Effects of Fluorouracil and Fluorouridine on Protein Synthesis in Rabbit Retina

John A. Leon, James M. Dritt, Richard H. Hopp, Richard P. Mills, and Ann H. Milam

5-Fluorouracil (5-FU) and its active metabolite 5-fluorouridine (FUR) are currently being evaluated for the treatment of proliferative vitreoretinopathy and the control of scarring after glaucoma filtering procedures. To test for retinal toxicity, the authors examined the effect of intravitreal injections of 5-FU and FUR on protein synthesis in rabbit retinal photoreceptors and ganglion cells. In addition, the toxic effect of subconjunctival 5-FU injections, after a trephine filtering procedure, on ganglion cell protein synthesis was examined. Albino rabbit eyes were given either unilateral intravitreal injections of 1 mg of 5-FU, 2.5 mg of 5-FU, or 0.1 mg of FUR, or subconjunctival injections of 3 mg of 5-FU twice daily after a trephine procedure. Quantitative autoradiography was used to study ganglion cells and photoreceptor outer segment renewal, and scintillation counting was used to quantify newly synthesized protein transported axonally from ganglion cell bodies to the superior colliculus (SC). Marked reduction of labeled protein reaching the SC was noted after either intravitreal 0.1 mg of FUR (41% inhibition after a single injection and 53% after two injections) or 2.5 mg of 5-FU (41% after one injection and 26% after two injections). This reduction was still present after 8 days in eyes receiving 0.1 mg of FUR (32%) and 2.5 mg of 5-FU (22%). Quantitative autoradiography of retinal photoreceptors and ganglion cells corroborated these data, demonstrating inhibition of outer segment renewal after one or two injections of either 0.1 mg of FUR or 2.5 mg of 5-FU. This inhibitory effect was statistically significant using the paired t-test for both drugs. No mean inhibition was observed after intravitreal 1 mg of 5-FU injections or after subconjunctival injections of 5-FU. Invest Ophthalmol Vis Sci 31:1709-1716, 1990

The drug 5-fluorouracil (5-FU) is potent antime- coma filtration procedures in animal models1' and in tabolite that inhibits intraocular fibroblast and retinal initial clinical studies.12"15 It is currently being evalu- pigment epithelium proliferation and contraction in ated in a multicenter clinical trial. animal models1"5 and in vitro.6"9 Currently 5-FU is The 5-FU alters DNA synthesis through inhibition under evaluation for treatment of proliferative vit- of the enzyme thymidylate synthetase and decreases reoretinopathy (PVR), and it was found to signifi- RNA function secondary to incorporation of 5-FU cantly reduce the incidence of traction retinal de- metabolites into newly synthesized RNA.16 The anti- tachment (TRD) produced by injection of cultured proliferative and anticontractile effects of 5-FU are fibroblasts into the vitreous chamber of animal eyes. probably due to production of altered RNA.17 Fluo- A dose of 1 mg of 5-FU injected intravitreally re- rouridine (FUR) is the key 5-FU metabolite that af- duced TRD in nonvitrectomized rabbit eyes.1'2'5 fects RNA synthesis and is up to 100 times more Multiple intravitreal injections of 0.5 mg of 5-FU effective than 5-FU in inhibiting fibroblast prolifera- into vitrectomized rabbit eyes have also been effica- tion in vitro.18 cious against PVR.3 A pilot study on patients with Toxicity of 5-FU and FUR has been assessed by PVR demonstrated a moderate success rate of 65% use of light and electron microscopy and by electro- for retinal reattachment after intravitreal 5-FU.10 physiology. We evaluated retinal toxicity of intravit- When applied topically or injected subconjunctivally, real and subconjunctival injection of 5-FU and FUR 5-FU also inhibits postoperative scarring after glau- in rabbits. Since ganglion cells and photoreceptors have high rates of protein synthesis, we measured the From the Department of Ophthalmology, University of Wash- effects of these drugs on protein synthesis and axonal ington School of Medicine, Seattle, Washington. transport in retinal ganglion cells and on outer seg- Supported in part by NIH Grants EY01730 and EY01311; the Lewis Berkowitz Family, National Retinitis Pigmentosa and ment (OS) renewal in photoreceptors. Chatlos Foundations; and in part by an award from Research to Prevent Blindness, Inc. (R.P.B.). A.H.M. is a Senior Scholar of Materials and Methods R.P.B. Reprint requests to Richard P. Mills, MD, Department of Oph- Fifty-three albino rabbits (2-3 kg) were used for thalmology RJ-10, University of Washington, Seattle, WA 98195. this study. This study was conducted in accordance

