|Year : 2011 | Volume
| Issue : 1 | Page : 24-27
EDTA decreases in vitro transcorneal permeation of fluconazole in phosphate buffer through excised sheep cornea
Sunil Thakral1, Munish Ahuja2
1 Gurukul College of Pharmacy, Suratgarh, Rajasthan, India
2 Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, India
|Date of Web Publication||15-Jul-2011|
5-M-24, Jawahar Nagar, Sri Ganganagar, Rajasthan
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: According to the World Health Organization, corneal diseases are a major cause of vision loss and blindness, second only to cataract in overall importance. Fungal keratitis is a major blinding eye disease in Asia. In epithelia, calcium has been implicated in the maintenance of intercellular matrix and therefore may be a key factor determining the size of potential paracellular routes for drug transport. Although the effects of chelating agents such as EDTA on the permeability of inorganic and organic solutes have been well documented in other epithelia, as well as the corneal endothelium, no definitive studies examining the effects of these compounds upon corneal epithelia have been reported. Materials and Methods: The corneal permeation studies were conducted using freshly excised sheep cornea, mounted between donor and receptor chambers of an all glass-modified Franz diffusion cell, containing 11 ml of ringer bicarbonate (pH 7.4, 34 o±1 o C). At the end of the experiment, each cornea (freed from sclera) was weighed, soaked in 1 ml of methanol, dried overnight at 90C and reweighed. From the difference in weights corneal hydration was calculated Results: Fluconazole ophthalmic solutions (0.2% w/v, pH 6.0) containing EDTA shows significant difference in P app 1.51×10 6 (cm/s) as compared to fluconazole ophthalmic solutions (0.2% w/v, pH 6.0) without EDTA showing 2.37×10 6 (cm/s). Conclusions: Use of ethylene diamine tetraacetate as chelating agent in fluconazole ophthalmic solutions significantly decreased the corneal permeability of fluconazole.
Keywords: Fungal keratitis, franz-diffusion cell, ocular, corneal permeability
|How to cite this article:|
Thakral S, Ahuja M. EDTA decreases in vitro transcorneal permeation of fluconazole in phosphate buffer through excised sheep cornea. J Pharm Negative Results 2011;2:24-7
|How to cite this URL:|
Thakral S, Ahuja M. EDTA decreases in vitro transcorneal permeation of fluconazole in phosphate buffer through excised sheep cornea. J Pharm Negative Results [serial online] 2011 [cited 2020 Feb 18];2:24-7. Available from: http://www.pnrjournal.com/text.asp?2011/2/1/24/82986
| Introduction|| |
Fungal eye infections are rare.  The number of fungal infections has increased dramatically, and those involving the eye pose a serious problem and treatment challenge to practicing physicians.  Fungal keratitis is a major blinding eye disease in Asia.  Fungal keratitis is a serious and painful corneal inflammation that results from infection by a fungal organism.  The symptoms of fungal keratitis are blurred vision; a red and painful eye that does not improve when contact lenses are removed, increased sensitivity to light, and excessive tearing or discharge. 
Ocular conditions are usually treated by topical administration of drug solutions administered as eye drops into the lower cul-de-sac. These conventional dosage forms account for around 90% of the available ophthalmic formulations, mainly due to their simplicity and convenience. 
Drugs are commonly applied to the eye for a localized action. A major problem in ocular therapeutics is the attainment of an optimal drug conc. at the site of action. Poor bioavailability of drugs from ocular dosage forms is due to the precorneal loss factors, physiological and anatomical constraints.  Consequently, after instillation of eye drops, typically less than 5% of an applied dose reaches the intraocular tissues.  This forces the clinician to recommend a frequent dosing at an extremely high conc., and pulse type dosing results in several side effects of ophthalmic products. 
