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Safety and efficacy of intracapsular tranilast microspheres in experimental posterior capsule opacification
LABORATORY SCIENCE
Safety and efficacy of intracapsular tranilast microspheres in experimental posterior capsule opacification Man Wang, MD, Jin-Jun Zhang, PhD, MD, Timothy L. Jackson, PhD, FRCOphth, Xinghuai Sun, MD, Wei Wu, MD, John Marshall, PhD
PURPOSE: To evaluate the safety and efficacy of a sustained-release agent designed to reduce posterior capsule opacification (PCO). SETTING: Department of Ophthalmology, EENT Hospital, Fudan University, Shanghai, Peoples Republic of China. METHODS: Free tranilast (TFree) was incorporated into polylactic acid microspheres and then tested using a rabbit model of PCO. Twenty-nine rabbits were randomized into 5 groups treated with balanced saline solution (BSS control); TFree; or 0.5, 1.0, or 2.0 mg tranilast microspheres (TMicro). Standard phacoemulsification cataract surgery, including manual aspiration of all visible soft lens matter, was performed in all groups. The selected test agent was then injected into the lens capsule. Postoperative clinical examinations were performed at 1, 3, 7, 14, 30, 60, and 90 days. Posterior capsule opacification was quantified using high-resolution computer image analysis at 1, 2, and 3 months. Histological examination was performed at 3 months. RESULTS: Eyes treated with TMicro had significantly less PCO than the eyes in the BSS and TFree groups. While the BSS control eyes had increased PCO over 3 months, eyes in the TMicro group had reduced PCO over time in a dose-dependent fashion. Histological examination showed reduced lens epithelial cell proliferation in the TMicro groups, with no manifest damage to the cornea, iris, or retina compared with the BSS controls. There was a transient increase in postoperative inflammation in all tranilast-treated groups compared with the BSS controls. CONCLUSION: Sustained-release intracapsular tranilast reduced PCO in an experimental model of PCO, suggesting further investigation of its therapeutic potential is justified. J Cataract Refract Surg 2007; 33:2122–2128 Q 2007 ASCRS and ESCRS
Cataract is the world’s leading cause of blindness. Although modern cataract surgery is an extremely effective treatment with a low incidence of serious complications, up to 15% to 50% of eyes develop posterior capsule opacification (PCO) within 3 to 5 years of surgery.1–5 In children and young adults, the incidence of PCO approaches 100%.6 Posterior capsule opacification degrades the image quality and may require laser capsulotomy. As such, it is the most common complication of cataract surgery. Furthermore, many patients in developing nations do not have easy access to laser capsulotomy.7 To date, most attempts at reducing PCO have concentrated on novel biomaterials and intraocular lens design. There are, however, many therapeutic agents that might reduce PCO. In vivo and in vitro studies show that antimetabolites such as mitomycin-C, 2122
Q 2007 ASCRS and ESCRS Published by Elsevier Inc.
5-fluorouracil, and daunomycin inhibit the proliferation of lens epithelial cells (LECs).8–10 Immunotoxins, such as the monoclonal antibody ricin A, and fibroblast growth factor 2-saporin also inhibit LEC proliferation, although ocular toxicity limits their therapeutic potential.11,12 Most of these drugs were administered by infusing a single dose into the capsular bag during surgery or by subconjunctival injection at the end of surgery. In both circumstances, the concentration of drug declined rapidly.10,13–15 Therefore, to achieve a therapeutic effect, high concentrations were often required, with the attendant risk for ocular toxicity.3,16–18 Tranilast, N-(3,4-dimethoxycinnamoyl)-anthranilic acid, is an antiallergy drug introduced to treat and prevent bronchial asthma and allergic rhinitis. Its effect was thought to be due to its ability to stabilize mast 0886-3350/07/$dsee front matter doi:10.1016/j.jcrs.2007.07.041
LABORATORY SCIENCE: INTRACAPSULAR TRANILAST MICROSPHERES IN PCO
cells, prevent degranulation, and reduce the production of histamine and 5-HTP as well as to reduce inflammatory cell activity and vascular permeability.19 More recent studies show an antifibrotic effect with inhibition of fibroblast proliferation and transformation to myofibroblasts, with reduced collagen production. These effects are mediated by a range of growth factors that are down-regulated by tranilast; these include transforming growth factor b (TGF-b), interleukin 1b, prostaglandin E2, vascular endothelial growth factor (VEGF), and bFGF.20,21 Tranilast also inhibits angiogenesis due to reduced migration and proliferation of microvascular endothelial cells, reduced cellular energy consumption, blocked calcium influx, and inhibition of tube formation, as well as reduced VEGF/ vascular permeability factor.22,23 Tranilast is also thought to have potential as an anticancer drug, with inhibition of malignant neurogliacytes that rely on TGF-b. Malignant cells exposed to tranilast show reduced proliferation, migration, and tissue invasion.24 Tranilast does not, however, appear to inhibit proliferation and collagenation in healthy tissue.20,21 Tranilast eyedrops (Rizaben) are widely used for allergic and seasonal conjunctivitis and are generally well tolerated.25,26 Taken together, these findings indicate that the main pharmacological effects of tranilast result from altered cell migration, differentiation, and proliferation, rather than from direct cytotoxicity. This suggests that tranilast has the potential to reduce PCO without an undue risk for ocular toxicity, and preliminary in vitro and in vivo studies appear to confirm this.22,27,28 Other potential applications include reducing corneal haze after excimer laser ablation, inhibiting scarring after glaucoma filtration surgery, and reducing proliferative vitreoretinopathy and retinal angiogenesis (T. Ishihashi, Y. Aoyama, ‘‘Effect of Tranilast on Conjunctival Bleb Scarring After Filtration Surgery’’ [Japanese], presented at the 103rd meeting of the Japan Ophthalmological Association, Chiba, Japan, April 1999. Abstract in Nippon Ganka Gakkai Zasshi 1999; 103, page 134).29–31
Accepted for publication July 16, 2007. From the Department of Ophthalmology (Wang, Zhang, Sun), EENT Hospital, and School of Pharmacy (Wu), Fudan University, Shanghai, Peoples Republic of China; and the GKT Department of Ophthalmology (Zhang, Jackson, Marshall), Rayne Institute, St. Thomas’ Hospital, London, United Kingdom. No author has a financial or proprietary interest in any material or method mentioned. Corresponding author: Dr. Man Wang, Eye and ENT Hospital, Fudan University, Number 83 FenYang Road, Shanghai, China. E-mail: [email protected].
