Abstract
purpose. To evaluate the efficacy of a biodegradable polymeric scleral plug containing the immunosuppressive agent, FK506, in a rabbit model for experimental uveitis.
methods. The scleral plugs were prepared by dissolving poly(dl-lactide-co-glycolide; PLGA) and FK506 (weight, 8.5 mg; length, 5 mm; 1% FK506). The release of FK506 was evaluated in vitro by spectrophotometry on days 1, 3, 7, 14, 21, and 35. In vivo, FK506 concentrations of the vitreous were measured by high performance liquid chromatography 2 and 4 weeks after intravitreous plug implantation in pigmented rabbits. Sixteen pigmented rabbits were immunized twice subcutaneously with 10 mg of Mycobacterium tuberculosis H37Ra antigen. Twelve days later, the right eyes of all rabbits were challenged with an intravitreal injection of 50 μg of antigen. After the first challenge, the 16 eyes of 16 pigmented rabbits were divided into two groups. Scleral plugs were implanted into the vitreous of the right eye of eight rabbits. Eight control rabbits received a sham device. The aqueous protein concentrations and cell counts were determined on postchallenge days 7, 14, and 28. To simulated chronic inflammation, the eyes were rechallenged with intravitreal antigen on day 14 and were observed for 1 month. Inflammation of the anterior chamber and the vitreous were graded clinically by two masked observers. Retinal function was evaluated by electroretinography (ERG) and histologic examination.
results. Clinical scores (anterior chamber cells, flare, and vitreous opacity) showed that treated eyes had significantly less inflammation than untreated eyes (P < 0.001). Quantitative analysis of inflammatory cells (P < 0.001) and protein concentrations (P < 0.0001) in the anterior chamber showed significant decreases in treated eyes. Histopathologic examination showed marked inflammation and tissue disorganization in the untreated eyes. No retinal toxicity was detected, histopathologically and electroretinographically. After antigen rechallenge, inflammation in experimental eyes was still less than in control eyes.
conclusions. Intravitreal sustained-release of FK506 from a biodegradable polymeric scleral plug was highly effective in suppressing the inflammation of experimental uveitis in a rabbit model for at least 6 weeks. This device may be useful in the management of patients with severe chronic uveitis.
Tacrolimus (FK506) is an immunosuppressive agent that has been isolated from the fermentation broth of
Streptomyces tsukubaensis.
1 2 It has a mechanism of action similar to that of cyclosporin A, but is more potent.
3 In vitro, FK506 inhibits the generation of cytotoxic T lymphocytes and the production of interleukin-2 and -3 and interferon-γ at levels approximately 100 times lower than that of cyclosporin A.
4 In vivo, FK506 showed a strong immunosuppressive effect in a variety of animal models of transplantation and in the treatment of experimental autoimmune uveoretinitis.
3 5
The use of FK506 is of special interest in ophthalmology, because it may be effective in the treatment of immune-mediated disease, such as corneal graft rejection,
6 keratitis, scleritis, ocular pemphigoid, and uveitis. In a previous study,
7 oral FK506 was used to treat patients with refractory uveitis including Behçet’s disease. The adverse effects
7 8 of systemically administered FK506, however, include nephrotoxicity, hypertension, hyperesthesia, muscular weakness, insomnia, tremor, photophobia, gastrointestinal symptoms, and central nervous system alterations. Long-term therapy is necessary for chronic uveitis, and the incidence of systemic side effects may be increased when FK506 is administered systemically. Furthermore, systemically administered drugs penetrate poorly into the intraocular tissues because of the blood–ocular barrier.
To avoid systemic side effects, topical application of an agent is preferred to the oral and intravenous routes. Unfortunately, topical application of drugs is limited by the lipophilic physical and chemical characteristics of the drug and vehicle and results in poor intravitreal penetration.
Therefore, intravitreal injection is becoming a more accepted method of delivering drugs directly to the vitreous and retina. Frequent intravitreal injections are essential to maintain drug concentrations within a therapeutic range in the posterior segment, and this approach may be associated with a risk of complications including cataract, retinal detachment, vitreous hemorrhage, and endophthalmitis. In addition, the half-life of a single intravitreally injected drug was demonstrated to be insufficiently long for an effective clinical response. Therefore, we have attempted to develop controlled drug delivery systems
9 10 for vitreoretinal tissue that are able to deliver a drug for long enough time after a single intravitreal injection. To avoid the complications mentioned herein, we prepared a scleral plug of biodegradable polymer containing FK506 as an intravitreal sustained-release device and investigated whether such an implantable biodegradable drug delivery device is effective in the treatment of severe panuveitis in a rabbit model.
Poly(dl-lactide-co-glycolide) with weight-average molecular weight of 63,000 and a 50:50 copolymer ratio of dl-lactic acid to glycolic acid (abbreviated as PLGA [50/50]-63,000) was purchased from Alkermes, Inc. (Cincinnati, OH).
The weight-average molecular weight was determined by gel permeation chromatography by the supplier. FR900506 (FK506) was kindly provided by Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan).