The last three decades have witnessed continued efforts aimed at improving the topical bioavailability of ophthalmic drugs. Investigations are being pursued along the following main lines:
- Prolongation of the ocular residence time of the medication (vehicle approach, muco-adhesives);
- Increase of the drug penetration characteristic (prodrug approach); and
- Enhancement of the corneal permeability (enhancer approach).
The last approach, which consists of increasing transitorily the permeability characteristics of the cornea with appropriate substances, known as penetration enhancers or absorption promoters, bears a strict analogy with techniques aimed at facilitating drug penetration through the skin and different epithelia (buccal, nasal, intestinal, rectal etc.). However, the unique characteristic and great sensitivity of the corneal/conjunctival tissues impose great caution in the selection of enhancers with regard to consideration to their capacity to affect the integrity of epithelial surfaces. 
The synthetic bis-triazole antifungal compound fluconazole exhibits outstanding physical and pharmacokinetic properties. Fluconazole is a stable, water-soluble, bis-triazole antifungal that has low molecular weight, high bioavailability, good ocular penetration when used either systemically or topically, and low toxicity. It is potentially useful as a topical ocular agent. It is quite effective against Candida species. 
Fungal cell membrane synthesis is a multi-step process that involves the conversion of squalene to ergosterol. Fluconazole prevent the synthesis of ergosterol, a major component of fungal plasma membranes, by inhibiting the cytochrome P-450-dependent enzyme lanosterol demethylase (also referred to as 14 α-sterol demethylase or P-450DM). Exposure of fungi to an azole causes depletion of ergosterol and accumulation of 14 α-methylated sterols. This interferes with the "bulk" functions of ergosterol in fungal membranes and disrupts both the structure of the membranes and several of its functions such as nutrient transport and chitin synthesis. The net effect is to inhibit fungal growth. 
| Materials and Methods|| |
Fluconazole was obtained as a gift sample from Aurobindo Pharmaceuticals Limited Research Center, Mandal (A.P.). Sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium bicarbonate, sodium dihydrogen orthophosphate dextrose and ethylene diamine tetraacetate sodium were purchased from Qualigens Fine Chemicals (Mumbai, India). All other chemicals purchased were of analytical grade and were used as received. Fresh whole eye ball of sheep were obtained from local butcher shop (Hisar, India), within half an hour of slaughtering the animal. The apparatus used in permeation studies was same as published elsewhere. 
Preparations of test formulations
Fluconazole ophthalmic solutions (0.2% w/v, pH 6.0) containing EDTA
Fluconazole 0.2% w/v ophthalmic solution in 0.0667M phosphate buffer (pH-6.0) made isotonic with mannitol and containing 0.01% EDTA was prepared.
Fluconazole ophthalmic solutions (0.2% w/v, pH 6.0) without EDTA
Fluconazole 0.2% w/v ophthalmic solution in 0.0667M phosphate buffer (pH-6.0) made isotonic with mannitol was prepared.
| In Vitro Transcorneal Permeation Study|| |
Whole eye ball of the sheep was obtained from local butcher shop within half an hour of slaughtering the animal, and was transported to the laboratory in cold (4°C) normal saline (0.9%) immediately. The corneal was carefully excised along with 2-4 mm of surrounding scleral tissues and washed with cold normal saline till free from proteins.
Fresh cornea was mounted by sandwiching the surrounding scleral tissue between clamped donor and receptor cells of modified version of Franz diffusion cell in such a way that its epithelial surface (apical) faced the donor compartment and endothelial surface faced the receptor compartment. Cell was placed on magnetic stirrer in holding position. The receptor compartment was filled with 11 ml of freshly prepared bicarbonate ringer solution (pH 7.4) and stirred using Teflon-coated magnetic stir bar. Drug solution (1 ml) was placed on the epithelial side of cornea in donor cell and stirring of the receptor fluid (jacketed with water at 34±1°C) was started. At appropriate intervals, 2 ml samples were withdrawn from the receptor compartment and withdrawn sample volume was replaced with equal volume of fresh bicarbonate ringer solution to ensure sink conditions. Withdrawn samples were analyzed spectrophotometrically (Varian-Cary 5000 UV-VIS-NIR) by measuring absorbance at λmax of 260 nm. Each experiment was continued for about 2 h and was performed at least in triplicate.
