Tacrolimus, previously known as FK 506, is a neutral macrolide compound isolated from the fermentation broth of a strain of soil fungus, Streptomyces tsukubaensis.1 This drug has been demonstrated to have potent immunosuppressive activity with immunopharmacological features similar to cyclosporine A. Although the action of tacrolimus resembles that of cyclosporine A, it is generally found to show comparable effectiveness both in vivo and in vitro at concentrations two or three orders of magnitude lower.2 Tacrolimus acts primarily on CD4+ T helper lymphocytes by inhibiting the production of lymphokines, especially interIeukin-2. This drug also inhibits the release of p roi n fiammato ry and vasoactive mediators from mast cells and basophils.3,4
In experimental studies in animals, tacrolimus prevented graft rejection and prolonged graft survival in kidney, liver, and heart transplantations.5'7 The immunosuppressive properties of tacrolimus have been confirmed in humans who underwent kidney, liver, and heart transplantation.8-10
Tacrolimus was used in the treatment of experimental autoimmune uveoretinitis in animals, and was shown to suppress the cellular and humoral immune responses to some extent.11 In humans, tacroiimus used systemically to treat refractory uveitis has been effective in a dosage-dependent manner. However, this therapy induced a variety of adverse side effects.12
Despite its systemic use in ophthalmology, there is no repon of its intravitreal use. This study was undertaken to investigate the ocular toxicity of intravitreally administered tacroiimus.
MATERIALS AND METHODS
The drug tacroiimus used in this study was commercially obtained from Prograf, Fujisawa Healthcare, Inc., Deerfìeld, IL and stored at 240C. The undiluted preparation had a tacroiimus concentration of 5 mg/mL. The drug was diluted in sterile balanced salt solution to concentrations of 10, 50, 100, 250, 500, and 1000 µg.
Twenty eyes of New Zealand pigmented rabbits, weighing 2 to 3 kg were used in this study. All animal care and euthanasia were in strict conformity with the Association for Research in Vision and Ophthalmology guidelines for use of animals in research. Before intravitreal injection, the eyes were examined with slitlamp bio microscopy, indirect ophthalmoscopy, and an electroretinography test (ERG) was performed.
The rabbits were anesthetized with an intramuscular injection of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (5 mg/kg). The pupils were dilated with 2.5% phenylephrme and 0.5% tropicamide. Topical anesthesia was achieved with 0.5% proparacaine hydrochloride. Before the intravitreal injection, the eyes were sterilized with 5% povidone-iodine topically. A paracentesis was performed with a 30-gauge needle to lower the intraocular pressure. The different doses of tacroiimus or balanced salt solution were then injected into the midvitreous cavity, 3 mm posterior to the limbus superonasally, using a 30-gauge needle on a 1-mL tuberculin syringe with the beveled side of the needle facing anteriorly. The precise location of the needle could be viewed through the dilated pupil. No apparent leakage of fluid from the injection site occurred during any of the injections. Immediately after the injection, biomicroscopy and indirect ophthalmoscopy were again performed to rule out injection complications. These examinations were repeated in all eyes on days 1 and 2 after injection and every other day thereafter for 14 days.
The eyes were divided in seven groups: group 1, 3 eyes given 10 pg of the drug in 0.1 mL of solution; group 2, 3 eyes given 50 pg of the drug in 0.1 mL of solution; group 3, 4 eyes given 100 pg of the drug in 0.1 mL of solution; group 4, 2 eyes given 250 ug of the drug in 0.1 mL of solution; group 5, 3 eyes given 500 µg of the drug in 0.1 mL of solution; group 6, 3 eyes given 1000 pg of the drug in 0.2 mL, and group 7, 2 eyes given 0. 1 mL of diluent as control. The contralateral eyes were left untouched.
The ERG tests were performed both before and 7 and 14 days after intravitreal injection. These tests were done using the UTAS-E 2000 system (LKC Technologies, Gaithersburg, MD).
The animals were anesthetized and their pupils were dilated as previously described. The rabbits were dark-adapted for 20 minutes before tests. Positive unipolar contact lenses were placed on both corneas with gonioscopic solution. The negative electrode was put into the subcutaneous space of the forehead and the ground electrode was clipped to the earlobe. Three sweeps were averaged for each step. Steps 1 and 2 were a scotopic white 24 dB single flash and a scotopic white O dB single flash, respectively. To perform step 4, the eyes were light-adapted three minutes at approximately 10 candle power and a photopic white O dB single flash ERG was performed.
Euthanasia was petformed following the final postinjection examination, 14 days after the intravitreal injection, with an intravenous dose of 100 mg/kg pentobarbital. The eyes were immediately enucleated and fixed in 2% parafo rmaldehyde and 3% glutaraldehyde. Following fixation for 24 hours, the eyes were hemìsected and each part was dehydrated, embedded in paraffin, serial sectioned, and stained with hematoxylin-eosin for light microscopy.