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Although PCO could be inhibited by tranilast, the therapeutic effect is likely to be limited by the lack of a satisfactory delivery system. Topical delivery has been hindered by a short half-life and poor tissue penetration, with less than 5% transcorneal drug delivery.32 This necessitates extended treatment periods in animal and human eyes.32 It is possible that intraocular delivery of a sustained-release preparation of tranilast could overcome many of the problems and realize the therapeutic potential of tranilast. Biodegradable microsphere delivery systems can be used to produce sustained release of therapeutic agents. The active agent is contained within a microsphere that degrades with time, reducing side effects, improving stability, and enhancing formulation flexibility. In addition, there is no need to surgically remove the delivery system once the drug is released as the microspheres break down into nontoxic products. Studies confirm that microsphere systems are nonirritating, nonmutagenic, and nonallergenic.33 Hence, intracapsular microspheres have the potential to reduce toxicity and produce sustained, local release of an agent designed to prevent PCO. In this study, free tranilast (TFree) was incorporated into microspheres and tested in a rabbit model of PCO to determine the agent’s therapeutic effect and potential toxicity. MATERIALS AND METHODS Preparation of Tranilast Microspheres Tranilast microspheres (TMicro) were prepared using an oil–water emulsion, after which the organic solvent from the inner organic droplets were extracted and evaporated as described previously.34 Briefly, TFree was micronized by sonification and suspended in an organic phase comprising 2 mL of polylactide dichloromethane solution. The outer water phase comprised 100 mL of 4% polyvinyl alcohol solution. Under continuous stirring using a mechanical stirrer (Ika) at 800 revolutions per minute, the organic phase was instilled gradually into the water phase through a 16-gauge needle. After 2 minutes of emulsification, the subsequent oil–water emulsion was diluted with 100 mL of distilled water, with the stirring rate reduced to 500 revolutions per minute. After another 2 minutes, 500 mL of distilled water was added and stirred for a further 1.5 hours to achieve complete extraction and evaporation of dichloromethane. The microspheres were collected through filtration under reduced pressure, washed with distilled water, and stored in a vacuum desiccator for 48 hours before use. Drug loading was adjustable and was tuned to a relatively low level (7.5%) in favor of a more sustained release. Tranilast microspheres were combined with 0.1 mL of 3% hydroxypropyl methylcellulose (HPMC) at the time of surgery to facilitate delivery and retention within the capsular bag.
Animals Thirty-seven New Zealand White rabbits of both sexes were used for the preliminary control and treatment
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experiments. Animals were approximately 10 to 12 weeks of age with an initial body weight of 1.8 to 2.0 kg. The rabbit was chosen because its crystalline lens, as in humans, has an anterior capsule with a single row of cubical epithelial cells from which lens fibers develop. The animals were kept under standardized conditions in separate cages and given tap water and food ad libitum. The experiments adhered to the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research.
Preliminary Control Experiments To ensure the microspheres were effective in providing sustained release, preliminary control experiments were performed in 8 rabbit eyes. Rates of drug elimination from the aqueous humor for TFree (n Z 4) and TMicro (n Z 4) were compared. In both groups, 0.1 mL HPMC 3% containing 0.15 mg TFree or TMicro was injected as a single dose directly into the anterior chamber through the limbus using a 1 mL syringe and 30-gauge needle. Anterior chamber samples (0.1 to 0.2 mL) were taken over 7 days using a 1 mL syringe and 30-gauge needle. The drug concentration was then determined by high-performance liquid chromatography.35,36 Table 1 shows the results in this experiment, which confirmed that the microspheres produced sustained drug release over 7 days and the nonencapsulated tranilast, over 2 days. There was a significant difference in anterior chamber tranilast concentrations at each time point (P!.05; n Z 4).
Treatment Groups Twenty-nine rabbits were divided into 5 groups: a control group (treated with balanced saline solution [BSS]) and 4 treatment groups. The treated animals received a single dose of TFree (treated control) or 0.5, 1.0, or 2.0 mg TMicro. Each group comprised 5 to 6 rabbits.
Surgical Technique Sterile surgical technique was used. Topical phenylephrine hydrochloride 1% and cyclopentolate hydrochloride 0.5% were administered 5 and 10 minutes before anesthesia to dilate the pupils. The animals were anesthetized with an intramuscular injection of xylazine hydrochloride 5 mg/kg body weight and ketamine hydrochloride 50 mg/kg body weight. Topical application of oxybuprocaine hydrochloride was used for local anesthesia. A corneal incision was made with a 3.2 mm calibrated blade (Alcon). Sodium hyaluronate
Table 1. Comparison of therapeutic concentrations and drug elimination of TFree and TMicro from the aqueous humor after direct aqueous humor injection in a rabbit model. Mean Concentration (mg/mL) G SD Group
Day 1
Day 3
Day 5
Day 7
TFree 1.18 G 0.64 0.046 G 0.005 U U TMicro 12.22 G 4.74 0.59 G 0.23 0.45 G 0.04 0.44 G 0.02 TFree Z free tranilast; TMicro Z tranilast microspheres; U Z undetectable concentration of tranilast in anterior chamber
3.0%–chondroitin sulfate 4.0% (Viscoat) (0.1 mL) and sodium hyaluronate 1.0% (Provisc) (0.1 mL) were injected into the anterior chamber. A continuous curvilinear capsulorhexis approximately 5.0 mm in diameter was created with a capsule forceps. The lens was then hydrodissected with BSS and the nucleus emulsified in the bag (Legacy 20000, Alcon). After the residual lens cortical material was removed by irrigation/aspiration, a single dose of the treatment solution (in 0.1 mL HPMC 3%) was injected into the capsule bag with a syringe. The wound was then closed with 10-0 nylon sutures. At the end of surgery, animals received a subconjunctival injection of 20 000 mg gentamicin and 2.5 mg dexamethasone. Postoperative medications included tobramycin 0.3%–dexamethasone 0.1% (TobraDex) and atropine 1% drops 3 times daily for a month.
Clinical Observation A surgeon masked to the groups performed slitlamp examinations at 0, 1, 3, 7, 14, 30, 60, and 90 days. The following were noted: corneal clarity, anterior chamber flare and cells, intraocular pressure (IOP), and abnormal findings.
Image Acquisition and Analysis The degree of PCO was quantified 1, 2, and 3 months postoperatively using previously described techniques.37 Briefly, the posterior capsule was photographed through a dilated pupil using a digital retroillumination camera (SLICPS 2000, LiuLiu Vision Technology) mounted on a slitlamp. The images were downloaded to a personal computer and analyzed using image-processing software (ShengTeng Information Technology). For each photograph, a 4.0 mm diameter region of interest was outlined by the computer in the center of the visual axis, after 3 points were manually chosen on the pupil border. The percentage and density of PCO in the assigned area were analyzed by the image-analysis software, which reverses color and increases color contrast. The area and density of PCO (Figure 1) were used to give an opacity index. The opacity index was calculated by the computer software using the following formula: Opacity indexZðROP =RROI Þ DPCO 100 where ROP is the area of opacity in the 4.0 mm diameter region of interest; RROI is the area of the 4.0 mm diameter region of interest, and DPCO is the mean value of the density of the PCO area (average positive density).
