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Year : 2006  |  Volume : 54  |  Issue : 4  |  Page : 237-240

Retinal toxicity of intravitreally injected plain and liposome formulation of fluconazole in rabbit eye

1 Ocular Pharmacology Division, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
2 Department of Pharmacology, All India Institute of Medical Sciences, New Delhi, India
3 Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India

Correspondence Address:
Thirumurthy Velpandian
Ocular Pharmacology Division, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0301-4738.27947

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Purpose: Candidal endophthalmitis is a sight-threatening ocular infection that most frequently occurs as a complication of candidemia. Fluconazole has been effective against Candida albicans in various animal models. Our objective was to evaluate retinal toxicity of plain and liposome formulation of fluconazole at various dose levels after intravitreal injection.
Materials and Methods:
Twelve New Zealand albino rabbits weighing 2-2.5 kg were used. Two rabbits were used for every dose level. Liposome formulation containing 100 and 200 g of fluconazole in sterile phosphate buffer solution and plain fluconazole at concentrations of 100, 200, 400 and 800 g in 0.1 ml of sterile normal saline were injected intravitreally into the right eyes. The left eyes received 0.1 ml normal saline or 0.1 ml of liposome formulation without fluconazole. One week later, the animals were sacrificed, their eyes enucleated and processed for light microscopy and scanning electron microscopy.
It showed that plain fluconazole at a concentration of 100 g and above caused retinal changes, with disorganization of the photoreceptor outer segments. However, liposome formulation of fluconazole (200 g/0.1 ml) did not show any significant microscopic changes of the retina.
The liposome formulation decreased the retinal toxicity of fluconazole up to the studied concentration of 200 g/0.1 ml.

Keywords: Fluconazole, liposome, retinal toxicity

How to cite this article:
Velpandian T, Narayanan K, Nag TC, Ravi AK, Gupta SK. Retinal toxicity of intravitreally injected plain and liposome formulation of fluconazole in rabbit eye. Indian J Ophthalmol 2006;54:237-40

How to cite this URL:
Velpandian T, Narayanan K, Nag TC, Ravi AK, Gupta SK. Retinal toxicity of intravitreally injected plain and liposome formulation of fluconazole in rabbit eye. Indian J Ophthalmol [serial online] 2006 [cited 2020 Dec 5];54:237-40. Available from: https://www.ijo.in/text.asp?2006/54/4/237/27947

Candidal endophthalmitis is a sight-threatening ocular infection that most frequently occurs as a complication of candidemia. While amphotericin B is considered as the gold standard for the treatment of most of the invasive fungal infections, the optimal management of candidal endophthalmitis has not been determined.[1] In order to reach direct, higher antifungal concentration in the vitreous, ophthalmologists need to opt for intravitreal injection. The retinal toxicity of commercial amphotericin B, when given as an intravitreal injection, has been reported to be reduced by liposome encapsulation.[2] Fluconazole is a fluorinated bis-triazole derivative that has been reported to be effective against Candida albicans in various experimental animal models and clinical settings. Various routes for the delivery of fluconazole have been employed for the treatment of candida endophthalmitis.[3],[4] Previous studies from our laboratory demonstrated that intravitreally injected plain fluconazole showed a relatively lesser half-life as compared to liposome formulation of fluconazole.[5] The aim of the present study was to evaluate the retinal toxicity of plain fluconazole at the concentrations of 100, 200, 400 and 800 g/0.1 ml as compared to liposome formulation of fluconazole 100 and 200 g/0.1 ml in rabbit eyes.

  Materials and Methods Top

Fluconazole was supplied free of charge by Torrent Pharmaceuticals, India. Soy-phosphatidylcholine (S-100) was provided free of charge by Lipoid GmbH, Germany. Other chemicals and drugs were purchased from their respective commercial sources.

New Zealand albino rabbits of either sex weighing 2-2.5 kg were used to investigate the retinal toxicity of plain and liposome encapsulated fluconazole. Twelve animals were procured from our institute's animal facility. They were housed in the departmental animal house, maintained at standard laboratory conditions and provided with food and water ad libitum . Their eyes were dilated with 2% homatropine hydrobromide. Anterior and posterior segments of the eye were examined thoroughly with the help of slit lamp biomicroscope (BC900 model, Haag-Streit, USA) and direct ophthalmoscope (Heine Ltd. USA) to rule out any abnormalities. This study was conducted in accordance with the provisions of the association for research in vision and ophthalmology guidelines on the use of animals in ocular research.