Biomicroscopy and Ophthalmoscopy
By biomicroscopy and indirect ophthalmoscopy, no gross evidence of a toxic reaction was seen during the 1 4-day period in any eye injected with 1 0 or 50 µg of tacrolimus. Similarly, serial exams showed normal findings in the control eyes that received intravitreal injection of balanced salt solution.
Figure 1. Fundus photograph of an eye injected with 1000 µg of tacrolimus, demonstrating white vitreous bodies.
Figure 2. Fundus photograph of an eye injected with 1000 pg of tacrolimus demonstrating occlusion of the temporal retinal vessels (arrow).
In group 3, 1 of 4 eyes that received 100 µg of the drug developed a vitreous reaction consisting of a few small white bodies moving on the vitreous. This reaction appeared within 24 hours of the injection and cleared 3 days later. Both eyes that received 250 pg developed the same vitreous reaction in the same time frame, but the small vitreous bodies persisted for 7 days after the intravitreal injection.
In group 5, all 3 eyes that received a dose of 500 ug of tacrolimus showed a lot of white vitreous opacities (Figure 1) that appeared within 24 hours and cleared 7 days later. These vitreous bodies were larger than the ones observed in the eyes that received 1 OO or 250 pg. Despite the presence of vitreous opacities, the vitreous body remained sufficiently clear to permit examination of the retina in all eyes. Central opacity of the posterior capsule of the lens was observed in 1 eye of this group.
Figure 3A. Electroretinograph (scotopic white O dB} of rabbit eyes before injection shows b-wave amplitude 302.67 µ? in the right eye and 333.79 µV in the left eye.
All 3 eyes that received 1000 pg developed the same type of vitreous opacities observed in group 5. These vitreous opacities were still present 10 days after the intravitreal injection. Within 3 days after the injection, we observed occlusion of the temporal retinal vessels in 1 eye that progressed to complete whitening and atrophy of these vessels (Figure 2) .
Examination of the electro reti nogram taken before intravitreal injection of the drug showed no abnormalities. By 7 days postinjection, a marked reduction in mean B-wave amplitude was noted in the 500 and 1000 pg groups in both scotopic and photopic conditions. No ERG evidence of a toxic reaction was apparent in the eyes that received 250 pg or less of tacrolimus (Figure 3).
Histological examination by light microscopy of the control eyes and all eyes that received 10, 50, 100 or 250 ug of tacrolimus exhibited normal retinal structures with no evidence of a toxic reaction (Figure 4). Fibrin was present in the vitreous in all eyes receiving 500 or 1000 pg. These eyes showed mild disorganization of the retina with loss of photoreceptor cells and vacuolation of the inner nuclear layer (Figure 5).
Figure 3B. Electroretinograph (scotopic white O dB) of a rabbit 7 days after intravitreal injection of 250 µg/0.1 mL tacrofimus shows no effect on the b-wave amplitude (right eye, 410.45 µV teft eye, 41 4.34 µV).
Figure 3C. Electroretinograph (scotopic white O dB) of rabbit eyes before injection shows b-wave amplitude 308.96 µV in the right eye and 286.99 µV in the left eye.
Figure 3D. Electroretinograph (scotopic white O dB) of the eyes of 2 rabbits injected intravitreally with 500 µg/0.1 ml_ tacrolimus shows marked reduction of b-wave amplitude (top, 116.09 pV; bottom, 95.58 µV).
Tacrolimus is a potent immunosuppressive agent that has a pharmacophysiologic action similar to that of cyclosporine A, suppressing lymphocytes reactions, the production of T-cell-mediated soluble factors, and the expression of interleukin-2 receptor.1 The immunosuppressive properties of tacrolimus have been confirmed in patients undergoing liver, kidney, and heart transplantations.8-10
Figure 4. Histologie section of an eye by light microscopy showing normal retinal structure after intravitreal injection of 250 µ9 of tacrolimus. (Hematoxylin & eosin, original magnification x200)
A previous study has shown that systemic tacrolimus was effective in treating refractory uveitis, including Behcet's disease, but it also caused a variety of adverse side effects.13 In some patients, side effects such as renai impairment, hyperglycemia, and meningitis-like symptoms required discontinuation of the therapy.11
This study is the first published work evaluating the possible ocular toxicity of intravitreal tacrolimus. Doses of 10 to 250 u.g were injected into the vitreous cavity of normal rabbit eyes without causing any changes that could be detected microscopically or by ERG. Although vitreous opacities were observed in 1 of 4 of the eyes that received 100 ug and both eyes that received 250 ug, these transient opacities that probably represent drug precipitates were reabsorbed quickly. Intravitreal doses of 500 and 1000 ug proved to be toxic to the retina, causing marked decrease in mean B-wave amplitude in the ERG. All eyes receiving these doses showed histologie toxic reactions characterized by disorganization of the retina with loss of photoreceptor cells. One eye injected with 1000 pg developed vascular occlusion. The encouraging results of this toxicity study allow further experimentation with tacrolimus in determining its effectiveness against experimentally induced uveitis.
Figure 5. Histologie section of an eye 14 days after the intravrtreal injection of 1000 pg of tacrolimus showing localized disorganization of the retina. (Hematoxylin & eosin, original magnification x200)
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