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with the ARVO Resolution on the Use of Animals in poral to the optic disc. Inhibition of protein synthesis Research. The eyes of 24 rabbits were anesthetized in ganglion cells was determined by comparing topically with proparacaine 0.5%, and the right eyes counts from a drug-treated eye with the companion (OD) received two intravitreal injections 24 hrs apart control eye injected with saline.21 of 1.0 mg of 5-FU, 2.5 mg of 5-FU, or 0.1 mg of FUR, diluted in 0.1 ml of 0.9% saline, doses previously Outer Segment Renewal in Photoreceptors thought to be nontoxic to rabbit or primate ret- 101819 inas. The needle tip was positioned just anterior Quantitative autoradiography was also used to as- 20 to the optic disc in the manner of Orcutt et al. A sess effects of 5-FU and FUR on protein synthesis in second group of seven rabbits underwent a bilateral retinal photoreceptors. Rod outer segment (ROS) re- Elliot trephine filtering procedure after sedation with newal occurs continuously, reflecting a high level of intramuscular ketamine and Rompun. A 1.0-mm protein synthesis in the rod inner segment and inser- trephine was used, straddling the limbus, followed by tion of newly synthesized protein as a band in the conjunctival coverage with a fornix-based flap. These ROS.22 Fifteen rabbits from the first group and 22 rabbits received subconjunctival injections OD of 3 rabbits from the third group were anesthetized with mg of 5-FU in 0.3 ml of 0.9% saline 180° from the sodium thiopental and perfused intravascularly with surgery wound. These injections were repeated twice 4% paraformaldehyde and 0.5% glutaraldehyde in daily for 7 days, according to the method of Gressel et 0.13 M phosphate buffer 72 hrs after intravitreal in- al," using intramuscular ketamine and Rompun for jection of 3H-leucine. The eyes were immediately anesthesia. Left eyes served as controls in all rabbits enucleated and immersed in the fixative for 24 hr. and received equivalent volumes of saline injected The anterior third of each eye was removed, and the intravitreally or subconjunctivally. Twenty-four retina was sectioned and processed as described hours after the last drug or saline injection, all eyes above for autoradiography. Autoradiographic grains 3 were injected intravitreally with 150 ^Ci H-leucine were counted per unit area of radioactive ROS band (New England Nuclear). A third group of 22 rabbits in a standardized segment of retina, just temporal to received only one injection of 2.5 mg of 5-FU or 0.1 the optic disc, to determine if photoreceptor protein mg of FUR into the midvitreous cavity OD under synthesis, as evidenced by OS renewal, had been af- ophthalmoscopic control. Again, the left eye served fected by prior drug treatment. as a control and received an equivalent volume of saline injected intravitreally. After drug exposure times of 1 day for 10 rabbits and 8 days for 12 rabbits, Protein Synthesis In all eyes were injected intravitreally with 3H-leucine. All rabbits showing evidence of leakage from the in- Ganglion Cells 30 jection site were excluded from the study. 21.8 i Protein Synthesis in Ganglion Cells 20-: 17.5 1 Quantitative light microscopic autoradiography 10- was used to determine the effects of 5-FU and FUR 21 1 on protein synthesis in ganglion cells of the retina. ^ 3 Four hours after intravitreal injection of H-leucine, C nine rabbits from the first group were killed with an -10- overdose of sodium thiopental; the eyes were imme- C retina with underlying choroid and sclera were em- -30- bedded in methacrylate (Sorvall). Sections 2.5 nm in