Plain fluconazole in the concentration of 100, 200, 400 and 800 g/0.1 ml was prepared in sterile saline. Liposome-entrapped fluconazole was prepared by reverse phase evaporation (RPE) process. Phosphatidylcholine, cholesterol and a-tocopherol were added in the molar ratio of 7:2:1. Fluconazole was added to this mixture in chloroform. The lipids along with fluconazole were dried using nitrogen to form a film coating. The film was dissolved using 9 ml of diethyl ether and 1 ml of phosphate buffered saline (PBS, pH 7.2) was added. After sonication for 2 min to form an emulsion, the mixture was placed in a rotary evaporator and the organic phase was removed by controlled suction. The formed gel was collapsed using a high-speed vortex. The liposome suspension of fluconazole was dialyzed against 1000 volume of sterile PBS for four hours at 4oC. The drug concentration of fluconazole in the final liposome formulation was determined by high performance liquid chromatography[6] and adjusted with sterile PBS to beget 100 and 200 mg/0.1 ml. The whole process was conducted with strict aseptic precautions to avoid microbial contamination at any stage of the preparation.

On the day of administration, rabbits were randomized into two batches of plain fluconazole treated (four groups IA, IB, IC and ID, each having two rabbits) and liposome formulation of fluconazole treated (two groups IIA and IIB, each having two rabbits). The right eyes (RE) of the rabbits were used as treated and left eyes (LE) served as control in both batches. Animals were sedated by injecting sodium pentobarbitone at a dose of 30 mg/kg body weight through marginal ear vein. The drug was injected intravitreally through sclera, 3 mm from limbus, with the help of 30-gauze needle fitted with tuberculin syringe after paracentesis of aqueous humor.

The RE of the groups I-A, I-B, I-C and I-D was injected with 0.1 ml of plain fluconazole at the concentration of 100, 200, 400 and 800 mg/0.1 ml respectively. The LE were injected with 0.1 ml sterile saline. The RE of the rabbits of the groups II-A and II-B were injected intravitreally with 0.1ml of liposome formulation of fluconazole 100 and 200 mg respectively. The LE of this batch received 0.1 ml of drug-free liposome.

One week after the intravitreal injection, the animals were sacrificed by administering excess of sodium pentobarbital and their eyes enucleated. Of the 24 eyes, 12 eyes from six rabbits (four RE, plain fluconazole treated and two RE, liposome encapsulated fluconazole treated and their six fellow LE, control) were immersion fixed in 10% formaldehyde for light microscopy. Other 12 eyes from six rabbits (four RE, plain fluconazole treated and two RE, liposome encapsulated fluconazole treated and their six fellow LE, control) were fixed in 3% glutaraldehyde and 1% paraformaldehyde mixture in 0.1 M phosphate buffer (pH 7.4)[7] for scanning electron microscopy. Prior to fixation, the cornea, lens and vitreous humor were removed. The retina was separated from the overlying choroids and sclera with the help of a binocular dissecting microscope.

The retinal cup thus obtained was briefly washed in distilled water and the temporal part of the retina from ora ciliaris to optic disc was cut for processing for light microscopy. The retinal tissue was dehydrated in ascending grades of ethanol, cleared in xylene, embedded in paraffin wax. Thick sections (7 m) were cut on a rotary microtome and stained with Harris hematoxylin and eosin. Photomicrographs were taken on Ilford Pan-100 films.

Following the primary fixation, the temporal part of the retinal tissue was washed overnight with 8% sucrose in 0.1 M phosphate buffer at 4oC. Then, the retinae were postfixed in 1% osmic acid in 0.1 M phosphate buffer. The osmicated tissues were dehydrated in ascending grades of acetone and critical point dried in liquid carbon dioxide. The dried retinal tissues were mounted on aluminium stub, sputter coated with colloidal gold (20 nm thick) and viewed under scanning electron microscope (Leo 435 SVP).

  Results Top

In the control groups, no damage was seen in the retina under light microscope [Figure - 1]a or scanning electron microscope [Figure - 2]a. In the treated animals, over the period of one-week treatment, plain fluconazole at it lowest dosage (100 g) had produced observable retinal changes at many places in the retina of the rabbits. The drug apparently showed its action at the outer retina, especially at the level of the photoreceptor layer [Figure - 1]b, [Figure - 2]b. Under light microscope, the photoreceptor layer appeared somewhat empty and the individual component of the photoreceptors could not be easily differentiated [Figure - 1]b. The outer nuclear layer appeared intact. No pycnotic or degenerative nuclei were evident in this layer. The inner nuclear layer appeared normal. Under scanning electron microscope, the photoreceptors appeared disoriented and their outer segments showed signs of degeneration. The long outer segments were either bent or convoluted [Figure - 2]b and in some cases, they were fragmented. The nature of changes observed in the retina in all doses with the plain fluconazole was similar and there was no dose-related change in the retinal pathology. In contrast, fluconazole treatment in liposome formulation at the concentrations of 100 and 200 g did not show any significant changes in the retinal anatomy [Figure - 3].