Light and Electron Microscopy After the 3-month clinical follow-up, all rabbits were humanely killed. The operated eyes were enucleated and placed in Karnovsky solution (paraformaldehyde and glutaraldehyde mixture). After the 12 o’clock position was marked on the sclera using tissue-marking dye, each globe was sectioned in the coronal plane just anterior to the equator. Histological sections were prepared and stained with hematoxylin–eosin and periodic acid-Schiff and then observed with a light microscope (Olympus). Photomicrographs were taken as required. All eyes had transmission electron microscopy (TEM) to detect ultrastructural damage. For TEM, the specimens were fixed in cacodylate glutaraldehyde 2%, postfixed in osmium tetroxide 2%, dehydrated through a graded series of ethanol, and embedded in EmBed 812 (EM Sciences). Ultrathin sections of the cornea and iris were examined using TEM (CM120, Philips).
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decreasing to 9.8% in the TMicro group (Figure 2). The inhibition of PCO was confirmed by histological examination. Figure 3, A, shows a rabbit eye treated with TMicro that had little lens capsule opacification 3 months postoperatively and a smooth posterior lens capsule observed on histology (Figure 3, B). In contrast, eyes treated with TFree showed some fibrous membrane in the pupil area (Figure 3, C) and significant LEC proliferation on histological examination (Figure 3, D).
Figure 1. Posterior capsule opacification measurement. A: Original slitlamp retroillumination digital image from a rabbit eye shows the pupil area and capsulorhexis. B: Raw image of A with the capsule mask applied, thereby defining the area of the posterior capsule within the 4.0 mm capsulorhexis. This area was used for subsequent image analysis. C: Contrast enhancement with reversing colors of the image within the capsulorhexis. D: Segmented image of A showing an area of PCO in red.
Statistical Analysis Raw data were analyzed using SPSS statistical software (version 11.0, SPSS, Inc.). The Student-Newman-Keuls test (q test) was applied to determine the significance of differences between groups; a P value less than 0.05 was considered statistically significant.
RESULTS Treatment Effect Although PCO was significantly reduced in both tranilast-treated groups (TFree and TMicro), the use of microspheres reduced PCO by 3.7-fold compared with the use of TFree. The mean opacity index was 39.31 G 20.591 (SEM) in the TMicro group (n Z 6) compared with 199.64 G 116.806 in the TFree group (n Z 5; P!.05) and 399.47 G 182.874 in the BSS control group (n Z 6; P!.05) (Table 2). Posterior capsule opacification in the TFree group was 36.5% of that in the BSS control, Table 2. Opacity index values 3 months postoperatively in the 3 groups. Opacity Index Rabbit
Control
TFree
TMicro
1 2 3 4 5 6
333.47 280.52 649.78 148.71 351.76 632.58
82.62 209.28 415.00 178.97 112.32 d
31.10 30.18 23.97 81.78 22.04 46.79
Control Z BSS; TFree Z free tranilast; TMicro Z tranilast microspheres
Time Dependency Over time, the PCO rates increased significantly in the control group and decreased significantly in the tranilast-treated groups. The mean opacity index in the BSS control group was 215.6 G 170.1 at 1 month, increasing to 399.5 G 182.9 at 3 months. Clinical observations were consistent with this finding, with posterior capsule fibrosis apparent in 2 of 6 eyes at 1 month and 4 of 6 eyes at 3 months. In contrast, eyes that received a single dose of TMicro had a time-dependent decrease in PCO (Figure 4). For example, the 6 eyes that received 1 mg TMicro had a mean opacity index of 41.6 G 25.1 at 1 month, 30.2 G 19.6 at 2 months, and 26.7 G 14.1 at 3 months (all P!.05). Dose Dependency Figure 4 shows that TMicro reduced PCO in a dosedependent fashion. For example, in the first month after surgery, the mean opacity index was 57.6 G 17.0 at a concentration of 0.5 mg, decreasing to 41.6 G 25.07 at 1.0 mg and 30.5 G 16.8 at 2.0 mg (all n Z 6 and P!.05). This dose-dependency persisted at 3 months. Side Effects There were no manifest systemic effects in that the animals did not show behavioral signs suggesting pain and gained weight normally. Biomicroscopic examination of all rabbit eyes revealed mild to moderate corneal haze daily for the first week after surgery. By 10 days postoperatively, the central haze had resolved, but a 1.0 to 2.0 mm area of haze persisted at the superior entry site. There were some differences in the inflammatory response between control eyes and treated eyes. Control eyes had postoperative inflammation in the form of iris hyperemia, fibroid exudates, and anterior chamber flare (Tyndall score C to CC), which peaked 1 day postoperatively and resolved in 5 to 7 days. All eyes treated with tranilast had elevated levels of postoperative anterior chamber flare (Tyndall score CC to CCC), fibroid exudates, and iris hyperemia, which peaked at 3 days and resolved in 7 to 10 days. Table 3 shows the IOP 1 day through 7 days postoperatively. The IOP was in the normal range (11 to
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Figure 2. Comparison of eyes treated with TFree (left column) and eyes treated with TMicro (right column). Posterior capsule opacification was assessed using image analysis to give an opacity index, expressed as a percentage of the negative control (mean G SEM; n Z 6). The results are from the 3-month time point and show a significant reduction in the group treated with TMicro compared with the TFree group (P!.05, paired t test).
Figure 4. Time and dose dependency of TMicro on rabbit PCO. The mean of the opacity index taken from controls and the 3 TMicro groups 1, 2, and 3 months after treatment are plotted. Note that over time, the opacity index increases in the control group but decreases in the treated groups (mean G SEM; n Z 6) (P!.05, all groups compared with control group).
24 mm Hg), except in the group treated with 2.0 mg TMicro, in which the IOP was slightly higher at 1 day. Histological examination did not reveal a significant difference between the control group and treated groups. No animals had evidence of endothelial cell loss or damage 3 months after surgery. There was loss of iris epithelial and stromal pigment with a vigorous melanophagic reaction, but no evidence of mitosis or cell destruction in the iris epithelium, in all groups.
There was no epithelial or stromal cell loss in the ciliary body. The retina and choroid did not show significant damage on microscopic examination (Figure 5).
Figure 3. High-resolution histology images of rabbit eyes treated with both forms of drug-delivery systems and the corresponding histological capsule sections 3 month after treatment. A: Retroillumination image of a rabbit eye treated by TMicro shows a relatively clear pupil (* Z focal area of opacification). B: Corresponding histological section of the lens capsule bag shows a smooth capsule and no significant LEC proliferation (original magnification 200). C: Image of a rabbit eye treated with TFree shows the development of PCO and focal opacities (*). The short arrow shows the light reflex. D: Histological section of the lens capsule in C shows significant LEC proliferation (arrowheads). The arrows point to the posterior lens capsule (original magnification 100).