thickness were processed for autoradiography, in- -40 cluding standardized dipping in photographic emul- 1.0mg. 5-FU 2.5mg. 5-FU 0.1 mg. FUR sion, exposure of all slides for 24 hr, and simulta- Drug Tested neous photographic processing. The slides were then dried and stained with Richardson's methylene blue- Fig. 1. Inhibitory effects of 5-FU and FUR on protein synthesis azure II mixture. Autoradiographic grains were in ganglion cells of the rabbit retina. Mild inhibition was found after two intravitreal injections of 2.5 mg 5-FU and 0.1 mg FUR. counted per unit cytoplasm of ganglion cells from a No reduction was found after intravitreal injection of 1.0 mg 5-FU. standardized segment of the retina immediately tem- The vertical bars represent means ± SEM.

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Fig. 2. Photomicrographs of rabbit retinas demonstrating incorporation of 3H-leucine into ganglion cell protein (originals X800). (A) Control retina. (B) Retina after two intravitreal injections of 0.1 mg FUR. Note moderate de- crease in amount of granules over ganglion cell somata.

Axonal Transport of Newly Synthesized Protein bits from the second group, and 22 rabbits from the third group were perfused with 4% paraformaldehyde Liquid scintillation counting was used to quantify and 0.5% glutaraldehyde in 0.13 M phosphate buffer 3H-leucine-labeled protein transported axonally by 72 hr after intravitreal injection of both eyes with ganglion cells to the contralateral superior colliculus 3H-leucine. Uniform samples were taken from both (SC).23 Albino rabbits have nearly total crossing of SC and the cerebellum, the latter sampled as a control optic axons at the optic chiasm, so that axons from a for blood-borne label, using a 3-mm trephine set at a drug-treated (right) eye terminate in the contralateral depth of 1.5 mm. The samples were digested over- (left) SC. Fifteen rabbits from the first group, 7 rab- night in 1 ml of Protosol at 50°C, mixed with 200 ml

