0.1% formic acid. A gradient was applied for 3 minutes to concentrations of 0% (A) and 100% (B). Afterward, a column reequilibration was performed for 3 minutes. The obtained data were analyzed using the t test at a significance level of P , 0.05 using SigmaPlot 11.0.
RESULTS AND DISCUSSION Entrapment Efficiency at Therapeutic Dosing
We first evaluated the efficiency by which CsA could be accumulated in aqueous solutions by means of encapsulation efficiency. The surfactant concentrations for CsA solutions ranged from 0.1% to 1.0% (wt/vol; Fig. 1) and the initial drug amount was set to 0.1% (wt/vol) to be comparable with Restasis (0.05% CsA).22 With at least 0.3% (wt/vol) and 0.5% (wt/vol) of AS and ACS, respectively, the encapsulation efficiency was 100% of the initial amount of the drug, but even at 0.3% (wt/vol) of ACS, it was still 91% 6 7.5%. Therefore, to prepare a formulation with 0.05% (wt/vol) CsA, the amount of surfactant could be reduced to 0.3% (wt/vol). In contrast, to prepare a 0.066% CsA solution, Di Tommaso et al12 needed 3.0% methoxy poly(ethylene glycol)-poly(hexyl-lactide) copolymer.
Micelle Characterization—Micelle Size and Charge Because of their great importance for the planned tissue intrusion, micelle size and zeta potential were determined. All micelles showed very small and uniform particle sizes (Fig. 2). Sympatens AS and ACS formed micelles of around 10 nm, and Solutol formed micelles of about 12 nm. It has been reported in the literature for other drug-loaded polymeric micelles that micelle size was nearly unaltered after drug loading occurred.23,24 CsA-loaded micelles, however, irrespective of the surfactant, were smaller than the drug-free
FIGURE 2. Micelle characterization: size, size distribution [polydispersity index (PdI) ( )] and Zeta potential of drugloaded and drug-free micellar systems of Sympatens AS ( ), Sympatens ACS ( ) and Solutol ( ); the drug loading was 1% (wt/vol) CsA.
control. All preparations had a narrow size distribution, with a polydispersity index (PdI) ,0.16 and a nearly neutral zeta potential with no significant differences between the drug-free and drug-loaded micelles. Although this indicates that the particles would not be able to stick to the negatively charged tissue surface via electrostatic interactions,25 this may be a major advantage over positively charged colloidal particles in terms of biocompatibility.14,26
Micelle Characterization—Micelle Shape and Distribution To visualize the micellar structures, and to confirm the results determined by dynamic light-scattering measurements, TEM photographs were taken of a 5% Sympatens AS aqueous solution (Figs. 3A, B). A homogeneous distribution of the colloidal structures with a size of 9.2 6 1.2 nm could be observed. The micelles were nearly spherical. These results agree well with the sizes determined by dynamic light scattering and the data presented by Di Tommaso et al.27
Drug Resorption In Situ
FIGURE 1. Encapsulation efficacy: encapsulation of an initial amount of 0.1% (wt/vol) CsA by micellar solutions of Sympatens AS ( ) and ACS ( ) at different concentrations. 2013 Lippincott Williams & Wilkins
To explore the potential of self-assembling colloidal systems to serve as a therapeutic formulation for the cornea, we investigated the CsA uptake from a 0.05% CsA micellar solution in porcine corneal tissue. We compared the micellar CsA solution with Restasis and with a 2% CsA olive oil solution (Fig. 4). The average www.corneajrnl.com |
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FIGURE 3. Micelle characterization: (A) + (B) TEM images for a 5% Sympatens ACS solution in water; (A) overview of the homogeneous preparation; (B) single micellar structures.
drug concentration in the cornea for the micellar CsA solution was 1557 6 407 ngCsA/gcornea. It was significantly higher than that reached with Restasis (545 6 137 ngCsA/gcornea; n = 3; P , 0.01) or the 2% CsA oily solution (452 6 142 ngCsA/gcornea; n = 3; P , 0.01). These values agree well with literature data. Daull et al25 observed comparable levels 20 minutes after a single administration of a cationic 0.05% CsA nanoemulsion and Restasis in rabbits. Even though the volume of 200 mL, which was needed in our study to ensure a complete wetting of the porcine cornea, was well above the volume of a typical eye drop (25–50 mL), the in situ model seemed to be quite suitable to compare the drug uptake from different types of formulations. The 2% CsA oily solution did not show any significant differences compared with that of Restasis. Despite its high drug loading, which was 40-fold higher than our micellar CsA solution and Restasis, the oily
FIGURE 4. In situ resorption of CsA by porcine cornea from 200 mL of sample solution over a period of 30 minutes. A micellar solution, Restasis and an olive oil solution were compared. Measurements were performed in triplicate, and the results are shown as average 6 SD [**P , 0.01 (t test); n.s, not significant].
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solution showed the lowest tissue levels. This seems to be caused by the expected partition of CsA, which is known to have a higher affinity for lipophilic vehicles than to the hydrophilic milieu of the cornea.11 In accordance with the therapeutic tissue levels of CsA reported by Kaswan et al (50–300 ng/gtissue)16 and the results of this in vitro investigation, the 0.05% micellar CsA solution developed by our group should easily be able to generate therapeutic levels in the cornea, which are significantly higher than those of Restasis. In comparison with the tissue levels reported by Daull et al25 and Di Tommaso et al,21,22 it would be possible to lower the number of applications per day compared with that of Restasis, which should be administered at least twice a day. Thus, the 0.05% micellar CsA solution would further enhance patient compliance. In summary, our self-assembling micellar solution is a highly promising approach to deliver high CsA concentrations to the corneal tissue. Only small amounts of the nonionic surfactants were necessary to create a drug loading at therapeutic dosage. The tremendous advantages of the small sized and neutrally charged micelles were their selfassembly and the drug loading directly in the aqueous medium by the use of only gentle stirring. In conclusion, we have shown a promising, simple, and effective approach for the application of an aqueous dispersion of cyclosporine A to treat inflammatory corneal diseases.
ACKNOWLEDGMENTS The authors thank the Bavarian Research Foundation (BFS) for financial support (BFS AZ 870-90). The authors are grateful to Elfriede Eckert (University Hospital Regensburg) for her help in the cellular tolerability studies, to Prof. Dr Rainhard Rachel (University of Regensburg) for his assistance with TEM and to Angelika Berié (University of Regensburg) for her help with the sample preparation for the TEM investigations. The authors also thank Mr Josef Kiermaier (University of Regensburg) for his support with UHPLC–MS analysis and Prof. Dr Frieder Kees (University of Regensburg) for his advice on tissue homogenization. 2013 Lippincott Williams & Wilkins
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