DISCUSSION Human28 and animal38 studies indicate that PCO can be inhibited by the long-term administration of tranilast eyedrops; however, this imposes considerable inconvenience to patients. Bioavailability is also low, with only 5% of topical treatment reaching the intraocular tissues.29,32 This is not unexpected as there are significant barriers to ocular penetration. First, the tear meniscus dilutes medications and its relatively low capacity may result in overspill of tears when eyedrops are instilled. Eyedrops can also cause ocular discomfort with reflex lacrimation that may remove most drugs from the ocular surface within 5 to 7 minutes. This may aggravate systemic absorption and side effects.39 Corneal structure further impairs absorption. The relatively lipophilic corneal epithelium, which has low porosity and high tortuosity due to tight annular junctions, is the main barrier to hydrophilic drugs, whereas the middle stromal layer, which consists mainly of water interspersed with collagen fibrils,
Table 3. Effects of TMicro on IOP. Mean IOP (mm Hg) G SEM TMicro Dose (mg) 2.0 1.0 0.5 0.0
Day 1
Day 3
Day 7
20.00 G 6.66 16.00 G 5.73 13.33 G 5.24 15.71 G 5.71
14.00 G 5.66 12.67 G 2.73 13.33 G 3.83 14.50 G 5.32
13.00 G 2.37 12.83 G 3.19 13.33 G 2.66 13.67 G 3.78
IOP Z intraocular pressure; TMicro Z tranilast microspheres
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this organic solvent induces anterior chamber reaction in the early period after surgery before it is completely metabolized. Although there was some clinical evidence of increased inflammation in the treated groups, this was transient and not associated with histological changes. This suggests tranilast may be safe for intraocular use; however, further safety testing is appropriate. In conclusion, this study indicates that intracapsular delivery of a sustained-release preparation of tranilast reduced PCO in rabbits for up to 3 months, with no histological evidence of tissue damage compared with control eyes. Further studies are needed to determine the lowest effective concentration and the longterm safety profile.
REFERENCES
Figure 5. Histology of cornea and retina 3 months after treatment with TMicro (1 mg). A: Control retina with a similar appearance to treated retina in B. Treated cornea (D) also appears similar to the control cornea (C). The arrows point to the retinal pigment epithelium and corneal endothelial cells (original magnification 40).
impedes the diffusion of lipophilic drugs. This suggests that an alternative delivery system is preferable. We present the results of one alternative, tranilast. Tranilast is a well-tolerated medication with multiple effects including inhibition of cell migration, proliferation, and fibrosis.33 Free tranilast was placed in microspheres and then delivered directly into the capsular bag at the end of cataract surgery. Local delivery has the potential to increase bioavailability at the site of action, while the use of microspheres produces sustained delivery that would be expected to increase the therapeutic effect and further reduce the need for high, single-dose treatment. The microsphere delivery system produced 90% less PCO than the negative control and 50% less PCO than unencapsulated free drugs. The presence of a dose-response curve supports the hypothesis that the effect was drug related. In contrast to the negative control, eyes treated with TMicro eyes had reduced PCO with time, suggesting sustained release of tranilast. Regarding the anterior chamber reactions in all groups (reaction was more severe and lasted longer in treated groups), we think that both the suspension and microspheres, acting as foreign objects, result in severe anterior chamber reaction. Moreover, during their manufacture (emulsion, evaporation, and extraction), a small amount of organic solvent remained in the microspheres. We must further study whether
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14. Clark DS, Emery JM, Munsel MF. Inhibition of posterior capsule opacification with an immunotoxin specific for lens epithelial cells: 24 month clinical results. J Cataract Refract Surg 1998; 24:1614–1620 ¨ ztu¨rk F, Kaynak S, et al. Prevention of posterior cap¨U ¨,O 15. Inan U sule opacification by intraoperative single-dose pharmacologic agents. J Cataract Refract Surg 2001; 27:1079–1087 16. Legler UFC, Apple DJ, Assia EI, et al. Inhibition of posterior capsule opacification: the effect of colchicine in a sustained drug delivery system. J Cataract Refract Surg 1993; 19:462–470 17. Tetz MR, Ries MW, Lucas C, et al. Inhibition of posterior capsule opacification by an intraocular-lens-bound sustained drug delivery system: an experimental animal study and literature review. J Cataract Refract Surg 1996; 22:1070–1078 18. Pandey SK, Cochener B, Apple DJ, et al. Intracapsular ring sustained 5-fluorouracil delivery system for prevention of posterior capsule opacification in rabbits: a histological study. J Cataract Refract Surg 2002; 28:139–148 19. Hiratochi M, Takamoto M, Tatemichi S, Sugane K. Inhibition of interleukin 5 production with no influence on interleukin 4 production by an anti-allergic drug, tranilast, in Toxocara canis-infected mice. Int J Immunopharmacol 2000; 22:463–471 20. Suzawa H, Kikuchi S, Ichikawa K, Koda A. Inhibitory action of tranilast, anti-allergic drug, on the release of cytokines and PGE2 from human monocytes-macrophages. Jpn J Pharmacol 1992; 60:85–90; Available at: http://www.journalarchive.jst. go.jp/jnlpdf.php?cdjournalZjphs1951&cdvolZ60&noissueZ2& startpageZ85&langZen&fromZjnltoc. Accessed July 30, 2007 21. Koyama S, Takagi H, Otani A, et al. Tranilast inhibits protein kinase C-dependent signalling pathway linked to angiogenic activities and gene expression of retinal microcapillary endothelial cells. Br J Pharmacol 1999; 127:537–545; Available at: http://www.nature.com/bjp/journal/v127/n2/pdf/0702564a.pdf. Accessed July 30, 2007 22. Isaji M, Miyata H, Ajisawa Y, et al. Tranilast inhibits the proliferation, chemotaxis and tube formation of human microvascular endothelial cells in vitro and angiogenesis in vivo. Br J Pharmacol 1997; 122:1061–1066; Available at: http://www.nature.com/ bjp/journal/v122/n6/pdf/0701493a.pdf. Accessed July 30, 2007 23. Isaji M, Miyata H, Ajisawa Y, Yoshimura N. Inhibition by tranilast of vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF)-induced increase in vascular permeability in rats. Life Science 1998; 63(4):PL71–PL74 24. Platten M, Wild-Bode C, Wick W, et al. N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast) inhibits transforming growth factor-b release and reduces migration and invasiveness of human malignant glioma cells. Int J Cancer 2001; 93:53–61 25. Nagata R, Arimura S, Samejima H, et al. [Frequent eye irritation study of a short-term and subacute toxicity of tranilast (N-50 ) ophthalmic solution in rabbits]. [Japanese] Kiso-to-Rinsho 1992; 26:3463–3469 26. Kogure F, Ishizaki M, Saiga T. Long-term clinical study of tranilast ophthalmic solution on vernal conjunctivitis. J Clin Ther Med 1993; 9:429–441