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of H2O2 and 40 ml of glacial acetic acid, followed by Outer Segment Renewal 10 ml of Scintiverse, and quantified (cpm/mg) using a Beckman LS 3801 liquid scintillation counter. The In Photoreceptors 47.7 percentage of inhibition of axonal transport was de- 44.8 termined from the ratio: 50- 1 c 40- % Inhibition o • 1 _ (cpm/mg right SC - C) - (cpm/mg left SC - C) 30- Ibiti (cpm/mg right SC - C) J= 20- c Results 10- 0- Protein Synthesis in Ganglion Cells •ceni -_ 1 a> -3.3 Intravitreal injections of 2.5 mg of 5-FU and 0.1 a. -10- mg of FUR produced inhibition of protein synthesis -20- in ganglion cell somata (Figs. 1, 2). A mean of 22% inhibition (range, 19-25%) was found after two daily -30- intravitreal injections of 0.1 mg of FUR, and 17% 1.0mg. 5-FU 2.5mg. 5-FU 0.1 mg. FUR inhibition of protein synthesis (range, 10-31%) was Drug Tested noted after two injections of 2.5 mg of 5-FU. Only the Fig. 3. Inhibitory effects of 5-FU and FUR on protein synthesis FUR results were statistically significant using the in rabbit retina photoreceptors after two intravitreal injections. paired t-test (P < 0.02). Intravitreal injections of 1.0 Marked inhibition was noted following intravitreal 0.1 mg FUR mg of 5-FU caused no significant inhibition of pro- and 2.5 mg 5-FU, with no effect following 1.0 mg 5-FU. The tein synthesis, with a mean negative inhibitory re- vertical bars represent means ± SEM. sponse of 15% (range, -52-+4%). given, the mean inhibitory effect was 41% (range, OS Renewal in Photoreceptors 18-71%) and 41% (range, 12-59%), respectively, which were both statistically significant (P < 0.05). Marked inhibition of incorporation of newly syn- The mean effect persisted, although somewhat di- thesized protein into the OS of retinal photoreceptors minished, when remeasured at 8 days, 32% (range, occurred after two intravitreal injections of 0.1 mg of —15—f-57%) for 0.1 mg of FUR and 22% (range, FUR (45%; range, 36-58%) and 2.5 mg of 5-FU -9-+57%) for 2.5 mg of 5-FU, neither of which was (48%; range, 35-55%) (Figs. 3, 4); both were statisti- statistically significant (Fig. 7). A smaller and not sta- cally significant (P < 0.3). A similar inhibitory effect tistically significant mean inhibitory effect of 13% was seen after a single intravitreal injection of 0.1 mg was noted after two intravitreal injections of 1 mg of of FUR (35%; range, 3-70%) or 2.5 mg of 5-FU (35%; 5-FU, and this dosage produced a wide range of range, —3-+69%), which persisted up to 8 days, for values (—64%-+78% inhibition). Twice-daily sub- both 0.1 mg of FUR (43%; range, 8-68%; P < 0.03) conjunctival injections of 5-FU for 1 week after a and 2.5 mg of 5-FU (25%; range, -19-+50%; P trephine filtering procedure did not produce signifi- < 0.10; Fig. 5). There was essentially no effect after cant reduction of axonally transported protein (-7%; injections of 1.0 mg of 5-FU (-3% inhibition), but a range, -34-+40%). wide range of values was obtained (—58-+54%). Discussion Axonal Transport The drug 5-FU is metabolized to 5-fluorodeoxy- Liquid scintillation counting of the SC revealed uridine monophosphate, a potent and irreversible in- greatest reduction of axonally transported 3H-leu- hibitor of thymidylate synthetase. The 5-FU and cine-labeled protein after two intravitreal injections FUR are also converted to FUR triphosphate which, of 0.1 mg of FUR, with an average of 53% inhibition when incorporated into RNA, inhibits the processing (range, 33-73%; Fig. 6). Less inhibition was found of messenger RNA and ribosomal maturation.1724 after two intravitreal injections of 2.5 mg of 5-FU Retinal tissue, being of neural origin, is comprised of (26%; range, 9-41%). When only one intravitreal in- nonreplicating cells so that drugs which inhibit DNA jection of 0.1 mg of FUR or 2.5 mg of 5-FU was synthesis would appear less likely to be toxic. How-

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f*.r'

Fig. 4. Photomicrographs of rabbit retinas demonstrating incorporation of 3H-leucine into band of newly synthesized protein in rod outer segments (originals X800). (A) Control retina. (B) Retina after two intravitreal injee£j,ons of 0.1 FUR. Note weak and disrupted band of granules in the outer segments.

ever, high rates of protein synthesis occur in several swelling and loss of substructure in axons and mito- cell types of the retina, particularly photoreceptor chondria of the outer plexiform layer, and loss of and ganglion cells. Therefore, one would expect that ribosomes in all neuron types.1'2425 Since the effec- the retinotoxic effects of 5-FU would be secondary to tiveness of an anti-PVR drug that acts by altering inhibition of RNA maturation and most deleterious nucleic acid function is based on a balance between to cells with high rates of protein synthesis. This is inhibition of intraocular cellular proliferation and consistent with previous studies, where toxic doses of toxic effects on the retina, an understanding of drug 5-FU produced ultrastructural changes suggesting in- effects on protein synthesis is essential. hibition of retinal protein synthesis, including OS Several studies evaluate the retinotoxicity of 5-FU disruption, swelling of inner segment mitochondria, in animal models using other methods.210-19'25-26 Blu-

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Outer Segment Renewal Axonal Transport In Photorecepters