The implants were placed in 2 mL of phosphate-buffered solution (pH 7.4) in a closed vial and were immersed in a shaking water bath at 37°C. On days 1, 3, 7, 14, 21, and 35, approximately 2 mL of the released medium was removed and replaced with the same quantity of fresh medium. The amount of FK506 released into the medium was measured by high-performance liquid chromatography (HPLC). All chromatograms were obtained using commercially available components. The analytical column used was a 4.6-mm (inner diameter) × 25-cm prepackaged column (model ODS-A303; YMC, Kyoto, Japan). A pump (model LC-10AS; Shimadzu, Kyoto, Japan) was used at a constant flow rate of 1.0 mL/min with a pulse dampener and degasser (model KT-16; Showa Denko, Tokyo, Japan). The eluent consisted of 30 mM PBS and 5 mM heptane sulfonate sodium salt in 2% acetonitrile-water (1:49; pH 2.0). The column oven (model CTO-10A; Shimadzu) was equipped and set at 45°C. A spectrophotometric detector (model SPD-6A; Shimadzu) was used at a wavelength of 254 nm.
We used pigmented rabbits weighing 1.5 to 2.0 kg each in the study. The animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rabbits were anesthetized with an intramuscular injection of pentobarbital sodium (20 mg/kg) before treatment. The pupils were dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride eye drops. The ocular surface was then anesthetized with a topical instillation of 0.4% oxybuprocaine hydrochloride.
After the sclera was exposed, a 1-mm sclerotomy was performed with a V-lance 2 mm from the limbus. The FK506-loaded scleral plug was inserted at the sclerotomy site. The conjunctival wound was sutured with 8-0 dexon. One drop of topical 0.3% gentamicin solution was instilled into the eye after surgery for the prevention of infection.
At weeks 2 and 4 after intravitreous implantation, FK506 concentrations in the vitreous were determined. Five rabbits were used at each time point. The intravitreal FK506 concentration was determined by the HPLC system. The rabbits were killed with an overdose of intravenous pentobarbital sodium, and the eyes were immediately enucleated. The eyes were stored at −85°C until processing. The retina and choroid separated from the vitreous while frozen and were homogenized after adding chilled acetonitrile (0.3 mL). The homogenate was centrifuged at 3000 rpm for 10 minutes (CF7D2; Hitachi, Ltd., Tokyo, Japan) and then the supernatant was dried under reduced pressure with a centrifugal concentrator (model VC-960; Taitec, Saitama, Japan). The residue was dissolved with 0.5 mL of the mobile phase eluent. Fifty milliliters of the solute was injected for analysis. Our methods provided FK506 sensitivity as low as 30 ng/mL.
A microparticulate suspension of
M. tuberculosis H37RA antigen was prepared by ultrasonicating a suspension of the crude extract in sterile balanced salt solution.
11 12 One day after scleral implantation, 50 μg of antigen suspended in 0.1 mL of balanced salt solution was injected into the vitreous cavity of the right eye of all rabbits under anesthesia (first challenge). To simulate chronic inflammation with exacerbations, eyes in the treated and untreated group were rechallenged with the same amount of intravitreal antigen on day 14 (second challenge). One drop of 1% atropine solution was then instilled into the eye to maintain postoperative cycloplegia.
In the treated group, eight eyes of eight pigmented rabbits were used. Animals were anesthetized with an intramuscular injection of 0.3 mL ketamine hydrochloride (100 mg/mL) and 0.1 mL xylazine hydrochloride (100 mg/mL) per kilogram body weight. After the sclera was exposed, a 1-mm sclerotomy was performed with a V-lance 2 mm from the limbus. The FK506-loaded scleral plug was inserted at the sclerotomy site. The conjunctival wound was sutured with 8-0 dexon.
In the untreated group, eight eyes of another eight rabbits received surgical implantation of the placebo plug. One drop of topical 0.3% gentamicin solution was instilled into the eye after surgery for the prevention of infection.
Three animals in each group were used at 1, 2, and 4 weeks after the first challenge for aqueous protein measurement and cell count. Animals were anesthetized, and 0.2 to 0.3 mL aqueous humor was aspirated from the right eye of each of the rabbits with a heparin-rinsed glass syringe connected to a 27-gauge needle. Aqueous cell count was measure by hemocytometer. One drop of aqueous was placed on a microscope slide and stained with Wright stain for differential cell count. The remaining aqueous was centrifuged at 250g for 5 minutes, and the supernatant was used for protein measurement. The protein content of the aqueous humor was determined using a Bio-Rad (Richmond, CA) assay kit with bovine serum albumin as a standard dilution reference curve, according to the manufacturer’s recommendation.
Eight weeks after the first challenge, selected eyes of treated and untreated rabbits were enucleated and processed for histopathologic analysis. The eyes were immediately fixed in phosphate-buffered 2.5% glutaraldehyde and 5% formaldehyde in 0.15 M phosphate buffered solution (pH 7.2) at 20°C. The specimens were dehydrated in a series of ascending concentrations of ethanol, cleared in xylene, and embedded in paraffin wax. Serial sections of the eye were cut at a thickness of 4.0 μm and mounted on glass slides. After being dewaxed in xylene, sections were hydrated in a series of descending concentrations of ethanol. The hydrated sections were stained with Mayer’s hematoxylin solution at 20°C for 10 minutes, rinsed in tap water for 15 minutes, immersed in 0.5% eosin solution at 20°C for 10 minutes, dehydrated in a series of ascending concentrations of ethanol, cleared in xylene, and mounted in synthetic resin solution (Harleco; Kokusai Shiyaku, Kobe, Japan). All sections were examined in detail by light microscopy (Provis AX 70 with U-photograph; Olympus Co., Japan).
In Vitro Release of FK506 from the Scleral Plugs and In Vivo Release in the Rabbit Eye