27. National Basic Drugs Leading Group. National Basic Drugs, published by People Health Publisher, China Public Health Bureau, August 1999; 680–681 28. Tobari I, Iwaki Y, Miyake K, et al. Effect of tranilast eyedrops in preventing posterior capsule opacification: preliminary report. J Cataract Refract Surg 1999; 25:1394–1399 29. Tani E, Katakami C, Negi A. Effect of various eye drops on corneal wound healing after superficial keratectomy in rabbits. Jpn J Ophthalmol 2002; 46:488–495 30. Ito S, Sakamoto T, Tahara Y, et al. The effect of tranilast on experimental proliferative vitreoretinopathy. Graefes Arch Clin Exp Ophthalmol 1999; 237:691–696 31. Takehana Y, Kurokawa T, Kitamura T, et al. Suppression of laser-induced choroidal neovascularization by oral tranilast in the rat. Invest Ophthalmol Vis Sci 1999; 40:459–466 32. Geroski DH, Edelhauser HF. Drug delivery for posterior segment eye disease. Invest Ophthalmol Vis Sci 2000; 41: 961–964 33. Herrero-Vanrell R, Refojo MF. Biodegradable microspheres for vitreoretinal drug delivery. Adv Drug Deliv Rev 2001; 52: 5–16 34. Kumar MT, Pandit JK, Balasubramaniam J. Novel therapeutic approaches for uveitis and retinitis. J Pharm Pharm Sci 2001; 4:248–254; Available at: http://www.ualberta.ca/wcsps/ JPPS4(3)/J.Balasubramaniam/retinitis.pdf. Accessed July 30, 2007 35. Sattari S, Jamali F. High performance liquid chromatographic determination of cyclooxygenase II inhibitor rofecoxib in rats and human plasma. J Pharm Pharm Sci 2000; 3:312–316; Available at: http://www.ualberta.ca/wcsps/JPPS3(3)/F.Jamali/ Jamali-Rofecoxib.pdf. Accessed July 30, 2007 36. Bohlooli S, Keyhanfar F, Mamoudian M. High performance liquid chromatography of mebudipine: application to pharmacokinetic study. J Pharm Pharm Sci 2001; 4:244–247; Available at: http://www.ualberta.ca/wcsps/JPPS4(3)/M.Mahmoudian(2)/ mebudipine.pdf. Accessed July 30, 2007 37. Friedman DS, Duncan DD, Munoz B, et al. Digital image capture and automated analysis of posterior capsular opacification. Invest Ophthalmol Vis Sci 1999; 40:1715–1726 38. Miyazawa Y, Iwaki Y, Yataka M, Tobari I. [Effect of different concentrations of tranilast on posterior capsule opacification]. [Japanese] Nippon Ganka Gakkai Zasshi 2001; 105:442–446 39. Le Bourlais C, Acar L, Zia H, et al. Ophthalmic drug delivery systemsdrecent advances. Prog Retin Eye Res 1998; 17: 33–58
First author: Man Wang, MD Department of Ophthalmology and EENT, Fudon University, Shanghai, China
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Safety and efficacy of intracapsular tranilast microspheres in experimental posterior capsule opacification Man Wang, MD, Jin-Jun Zhang, PhD, MD, Timothy L. Jackson, PhD, FRCOphth, Xinghuai Sun, MD, Wei Wu, MD, John Marshall, PhD
PURPOSE: To evaluate the safety and efficacy of a sustained-release agent designed to reduce posterior capsule opacification (PCO). SETTING: Department of Ophthalmology, EENT Hospital, Fudan University, Shanghai, Peoples Republic of China. METHODS: Free tranilast (TFree) was incorporated into polylactic acid microspheres and then tested using a rabbit model of PCO. Twenty-nine rabbits were randomized into 5 groups treated with balanced saline solution (BSS control); TFree; or 0.5, 1.0, or 2.0 mg tranilast microspheres (TMicro). Standard phacoemulsification cataract surgery, including manual aspiration of all visible soft lens matter, was performed in all groups. The selected test agent was then injected into the lens capsule. Postoperative clinical examinations were performed at 1, 3, 7, 14, 30, 60, and 90 days. Posterior capsule opacification was quantified using high-resolution computer image analysis at 1, 2, and 3 months. Histological examination was performed at 3 months. RESULTS: Eyes treated with TMicro had significantly less PCO than the eyes in the BSS and TFree groups. While the BSS control eyes had increased PCO over 3 months, eyes in the TMicro group had reduced PCO over time in a dose-dependent fashion. Histological examination showed reduced lens epithelial cell proliferation in the TMicro groups, with no manifest damage to the cornea, iris, or retina compared with the BSS controls. There was a transient increase in postoperative inflammation in all tranilast-treated groups compared with the BSS controls. CONCLUSION: Sustained-release intracapsular tranilast reduced PCO in an experimental model of PCO, suggesting further investigation of its therapeutic potential is justified. J Cataract Refract Surg 2007; 33:2122–2128 Q 2007 ASCRS and ESCRS
Cataract is the world’s leading cause of blindness. Although modern cataract surgery is an extremely effective treatment with a low incidence of serious complications, up to 15% to 50% of eyes develop posterior capsule opacification (PCO) within 3 to 5 years of surgery.1–5 In children and young adults, the incidence of PCO approaches 100%.6 Posterior capsule opacification degrades the image quality and may require laser capsulotomy. As such, it is the most common complication of cataract surgery. Furthermore, many patients in developing nations do not have easy access to laser capsulotomy.7 To date, most attempts at reducing PCO have concentrated on novel biomaterials and intraocular lens design. There are, however, many therapeutic agents that might reduce PCO. In vivo and in vitro studies show that antimetabolites such as mitomycin-C, 2122
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5-fluorouracil, and daunomycin inhibit the proliferation of lens epithelial cells (LECs).8–10 Immunotoxins, such as the monoclonal antibody ricin A, and fibroblast growth factor 2-saporin also inhibit LEC proliferation, although ocular toxicity limits their therapeutic potential.11,12 Most of these drugs were administered by infusing a single dose into the capsular bag during surgery or by subconjunctival injection at the end of surgery. In both circumstances, the concentration of drug declined rapidly.10,13–15 Therefore, to achieve a therapeutic effect, high concentrations were often required, with the attendant risk for ocular toxicity.3,16–18 Tranilast, N-(3,4-dimethoxycinnamoyl)-anthranilic acid, is an antiallergy drug introduced to treat and prevent bronchial asthma and allergic rhinitis. Its effect was thought to be due to its ability to stabilize mast 0886-3350/07/$dsee front matter doi:10.1016/j.jcrs.2007.07.041