One Day • One Day Eight Days E3 Eight Days

0.1mg. FUR 2.5mg. 5-FU 0.1m,, 2.5nu Drug Tested Drug Tested Fig. 7. Inhibitory effects of 5-FU and FUR on axonal transport of Fig. 5. Inhibitory effects of 5-FU and FUR on protein synthesis newly synthesized protein by retinal ganglion cells. Scintillation in retinal photoreceptors after a single intravitreal injection. counting of superior colliculi revealed a similar reduction 24 hr Marked inhibition was found on the first day following intravitreal after a single intravitreal injection of either 0.1 mg FUR or 2.5 mg 0.1 mg FUR and 2.5 mg 5-FU. The inhibitory effect was persistent 5-FU. The results at 8 days demonstrated persistence of this inhibi- when measured at 8 days after injection of either drug. The vertical tory effect after injection of either drug. The vertical bars represent bars represent means ± SEM. means ± SEM.

menkranz and associates10 found that 5-FU was not tion of 1.0 mg of 5-FU.2'25 The FUR appeared nontoxic to rabbit retina, as assessed by light and electron toxic to the retina after an intravitreal injection of 0.1 microscopy and electrophysiology after a single in- mg, while doses of 1 mg and 10 mg of FUR were toxic travitreal dose of 2.5 mg in nonvitrectomized eyes or to the retina and retinal pigment epithelium, as as- after subconjunctival injections of 10 mg daily for 7 sessed by light microscopy and electroretinography.18 days. Barrad et al19 found 1.0 mg of intravitreal 5-FU Our data showed marked inhibition of protein syn- to be nontoxic in primate eyes. However, other stud- thesis after one or two intravitreal injections of 0.1 ies note retinal toxicity in rabbits using light and mg of FUR and 2.5 mg of 5-FU using three different electron microscopy after a single intravitreal injec- methods for evaluating protein synthesis in the rabbit retina. This effect was statistically significant for 0.1 mg of FUR and 2.5 mg of 5-FU with at least two of Axonal Transport the three methods used, and was persistent for both drugs when tissue was reevaluated 8 days after the injection. After two intravitreal injections of 1.0 mg of 5-FU, no net inhibitory effect on protein synthesis c was found in ganglion cell somata and photoreceptor .2 40 OS, and only a small negative effect was noted on axonal transport. However, the results were widely Sub-conjunctival Intravitreal scattered after injections of 1.0 mg of 5-FU, especially compared with the more consistent results obtained from the other doses and injections. Therefore, although the results could indicate that 1.0 mg is a nontoxic dose of 5-FU, the variability could also indicate that this concentration of 5-FU is close to the 3.0mg. 5-FU 1.0mg. 5-FU 2.5mg. 5-FU 0.1mg. FUR threshold for drug toxicity of the rabbit retina. Drug Tested Historically, toxicity studies using intravitreal in- Fig. 6. Inhibitory effects of 5-FU and FUR on axonal transport of jections in nonvitrectomized eyes yield results with newly synthesized protein by retinal ganglion cells. Scintillation significant levels of variability. Injection techniques, counting of superior colliculi revealed the greatest mean reduction of axonally transported 3H-leucine-labeled protein after two intra- differential transport of tritiated amino acids or phar- vitreal injections of 0.1 mg FUR with less inhibition noted after 2.5 macologic agents across the retina at various ana- mg 5-FU. The vertical bars represent means ± SEM. tomic points, occasional leakage of material from the