  Discussion Top

Fungal endophthalmitis can be endogenous or blood-born or exogenous, as after ophthalmic surgery or penetrating intraocular trauma or from a mycotic corneal ulcer. Systemic administration of antifungal agents is useful if they penetrate the vitreous body in sufficient concentrations to inhibit the growth of fungi. However, it is not possible to achieve the desired concentrations in the vitreous body due to the blood retinal barrier and the inherent nature of the drug. Intravitreal administration is a suitable route to provide sufficient levels of the drug inside the vitreous. Among the drugs meant for intraocular administration amphotericin B has been considered as a therapeutic choice. Despite the fact that amphotericin B is very effective against Candida albicans, its retinal toxicity poses a major concern.

Oral fluconazole has been successfully used in the management of mild fungal endophthalmitis. In more severe cases, additional vitrectomy and intraocular amphotericin B injection were considered as treatment modalities.[8] The retinal toxicity of intravitreal fluconazole at a dose of 100 g/ml was studied in rabbits by Schulman and coworkers[9] using biomicroscopy, ophthalmoscopy and electroretinography. The authors have suggested that fluconazole has potential application in the treatment of exogenous fungal endophthalmitis. The intravitreal pharmacokinetics studies of plain fluconazole in rabbit eyes showed a vitreous half-life of 3.08 h whereas in the form of liposome formulation it was extended to 23.4 h.[6] Subsequently, when we examined its efficacy in the model of exogenous candidal endophthalmitis we found lesser efficacy with liposomal formulation of fluconazole as compared to plain fluconazole at the concentration of 100 and 200 g/0.1 ml.[6] The present study was conducted to evaluate the advantage of using liposome formulation in terms of retinal toxicity of intravitreally injected fluconazole, using light and scanning electron microscopy. The result of the present study clearly indicates that even with the low dose (100 g) plain fluconazole has produced observable changes in the retinal architecture of the rabbit. For the comparative evaluation, liposome formulation of fluconazole was studied at the dose of 100 and 200 g/0.1 ml, which however did not show any significant retinal changes in one week. It is unlikely that the changes could have developed after a longer interval. Despite our best attempts, making a liposome formulation containing 400 g/0.1 ml of fluconazole was not possible. The liposome encapsulated form of fluconazole might have been associated with slower release of fluconazole as evidenced by our previous results.[6] The slow release of fluconazole from its liposome formulation may be accountable for its reduced retinal toxicity as evidenced by the findings. White vitreous body was noticed in the vitreous humor of all eyes in the liposome-injected groups seven days after their intravitreal injection, they were empty lipid bodies of the liposome. This kind of observation was already reported by Tremblay et al ,[2] when they studied the retinal toxicity of amphotericin B enclosed liposome upon intravitreal injection. Their study also supports our finding that liposome formulation reduces the retinal toxicity of intravitreally injected drugs. Moreover, it is most unlikely that liposome formulation per se can cause any retinal toxicity since the lipids used for the formulation are commonly found in mammalian cells and known to have biocompatibility.

Mochizuki et al.[10] reported that systemic administration of 25 mg/kg of fluconazole to the rabbit resulted in the vitreous levels of 20.63 g/ml and did not evoke any significant changes in electroretinogram (ERG) up to the studied period of eight days. Schulman et al.[9] conducted a toxicity study eight days after the intravitreal injection of fluconazole at the dose of 100 g using biomicroscopy, ophthalmoscopy and electroretinography. This study also did not reveal any changes in ERG. Cheng et al.[11] too did not find any significant changes in ERG after 2, 4, 8 and 24 h of intravitreal infusion of fluconazole (20 ml of 2 mg/ml). Similarly, the results from our laboratory also showed no significant changes in electroretinography after intravitreal administration of fluconazole at the dose of 100 and 200 g in the rabbits (unpublished data). Therefore, it may be concluded that the present observation of ultrastructural changes noted eight days after intravitreal plain fluconazole administration may not be functionally significant enough to change the ERG response. Usage of drug delivery system for the delivery of fluconazole was reported by Miyamoto et al .[12] They have evaluated the feasibility of using a biodegradable polymeric scleral implant containing fluconazole as a potential intravitreal-controlled drug delivery system. Singh et al.[13] demonstrated the usefulness of liposomal formulation of fluconazole in a limited in vitro assay. They incorporated fluconazole into multilamellar (MLV) and large unilamellar liposomes (LUV) and reported that both MLV and LUV were stable up to 72 h in saline but were less stable in the high-resolution medium. Moreover, they have also reported that the MLV-entrapped fluconazole was found to be four-fold more active than the LUV-entrapped fluconazole against Candida pseudotropicalis and over six-fold more active against C. albicans.