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cells, prevent degranulation, and reduce the production of histamine and 5-HTP as well as to reduce inflammatory cell activity and vascular permeability.19 More recent studies show an antifibrotic effect with inhibition of fibroblast proliferation and transformation to myofibroblasts, with reduced collagen production. These effects are mediated by a range of growth factors that are down-regulated by tranilast; these include transforming growth factor b (TGF-b), interleukin 1b, prostaglandin E2, vascular endothelial growth factor (VEGF), and bFGF.20,21 Tranilast also inhibits angiogenesis due to reduced migration and proliferation of microvascular endothelial cells, reduced cellular energy consumption, blocked calcium influx, and inhibition of tube formation, as well as reduced VEGF/ vascular permeability factor.22,23 Tranilast is also thought to have potential as an anticancer drug, with inhibition of malignant neurogliacytes that rely on TGF-b. Malignant cells exposed to tranilast show reduced proliferation, migration, and tissue invasion.24 Tranilast does not, however, appear to inhibit proliferation and collagenation in healthy tissue.20,21 Tranilast eyedrops (Rizaben) are widely used for allergic and seasonal conjunctivitis and are generally well tolerated.25,26 Taken together, these findings indicate that the main pharmacological effects of tranilast result from altered cell migration, differentiation, and proliferation, rather than from direct cytotoxicity. This suggests that tranilast has the potential to reduce PCO without an undue risk for ocular toxicity, and preliminary in vitro and in vivo studies appear to confirm this.22,27,28 Other potential applications include reducing corneal haze after excimer laser ablation, inhibiting scarring after glaucoma filtration surgery, and reducing proliferative vitreoretinopathy and retinal angiogenesis (T. Ishihashi, Y. Aoyama, ‘‘Effect of Tranilast on Conjunctival Bleb Scarring After Filtration Surgery’’ [Japanese], presented at the 103rd meeting of the Japan Ophthalmological Association, Chiba, Japan, April 1999. Abstract in Nippon Ganka Gakkai Zasshi 1999; 103, page 134).29–31
Accepted for publication July 16, 2007. From the Department of Ophthalmology (Wang, Zhang, Sun), EENT Hospital, and School of Pharmacy (Wu), Fudan University, Shanghai, Peoples Republic of China; and the GKT Department of Ophthalmology (Zhang, Jackson, Marshall), Rayne Institute, St. Thomas’ Hospital, London, United Kingdom. No author has a financial or proprietary interest in any material or method mentioned. Corresponding author: Dr. Man Wang, Eye and ENT Hospital, Fudan University, Number 83 FenYang Road, Shanghai, China. E-mail: [email protected].
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Although PCO could be inhibited by tranilast, the therapeutic effect is likely to be limited by the lack of a satisfactory delivery system. Topical delivery has been hindered by a short half-life and poor tissue penetration, with less than 5% transcorneal drug delivery.32 This necessitates extended treatment periods in animal and human eyes.32 It is possible that intraocular delivery of a sustained-release preparation of tranilast could overcome many of the problems and realize the therapeutic potential of tranilast. Biodegradable microsphere delivery systems can be used to produce sustained release of therapeutic agents. The active agent is contained within a microsphere that degrades with time, reducing side effects, improving stability, and enhancing formulation flexibility. In addition, there is no need to surgically remove the delivery system once the drug is released as the microspheres break down into nontoxic products. Studies confirm that microsphere systems are nonirritating, nonmutagenic, and nonallergenic.33 Hence, intracapsular microspheres have the potential to reduce toxicity and produce sustained, local release of an agent designed to prevent PCO. In this study, free tranilast (TFree) was incorporated into microspheres and tested in a rabbit model of PCO to determine the agent’s therapeutic effect and potential toxicity. MATERIALS AND METHODS Preparation of Tranilast Microspheres Tranilast microspheres (TMicro) were prepared using an oil–water emulsion, after which the organic solvent from the inner organic droplets were extracted and evaporated as described previously.34 Briefly, TFree was micronized by sonification and suspended in an organic phase comprising 2 mL of polylactide dichloromethane solution. The outer water phase comprised 100 mL of 4% polyvinyl alcohol solution. Under continuous stirring using a mechanical stirrer (Ika) at 800 revolutions per minute, the organic phase was instilled gradually into the water phase through a 16-gauge needle. After 2 minutes of emulsification, the subsequent oil–water emulsion was diluted with 100 mL of distilled water, with the stirring rate reduced to 500 revolutions per minute. After another 2 minutes, 500 mL of distilled water was added and stirred for a further 1.5 hours to achieve complete extraction and evaporation of dichloromethane. The microspheres were collected through filtration under reduced pressure, washed with distilled water, and stored in a vacuum desiccator for 48 hours before use. Drug loading was adjustable and was tuned to a relatively low level (7.5%) in favor of a more sustained release. Tranilast microspheres were combined with 0.1 mL of 3% hydroxypropyl methylcellulose (HPMC) at the time of surgery to facilitate delivery and retention within the capsular bag.
Animals Thirty-seven New Zealand White rabbits of both sexes were used for the preliminary control and treatment
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experiments. Animals were approximately 10 to 12 weeks of age with an initial body weight of 1.8 to 2.0 kg. The rabbit was chosen because its crystalline lens, as in humans, has an anterior capsule with a single row of cubical epithelial cells from which lens fibers develop. The animals were kept under standardized conditions in separate cages and given tap water and food ad libitum. The experiments adhered to the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research.
Preliminary Control Experiments To ensure the microspheres were effective in providing sustained release, preliminary control experiments were performed in 8 rabbit eyes. Rates of drug elimination from the aqueous humor for TFree (n Z 4) and TMicro (n Z 4) were compared. In both groups, 0.1 mL HPMC 3% containing 0.15 mg TFree or TMicro was injected as a single dose directly into the anterior chamber through the limbus using a 1 mL syringe and 30-gauge needle. Anterior chamber samples (0.1 to 0.2 mL) were taken over 7 days using a 1 mL syringe and 30-gauge needle. The drug concentration was then determined by high-performance liquid chromatography.35,36 Table 1 shows the results in this experiment, which confirmed that the microspheres produced sustained drug release over 7 days and the nonencapsulated tranilast, over 2 days. There was a significant difference in anterior chamber tranilast concentrations at each time point (P!.05; n Z 4).
Treatment Groups Twenty-nine rabbits were divided into 5 groups: a control group (treated with balanced saline solution [BSS]) and 4 treatment groups. The treated animals received a single dose of TFree (treated control) or 0.5, 1.0, or 2.0 mg TMicro. Each group comprised 5 to 6 rabbits.