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injection site, and interanimal variability all compli- view of this manuscript; Dan Possin, Ingrid KJock, and cate the interpretation of data. Faridah Dahlan for technical assistance; and Brad Clifton, Orcutt et al20 described a two-person injection Ron Jones, Chuck Stephens, and Pat Siedlak for photographic help. technique that produces equal transport of radioac- tivity to the SCs of rabbits after bilateral intravitreal References injections. Their technique of injecting over a standardized region of the retina yielded reproducible re- 1. Blumenkranz MS, Ophir A, Claflin AJ, and Hajek A: Fluoro- uracil for the treatment of massive periretinal proliferation. sults with acceptable variability (1.5% to ± 4.8). Our Am J Ophthalmol 94:458, 1982. data and that of Orcutt et al (personal communica- 2. Binder S, Riss B, Skorpik Ch, and Kulnig W: Inhibition of tion) demonstrated significant interanimal variabil- experimental intraocular proliferation with intravitreal 5-fluo- ity. However, the reproducibility of results with min- rouracil. Graefes Arch Clin Exp Ophthalmol 221:126, 1983. imal variability between eyes of the same rabbit argue 3. Stern WH, Lewis GP, Erickson PA, Guerin CJ, Anderson DH, Fisher SK and O'Donnell JJ: Fluorouracil therapy for prolifer- that the technique is reproducible. Interanimal vari- ative vitreoretinopathy after vitrectomy. Am J Ophthalmol ability did not affect the statistical outcome since 96:33, 1983. both the treated and control eyes of an animal were 4. Hatchell DC, McAdoo T, Sheta S, King, III RT, and Barto- affected equally. lome J V: Quantification of cellular proliferation in experimental proliferative vitreoretinopathy. Arch Ophthalmol 106:669, Use of 5-FU to reduce postoperative scarring after 1988. a glaucoma filtering procedure presently requires nu- 5. Joondeph BC, Peyman GA, Khoobehi B, and Yue BY: Lipo- merous subconjunctival injections of the drug over some-encapsulated 5-fluorouracil in the treatment of prolifera- several weeks.13'27 A single subconjunctival injection tive vitreoretinopathy. Ophthalmic Surg 19:252, 1988. of 6.25 mg of 5-FU in a rabbit produced an intravit- 6. Fiscella R, Peyman GA, Elvert J, and Yue BY: In vitro evalua- real peak level of 0.5 /ug/ml,28 which appears small tion of cellular inhibitory potential of various antineoplastic drugs and dexamethosene. Ophthalmic Surg 15:411, 1984. when compared with the peak vitreous level of 664 7. Blumenkranz MS, Claflin A, and Hajek AS: Selection of thera- ^g/ml after an intravitreal injection of 1 mg of peutic agents for intraocular proliferative disease: Cell culture 5-FU.29 However, toxicity might accrue after multi- evaluation. Arch Ophthalmol 102:598, 1984. ple injections to produce sustained drug levels, espe- 8. van Bockxmeer FM, Martin CE, and Constable IJ: Models for cially after a filtering procedure which may increase assessing scar tissue inhibitors. Retina 5:47, 1985. 9. Rivera AH, Hajek AS, Fantes F, Malick KS, and Parish, II RK: intraocular drug levels by breakdown of the blood- Trifluorothymidine and 5-fluorouracil: Antiproliferative activ- retinal barrier and/or by producing a direct route of ity in tissue culture. Can J Ophthalmol 22:13, 1987. drug entry into the eye. We found no inhibition of 10. Blumenkranz M, Hernandez E, Ophir A, and Norton EWD: protein synthesis in the rabbit retina after repeated 5-Fluorouracil: New applications in complicated retinal de- subconjunctival injections of 5-FU following a tre- tachment for an established antimetabolite. Ophthalmology 91:122, 1984. phine filtering procedure. This corroborates previous 11. Gressel MG, Parrish RK, and Folberg R: 5-Fluorouracil and findings in nonoperated rabbit eyes; there was no tox- glaucoma filtering surgery: I. An animal model. Ophthalmol- icity to the retina after subconjunctival injections of ogy 91:378, 1984. 10 mg of 5-FU given daily for 7 days." 12. Heuer DK, Parrish RK, Gressel MG, Hodapp E, Palmberg PF, In summary, 5-FU injected subconjunctivally after and Anderson DR: 5-Fluorouracil and glaucoma filtering surgery: II. A pilot study. Ophthalmology 91:384, 1984. a trephine filtering procedure did not inhibit retinal 13. Heuer DK, Parrish RK, Gressel MG, Hodapp E, Desjordins protein synthesis in this animal model. On the other DC, Skuta GL, Palmberg PF, Nevarez JA, and Rockwood EJ: hand, this study documented statistically significant 5-Fluorouracil and glaucoma filtering surgery: III. Interme- inhibition by intravitreally injected 5-FU and FUR of diate follow-up of a pilot study. Ophthalmology 93:1537,1986. protein synthesis in photoreceptors and ganglion cells 14. Rockwood EJ, Parrish RK, Heuer DK, Skuta GL, Hodapp E, Palmberry PF, Gressel MG, and Fever W: Glaucoma filtering of rabbit retina. This protein synthesis inhibition per- surgery with 5-fluorouracil. Ophthalmology 94:1071, 1987. sisted at least 8 days after injection of 5-FU and FUR, 15. Ruderman JM, Welch DB, Smith MF, and Shoch DE: A pro- suggesting a long-term effect at dosage levels noted in spective, randomized study of 5-fluorouracil and filtration some studies to be "nontoxic." Further studies are surgery. Trans Am Ophthalmol Soc 85:238, 1987. indicated to determine the long-term significance of 16. Wilkinson DS and Pitot HC: Inhibition of ribosomal ribonu- cleic acid maturation in Novikoflf hepatoma cells by 5-fluoro- this inhibition of rabbit retinal protein synthesis. uracil and 5-fluorouridine. J Biol Chem 248:63, 1973. Key words: fluorouracil, fluorouridine, glaucoma, prolifer- 17. Heath TD, Lopez NG, Lewis GP, and Stern WH: Fluoropyri- ative vitreoretinopathy midine treatment of ocular cicatricial disease. Invest Ophthal- mol Vis Sci 27:940, 1986. Acknowledgments 18. Blumenkranz MS, Hartzer MK, and Hajek A: Selection of therapeutic agents for intraocular proliferative disease: II. Dif- The authors thank Doctors Robert E. Kalina, James L. fering antiproliferative activity of the fluoropyrimidines.Arc h Kinyoun, and the late D. Frank Milam, Jr, for critical re- Ophthalmol 105:396, 1987.