At the end of the experiment, each cornea (freed from sclera) was weighed, soaked in 1 ml methanol, dried overnight at 90°C and reweighed. From the difference in weights, the corneal hydration was calculated.
Calculation of apparent permeability coefficient
The apparent permeability coefficient was calculated using the following equation:
where ΔQ/Δt (μg/min) is the flux across corneal tissue, A is the exposed surface area of corneal tissue (0.786 cm2 ), C 0 is the initial drug conc. (μg/ml) in the donor compartment and 60 is included to convert minutes to seconds. The flux across the cornea was determined from the slope of the regression line obtained from the linear part of the curve between the cumulative amount permeated (Q) vs. time (t) plot.
| Statistical Analysis|| |
Statistical calculation were done by one-way analysis of variance (ANOVA) followed by Dunnett's test. A P value <0.05 was considered significant.
| Results and Discussion|| |
The first barrier to intraocular entry via the corneal and non-corneal pathways is the cornea and conjunctiva, respectively. These epithelial tissues are known to have tight junctions that act as a barrier in the paracellular spaces. Because of this barrier structure in the epithelia, insufficient drug may be absorbed after instillation. Thus, subconjunctival and intravitreous injections are generally used in ocular pharmacotherapy. These invasive methods may not be acceptable to many patients and could potentially increase the risk of infection. In order to obtain a simpler and more acceptable form of application, the development of effective instilled formulations would be a major improvement. A number of ocular penetration enhancers, including calcium chelator (EDTA) and bile salts, have already been investigated, and it was found that those enhancers increased the apparent permeability coefficient (Papp) of FITC-Dextran (MW: 4000, FD-4) from 2.9 to 15.5-fold in excised cornea and conjunctiva.  Penetration of molecules through the cornea is mainly limited by the outermost epithelial cell layer containing tight junctions. Absorption enhancers increase transitorily the permeability characteristics of physiological membranes and are used to facilitate drug penetration through the skin, the cornea and different epithelia (buccal, nasal, intestinal, and rectal). The use of absorption promoters was thought to be helpful in the formulation of ophthalmic preparations to increase therapeutic action of a drug or achieve an equivalent effect with a lower concentration of the active ingredients. 
[Table 1] and [Figure 1] compare the effect of EDTA on corneal permeation of fluconazole. EDTA, a known calcium-chelating agent has been shown to act on cell junction by interfering with calcium ions and altering intracellular integrity. EDTA also disrupts plasma membrane and consequently increases intercellular permeability.  EDTA has been reported to increase corneal absorption of various drugs through intact corneas.  However in our study use of EDTA in the formulation caused a significant decrease in apparent corneal permeability of fluconazole.
|Figure 1: Effect of EDTA on in vitro transcorneal permeation of fluconazole|
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|Table 1: Comparative corneal permeation of fluconazole from 0.2% ophthalmic solutions containing EDTA in phosphate buffer (pH-6.0, 0.0667M)|
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| Conclusions|| |
Use of ethylene diamine tetraacetate as chelating agent in fluconazole ophthalmic solutions significantly decreased the corneal permeability of fluconazole.
| Acknowledgments|| |
The authors are grateful to Professors and Head of Department and Librarian of Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, for providing laboratory and library facility. Also, the authors wish to thank Gurukul College of Pharmacy, Suratgarh, for providing guidance and library facilities.