Surgical Technique Sterile surgical technique was used. Topical phenylephrine hydrochloride 1% and cyclopentolate hydrochloride 0.5% were administered 5 and 10 minutes before anesthesia to dilate the pupils. The animals were anesthetized with an intramuscular injection of xylazine hydrochloride 5 mg/kg body weight and ketamine hydrochloride 50 mg/kg body weight. Topical application of oxybuprocaine hydrochloride was used for local anesthesia. A corneal incision was made with a 3.2 mm calibrated blade (Alcon). Sodium hyaluronate
Table 1. Comparison of therapeutic concentrations and drug elimination of TFree and TMicro from the aqueous humor after direct aqueous humor injection in a rabbit model. Mean Concentration (mg/mL) G SD Group
Day 1
Day 3
Day 5
Day 7
TFree 1.18 G 0.64 0.046 G 0.005 U U TMicro 12.22 G 4.74 0.59 G 0.23 0.45 G 0.04 0.44 G 0.02 TFree Z free tranilast; TMicro Z tranilast microspheres; U Z undetectable concentration of tranilast in anterior chamber
3.0%–chondroitin sulfate 4.0% (Viscoat) (0.1 mL) and sodium hyaluronate 1.0% (Provisc) (0.1 mL) were injected into the anterior chamber. A continuous curvilinear capsulorhexis approximately 5.0 mm in diameter was created with a capsule forceps. The lens was then hydrodissected with BSS and the nucleus emulsified in the bag (Legacy 20000, Alcon). After the residual lens cortical material was removed by irrigation/aspiration, a single dose of the treatment solution (in 0.1 mL HPMC 3%) was injected into the capsule bag with a syringe. The wound was then closed with 10-0 nylon sutures. At the end of surgery, animals received a subconjunctival injection of 20 000 mg gentamicin and 2.5 mg dexamethasone. Postoperative medications included tobramycin 0.3%–dexamethasone 0.1% (TobraDex) and atropine 1% drops 3 times daily for a month.
Clinical Observation A surgeon masked to the groups performed slitlamp examinations at 0, 1, 3, 7, 14, 30, 60, and 90 days. The following were noted: corneal clarity, anterior chamber flare and cells, intraocular pressure (IOP), and abnormal findings.
Image Acquisition and Analysis The degree of PCO was quantified 1, 2, and 3 months postoperatively using previously described techniques.37 Briefly, the posterior capsule was photographed through a dilated pupil using a digital retroillumination camera (SLICPS 2000, LiuLiu Vision Technology) mounted on a slitlamp. The images were downloaded to a personal computer and analyzed using image-processing software (ShengTeng Information Technology). For each photograph, a 4.0 mm diameter region of interest was outlined by the computer in the center of the visual axis, after 3 points were manually chosen on the pupil border. The percentage and density of PCO in the assigned area were analyzed by the image-analysis software, which reverses color and increases color contrast. The area and density of PCO (Figure 1) were used to give an opacity index. The opacity index was calculated by the computer software using the following formula: Opacity indexZðROP =RROI Þ DPCO 100 where ROP is the area of opacity in the 4.0 mm diameter region of interest; RROI is the area of the 4.0 mm diameter region of interest, and DPCO is the mean value of the density of the PCO area (average positive density).
Light and Electron Microscopy After the 3-month clinical follow-up, all rabbits were humanely killed. The operated eyes were enucleated and placed in Karnovsky solution (paraformaldehyde and glutaraldehyde mixture). After the 12 o’clock position was marked on the sclera using tissue-marking dye, each globe was sectioned in the coronal plane just anterior to the equator. Histological sections were prepared and stained with hematoxylin–eosin and periodic acid-Schiff and then observed with a light microscope (Olympus). Photomicrographs were taken as required. All eyes had transmission electron microscopy (TEM) to detect ultrastructural damage. For TEM, the specimens were fixed in cacodylate glutaraldehyde 2%, postfixed in osmium tetroxide 2%, dehydrated through a graded series of ethanol, and embedded in EmBed 812 (EM Sciences). Ultrathin sections of the cornea and iris were examined using TEM (CM120, Philips).
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decreasing to 9.8% in the TMicro group (Figure 2). The inhibition of PCO was confirmed by histological examination. Figure 3, A, shows a rabbit eye treated with TMicro that had little lens capsule opacification 3 months postoperatively and a smooth posterior lens capsule observed on histology (Figure 3, B). In contrast, eyes treated with TFree showed some fibrous membrane in the pupil area (Figure 3, C) and significant LEC proliferation on histological examination (Figure 3, D).
Figure 1. Posterior capsule opacification measurement. A: Original slitlamp retroillumination digital image from a rabbit eye shows the pupil area and capsulorhexis. B: Raw image of A with the capsule mask applied, thereby defining the area of the posterior capsule within the 4.0 mm capsulorhexis. This area was used for subsequent image analysis. C: Contrast enhancement with reversing colors of the image within the capsulorhexis. D: Segmented image of A showing an area of PCO in red.
Statistical Analysis Raw data were analyzed using SPSS statistical software (version 11.0, SPSS, Inc.). The Student-Newman-Keuls test (q test) was applied to determine the significance of differences between groups; a P value less than 0.05 was considered statistically significant.