Su et al.[4] evaluated the use of intravitreal fluconazole at the dose of 5-10 g/ml in patients suffering from endogenous endophthalmitis. With that dose the investigators reported that there was no ocular toxicity. However, the intravitreal dose used in the present study was much higher than the dose used by Sue et al .

  Conclusion Top

The present study reports the retinal ultrastructural changes after intravitreal administration of fluconazole in rabbits and it is prevented by liposome formulation up to the studied concentration of 200 g/0.1 ml. In view of these observations care should be exercised while considering plain fluconazole at the concentration of 100 g/0.1 ml or above for intraocular administration. There were no retinal alterations seen eight days after the injection of liposome-enclosed fluconazole. However, further experimental studies are necessary to ensure the safety and efficacy of liposome-enclosed fluconazole formulation.

  Acknowledgments Top

We acknowledge the Department of Biotechnology (DBT), India, for providing the financial assistance (Grant no. BT/PRO236/09/049/96). The electron microscope work was carried out at the SAIF (DST), All India Institute of Medical Sciences, New Delhi and is acknowledged.

  References Top

Akler ME, Vellend H, McNeely DM, Walmsley SL, Gold WL. Use of fluconazole in the treatment of candidal endophthalmitis. Clin Infect Dis 1995;20:657-64.   Back to cited text no. 1
Tremblay C, Barza M, Szoka F, Lahav M, Baum J. Reduced toxicity of liposome-associated amphotericin B injected intravitreally in rabbits. Invest Ophthalmol Vis Sci 1985;26:711-8.  Back to cited text no. 2
Gupta A, Gupta V, Dogra MR. Chakrabarti A, Ray P, Ram J, et al . Fungal endophthalmitis after a single intravenous administration of presumably contaminated dextrose infusion fluid. Retina 2000;20:262-8.   Back to cited text no. 3
Su CY, Lin CP, Wang HZ, Su MY, Tsai RK, Wu KY, et al . Intraocular use of fluconazole in the management of ocular fungal infection. Kaohsiung J Med Sci 1999;15:218-25.  Back to cited text no. 4
Gupta SK, Dhingra N, Velpandian T, Jaiswal J. Efficacy of fluconazole and liposome entrapped fluconazole for C. albicans induced experimental mycotic endophthalmitis in rabbit eyes. Acta Ophthalmol Scand 2000;78:448-50.  Back to cited text no. 5
Gupta SK, Velpandian T, Dhingra N, Jaiswal J. Intravitreal pharmacokinetics of plain and liposome-entrapped fluconazole in rabbit eyes. J Ocul Pharmacol Ther 2000;16:511-8.   Back to cited text no. 6
Hayat MA. Principles and techniques of electron microscopy: Biological applications. Vol.1. Van Nostrand Co: New York; 1970.  Back to cited text no. 7
Mora P, Bovey EH, Guex-Crosier Y. Mycotic endophthalmitis: Management and treatment (9 year study). Klin Monatsbl Augenheilkd 2002;219:221-5.   Back to cited text no. 8
Schulman JA, Peyman G, Fiscella R, Small G, Coats M, Wajszczuk CP, et al . Toxicity of intravitreal injection of fluconazole in the rabbit. Can J Ophthalmol 1987;22:304-6.   Back to cited text no. 9
Mochizuki K, Yamashita Y, Torisaki T, Komatsu K, Tanahashi T, Sakai H. Intraocular penetration and effect on the retina of fluconazole. Lens Eye Toxic Res 1992;9:537-46.  Back to cited text no. 10
Cheng CK, Yang CH, Hsueh PR, Liu CM, Lu HY. Vitrectomy with fluconazole infusion: Retinal toxicity, pharmacokinetics and efficacy in the treatment of experimental candidal endophthalmitis. J Ocul Pharmacol Ther 2004;20:430-8.   Back to cited text no. 11
Miyamoto H, Ogura Y, Hashizoe M, Kunou N, Honda Y, Ikada Y. Biodegradable scleral implant for intravitreal controlled release of fluconazole. Curr Eye Res 1997;16:930-5.   Back to cited text no. 12
Singh M, Singh MP, Maiti SN, Gandhi A, Micetich RG, Atwal H. Preparations of liposomal fluconazole and their in vitro antifungal activity. J Microencapsul 1993;10:229-36.   Back to cited text no. 13


  [Figure - 1], [Figure - 2], [Figure - 3]

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