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19. Barrad A, Peymkan GA, Case J, Fisherman G, Thomas A, and tured cells: Protection from 5-fluorouracil cytotoxicity by pu- Fiscella R: Evaluation of intravitreal 5-fluorouracil, vincris- rines. Mol Pharmacol 15:357, 1979. tine, VP161, doxorubicine and thiotepa in primate eyes. Oph- 25. Kulnig W, Binder S, Riss B, and Skorpik CH: Inhibition of thalmic Surg 15:767, 1984. experimental intraocular proliferation with intravitreous 5-flu- 20. Orcutt JC, Possin D, and Bunt-Milam AH: Standardized in- orouracil: A transmission electron-microscopic study in rab- travitreal injections: Evaluation of the effect of anesthesia on bits. Ophthalmologica 188:248, 1984. rapid axonal transport in the optic nerve. Exp Eye Res 47:621, 26. Stern WH, Guerin CJ, Erickson PA, Lewis GP, Anderson DH, 1988. and Fisher SK: Ocular toxicity of fluorouracilafte r vitrectomy. 21. Bunt AH: Protein synthesis in ganglion cells of the rabbit ret- Am J Ophthalmol 96:43, 1983. ina after intravitreous injection of vinblastine. Invest Ophthal- 27. Heuer DK, Gressel MG, Parrish RK, Folberg R, Dillberger JE, mol 12:467, 1973. and Altman NH: Topical fluorouracil: II. Postoperative ad- 22. Bok D: Retinal photoreceptor-pigment epithelium interac- ministration in an animal model of glaucoma filtering surgery. tions. Invest Ophthalmol Vis Sci 26:1659, 1985. Arch Ophthalmol 104:132, 1986. 23. Bunt-Milam AH: The effects of drugs on axonal transport. In 28. Rootman J, Tisdall J, Gudauskas G, and Osfry A: Intraocular Applied Pharmacology in the Medical Treatment of Glau- penetration of subconjunctivally administered l4C-fluoroura- comas, Drance S and Neufeld A, editors. New York, Grune & cil in rabbits. Arch Ophthalmol 97:2375, 1979. Stratton, 1984, pp. 233-253. 29. Jams G, Blumenkranz M, Hernandez E, and Sossi N: Clear- 24. Ullman B and Kirsch J: Metabolism of 5-fluorouracil in cul- ance of intravitreal fluorouracil. Ophthalmology 92:91, 1985.

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