RESULTS Treatment Effect Although PCO was significantly reduced in both tranilast-treated groups (TFree and TMicro), the use of microspheres reduced PCO by 3.7-fold compared with the use of TFree. The mean opacity index was 39.31 G 20.591 (SEM) in the TMicro group (n Z 6) compared with 199.64 G 116.806 in the TFree group (n Z 5; P!.05) and 399.47 G 182.874 in the BSS control group (n Z 6; P!.05) (Table 2). Posterior capsule opacification in the TFree group was 36.5% of that in the BSS control, Table 2. Opacity index values 3 months postoperatively in the 3 groups. Opacity Index Rabbit
Control
TFree
TMicro
1 2 3 4 5 6
333.47 280.52 649.78 148.71 351.76 632.58
82.62 209.28 415.00 178.97 112.32 d
31.10 30.18 23.97 81.78 22.04 46.79
Control Z BSS; TFree Z free tranilast; TMicro Z tranilast microspheres
Time Dependency Over time, the PCO rates increased significantly in the control group and decreased significantly in the tranilast-treated groups. The mean opacity index in the BSS control group was 215.6 G 170.1 at 1 month, increasing to 399.5 G 182.9 at 3 months. Clinical observations were consistent with this finding, with posterior capsule fibrosis apparent in 2 of 6 eyes at 1 month and 4 of 6 eyes at 3 months. In contrast, eyes that received a single dose of TMicro had a time-dependent decrease in PCO (Figure 4). For example, the 6 eyes that received 1 mg TMicro had a mean opacity index of 41.6 G 25.1 at 1 month, 30.2 G 19.6 at 2 months, and 26.7 G 14.1 at 3 months (all P!.05). Dose Dependency Figure 4 shows that TMicro reduced PCO in a dosedependent fashion. For example, in the first month after surgery, the mean opacity index was 57.6 G 17.0 at a concentration of 0.5 mg, decreasing to 41.6 G 25.07 at 1.0 mg and 30.5 G 16.8 at 2.0 mg (all n Z 6 and P!.05). This dose-dependency persisted at 3 months. Side Effects There were no manifest systemic effects in that the animals did not show behavioral signs suggesting pain and gained weight normally. Biomicroscopic examination of all rabbit eyes revealed mild to moderate corneal haze daily for the first week after surgery. By 10 days postoperatively, the central haze had resolved, but a 1.0 to 2.0 mm area of haze persisted at the superior entry site. There were some differences in the inflammatory response between control eyes and treated eyes. Control eyes had postoperative inflammation in the form of iris hyperemia, fibroid exudates, and anterior chamber flare (Tyndall score C to CC), which peaked 1 day postoperatively and resolved in 5 to 7 days. All eyes treated with tranilast had elevated levels of postoperative anterior chamber flare (Tyndall score CC to CCC), fibroid exudates, and iris hyperemia, which peaked at 3 days and resolved in 7 to 10 days. Table 3 shows the IOP 1 day through 7 days postoperatively. The IOP was in the normal range (11 to
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Figure 2. Comparison of eyes treated with TFree (left column) and eyes treated with TMicro (right column). Posterior capsule opacification was assessed using image analysis to give an opacity index, expressed as a percentage of the negative control (mean G SEM; n Z 6). The results are from the 3-month time point and show a significant reduction in the group treated with TMicro compared with the TFree group (P!.05, paired t test).
Figure 4. Time and dose dependency of TMicro on rabbit PCO. The mean of the opacity index taken from controls and the 3 TMicro groups 1, 2, and 3 months after treatment are plotted. Note that over time, the opacity index increases in the control group but decreases in the treated groups (mean G SEM; n Z 6) (P!.05, all groups compared with control group).
24 mm Hg), except in the group treated with 2.0 mg TMicro, in which the IOP was slightly higher at 1 day. Histological examination did not reveal a significant difference between the control group and treated groups. No animals had evidence of endothelial cell loss or damage 3 months after surgery. There was loss of iris epithelial and stromal pigment with a vigorous melanophagic reaction, but no evidence of mitosis or cell destruction in the iris epithelium, in all groups.
There was no epithelial or stromal cell loss in the ciliary body. The retina and choroid did not show significant damage on microscopic examination (Figure 5).
Figure 3. High-resolution histology images of rabbit eyes treated with both forms of drug-delivery systems and the corresponding histological capsule sections 3 month after treatment. A: Retroillumination image of a rabbit eye treated by TMicro shows a relatively clear pupil (* Z focal area of opacification). B: Corresponding histological section of the lens capsule bag shows a smooth capsule and no significant LEC proliferation (original magnification 200). C: Image of a rabbit eye treated with TFree shows the development of PCO and focal opacities (*). The short arrow shows the light reflex. D: Histological section of the lens capsule in C shows significant LEC proliferation (arrowheads). The arrows point to the posterior lens capsule (original magnification 100).
DISCUSSION Human28 and animal38 studies indicate that PCO can be inhibited by the long-term administration of tranilast eyedrops; however, this imposes considerable inconvenience to patients. Bioavailability is also low, with only 5% of topical treatment reaching the intraocular tissues.29,32 This is not unexpected as there are significant barriers to ocular penetration. First, the tear meniscus dilutes medications and its relatively low capacity may result in overspill of tears when eyedrops are instilled. Eyedrops can also cause ocular discomfort with reflex lacrimation that may remove most drugs from the ocular surface within 5 to 7 minutes. This may aggravate systemic absorption and side effects.39 Corneal structure further impairs absorption. The relatively lipophilic corneal epithelium, which has low porosity and high tortuosity due to tight annular junctions, is the main barrier to hydrophilic drugs, whereas the middle stromal layer, which consists mainly of water interspersed with collagen fibrils,
Table 3. Effects of TMicro on IOP. Mean IOP (mm Hg) G SEM TMicro Dose (mg) 2.0 1.0 0.5 0.0
Day 1
Day 3
Day 7
20.00 G 6.66 16.00 G 5.73 13.33 G 5.24 15.71 G 5.71
14.00 G 5.66 12.67 G 2.73 13.33 G 3.83 14.50 G 5.32
13.00 G 2.37 12.83 G 3.19 13.33 G 2.66 13.67 G 3.78
IOP Z intraocular pressure; TMicro Z tranilast microspheres
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this organic solvent induces anterior chamber reaction in the early period after surgery before it is completely metabolized. Although there was some clinical evidence of increased inflammation in the treated groups, this was transient and not associated with histological changes. This suggests tranilast may be safe for intraocular use; however, further safety testing is appropriate. In conclusion, this study indicates that intracapsular delivery of a sustained-release preparation of tranilast reduced PCO in rabbits for up to 3 months, with no histological evidence of tissue damage compared with control eyes. Further studies are needed to determine the lowest effective concentration and the longterm safety profile.
REFERENCES
Figure 5. Histology of cornea and retina 3 months after treatment with TMicro (1 mg). A: Control retina with a similar appearance to treated retina in B. Treated cornea (D) also appears similar to the control cornea (C). The arrows point to the retinal pigment epithelium and corneal endothelial cells (original magnification 40).
impedes the diffusion of lipophilic drugs. This suggests that an alternative delivery system is preferable. We present the results of one alternative, tranilast. Tranilast is a well-tolerated medication with multiple effects including inhibition of cell migration, proliferation, and fibrosis.33 Free tranilast was placed in microspheres and then delivered directly into the capsular bag at the end of cataract surgery. Local delivery has the potential to increase bioavailability at the site of action, while the use of microspheres produces sustained delivery that would be expected to increase the therapeutic effect and further reduce the need for high, single-dose treatment. The microsphere delivery system produced 90% less PCO than the negative control and 50% less PCO than unencapsulated free drugs. The presence of a dose-response curve supports the hypothesis that the effect was drug related. In contrast to the negative control, eyes treated with TMicro eyes had reduced PCO with time, suggesting sustained release of tranilast. Regarding the anterior chamber reactions in all groups (reaction was more severe and lasted longer in treated groups), we think that both the suspension and microspheres, acting as foreign objects, result in severe anterior chamber reaction. Moreover, during their manufacture (emulsion, evaporation, and extraction), a small amount of organic solvent remained in the microspheres. We must further study whether
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LABORATORY SCIENCE: INTRACAPSULAR TRANILAST MICROSPHERES IN PCO
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First author: Man Wang, MD Department of Ophthalmology and EENT, Fudon University, Shanghai, China
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