| References|| |
|1.||Manzouri B, Wyse RK, Vafidis GC. Pharmacotherapy of fungal eye infections. Expert Opin Pharmacother 2001;2:1849-57. |
|2.||Urbak SF, Degn T. Fluconazole in the management of fungal ocular infections. Ophthalmologica 1994;208:147-56. |
|3.||Srinivasan M. Fungal Keratitis. Curr Opin in Ophthalmol 2004;15:321-7. |
|4.||Available from: http://www.drug-attorneys.com/definitions/fungal-keratitis.cfm. |
|5.||Available from: http://en.wikipedia.org/wiki/Fungal_keratitis. Fungal Keratitis. |
|6.||Alany RG, Rades T, Nicoll J, Tucker IG, Davies NM. W/O microemulsions for ocular delivery: Evaluation of ocular irritation and precorneal retention. J Control Release 2006;111:145-52. |
|7.||Hamalainen KM, Ranta VP, Auriola S, Urtti A. Enzymatic and permeation barrier of [D-Ala2]-Met-enkephalinamide in the anterior membrane of the albino rabbit eye. Eur J Pharm Sci 2000;9:265-70. |
|8.||Jarvinen K, Jarvinen T, Urtti A. Ocular absorption following topical delivery. Adv Drug Deliv Rev 1995;16:3-19. |
|9.||Kaur IP, Garg A, Singla AK, Aggarwal D. Vesicular system in ocular drug delivery: An overview. Int J Pharm 2004;269:1-14. |
|10.||Saettone MF, Chetoni P, Cerbai R, Mazzanti G, Braghiroli L. Evaluation of ocular permeation enhancers: In vitro effects on corneal transport of four â-blockers, and in vitro / in vivo toxic activity. Int J Pharm 1996;142:103-13. |
|11.||Abbasoglu OE, Hosal BM, Sener B, Erdemoglu N, Gursel E. Penetration of Fluconazole into Human Aqueous Humor. Exp Eye Res 2001;72:147-51. |
|12.||Sheehan DJ, Hitchcock CA, Sibley CM. Current and Emerging Azole Antifungal Agents. Clin Microbiol Rev 1999;12:40-79. |
|13.||Malhotra M, Majumdar DK. In vitro transcorneal permeation of ketorlac tromethamine from buffered and unbuffered aqueous ocular drops. Indian J Exp Biol 1997;35:941-5. |
|14.||Kikuchi T, Suzuki M, Kusai A, Iseki K, Sasaki H, Nakashima K. Mechanism of permeability-enhancing effect of EDTA and boric acid on the corneal penetration of 4-[1-hydroxy-1-methylethyl]-2-propyl-1-[4-[2-[tetrazole-5-yl] phenyl] phenyl] methylimidazole-5-carboxylic acid monohydrate (CS-088). Int J Pharm 2005;299:107-14. |
|15.||Monti D, Chetoni P, Burgalassi S, Najarro M, Saettone MF. Increased corneal hydration indused by potential ocular penetration enhancers: Assessment by differential scanning calorimetry (DSC) and by desiccation. Int J Pharm 2002;232:139-47. |
|16.||Toropainen E, Ranta VP, Vellonen KS, Palmgren J, Talvitie A, Laavola M, et al. Paracellular and passive transcellular permeability in immortalized human corneal epithelial cell culture model. Eur J Pharm Sci 2003;20:99-106. |
|17.||Nemoto E, Takahashi H, Kobayashi D, Ueda H, Morimoto Y. Effects of Poly-L-arginine on the Permeation of Hydrophilic Compounds through Surface Ocular Tissues. Biol Pharm Bull 2006;29:155-60. |
|18.||Furrer P, Mayer JM, Plazonnet B, Gurny R. Ocular Tolerance of Absorption Enhancers in Ophthalmic Preparations. AAPS Pharm Sci 2002;4:1-5. |
|19.||Grass GM, Wood RW, Robinson JR. Effects of calcium chelating agents on corneal permeability. Invest Ophthalmol Vis Sci 1985;26:110-5. |
|20.||Rojanasakul Y, Liaw J, Robinson JR. Mechanism of some penetration enhancers in the cornea: Laser scanning confocal microscopic and electrophysiology. Int J Pharm 1990;66:131-6 |