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Trifluridine, Cidofovir, and Penciclovir in the Treatment of Experimental Herpetic Keratitis
Herbert E. Kaufman, MD;
Emily D. Varnell, BS;
Hilary W. Thompson, PhD
Arch Ophthalmol. 1998;116:777-780.
ABSTRACT
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Objective To compare trifluridine eyedrops, cidofovir eyedrops, and penciclovir ophthalmic ointment for the treatment of herpes simplex virus type 1 keratitis.
Methods New Zealand white rabbits were infected with the McKrae strain of herpes simplex virus type 1. Three days after viral inoculation, the rabbits were randomly assigned to treatment with 1% trifluridine, 0.2% cidofovir, 3% penciclovir ointment, or phosphate-buffered saline (for control) on various schedules. The severity of keratitis was graded in a masked manner.
Results Treatment with any of the antiviral drugs resulted in significantly less severe keratitis than treatment with phosphate-buffered saline. There was no statistically significant difference between eyes given trifluridine 2, 4, or 7 times a day and eyes given cidofovir 2 times a day (P=.06, P =.43, and P=.19, respectively, using the F test of the analysis of variance). Cidofovir given twice a day was significantly more effective than penciclovir given either 2 or 4 times a day (P<.001 and P =.002, respectively). Even with once-a-day dosage, all 3 drugs were significantly more effective than phosphate-buffered saline (P<.001 for all). There was no significant difference between once-a-day trifluridine and cidofovir treatments (P=.17). Trifluridine administered 5 times a day was as effective as 1% cidofovir. A similar degree of punctate keratitis was seen after 4 to 5 days in eyes treated with trifluridine at the highest frequency, 1% cidofovir, or penciclovir ointment.
Conclusion Trifluridine treatment was highly effective in this rabbit model, even when given only once a day. Treatment with cidofovir was as effective as that with trifluridine.
Clinical Relevance Cidofovir and penciclovir treatments may prove to be effective against epithelial keratitis. Clinical trials of trifluridine, cidofovir, and penciclovir with lower treatment frequencies appear to be warranted.
INTRODUCTION
THE RABBIT provides a model of herpes simplex virus keratitis, which involves the multiplication of the virus in the rabbit corneal epithelium with the production of dendrites and is similar to the infection in humans. Studies using this model have been predictive of results in humans, and the relative potencies of drugs determined in this model have paralleled those seen in human studies.1-4 The purpose of our study was to compare 2 of the newer antiviral drugs—penciclovir and cidofovir—with trifluridine, which one of us (H.E.K.) introduced as an effective antiviral agent 34 years ago,3 and to determine the frequency of drug administration needed for effective therapy. There are theoretical reasons suggesting that these newer drugs may persist for a longer period in ocular tissues and could, therefore, be administered less frequently than trifluridine.
Trifluridine is a substituted nucleoside similar to thymidine.5 For the drug to become active, it must be phosphorylated by cellular thymidine kinase (a relatively rate-limiting step) and then, in addition, by the more rapidly acting cellular enzymes. The primary action of trifluridine is the inhibition of DNA polymerase; it may also act within the cell to block the synthesis of thymidylic synthetase.6 As approved by the US Food and Drug Administration, the package insert suggests the application of 1 drop of trifluridine every 2 hours during waking hours, up to a maximum daily dose of 9 drops. Trifluridine has been found to inhibit the growth of adenovirus in tissue culture.7
Penciclovir (9-[4-hydroxy-3-hydroxymethylbutyl]guanine) (and its better-absorbed oral prodrug, famciclovir, which is hydrolyzed to penciclovir) is a nucleoside similar to acyclovir. Famciclovir has been approved by the Food and Drug Administration for use in treating herpes zoster and appears to reduce postzoster pain.8 Like trifluridine, penciclovir is initially phosphorylated by cellular thymidine kinase and then rapidly phosphorylated to the triphosphate.9 Also like trifluridine, penciclovir is an inhibitor of DNA polymerase, but it is more selective than trifluridine. Although the active triphosphate of penciclovir is somewhat less inhibitory toward DNA polymerase than the acyclovir triphosphate, penciclovir persists in the cell for a substantially longer period and, therefore, requires less frequent administration. For this reason, it seemed like a good candidate for a long-acting topical antiviral agent.
Cidofovir (1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine), unlike trifluridine and penciclovir, is a phosphonate. Like foscarnet, cidofovir already has the first phosphate in place and then rapidly acquires 2 additional phosphates without the need for thymidine kinase.10-11 Phosphonates inhibit DNA polymerase, but changes in thymidine kinase do not cause resistance to develop.12 Cidofovir is significantly toxic when given in high concentrations and when given frequently as a topical medication, and it is highly nephrotoxic when given parenterally.13 To be used parenterally, cidofovir requires the concomitant administration of probenecid to reduce active secretion of the drug into the renal tubules. Like penciclovir, cidofovir also seems to persist in tissues for a long time.14-17
The purpose of this study was to compare the effectiveness of these 3 drugs—trifluridine, cidofovir, and penciclovir—in a variety of dosage regimens for the treatment of herpes keratitis in the rabbit eye.
MATERIALS AND METHODS
MATERIALS
New Zealand white rabbits (2-3 kg) of both sexes were used. All animals were handled in accordance with the National Institutes of Health guidelines on the care and use of animals in research; the Louisiana State University Medical Center, New Orleans, Institutional Animal Care and Use Committee guidelines and approvals; and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Drugs used in this study included 1% trifluridine drops (Viroptic, Glaxo Wellcome Inc, Research Triangle Park, NC), 0.2% or 1% cidofovir solution formulated in our laboratory from cidofovir powder, and 3% penciclovir ointment formulated in our laboratory from penciclovir powder. Control eyes were treated with phosphate-buffered saline (PBS).
METHODS
The McKrae strain of herpes simplex virus type 1 was propagated in primary rabbit kidney cells and titered in CV-1 cells. Both corneas of each rabbit were anesthetized with proparacaine hydrochloride. The superficial corneal epithelium was lightly scarified with a 27-gauge needle, and 25 µL of virus suspension was dropped onto the cornea to synchronize the initial appearance and severity of keratitis. The eyelids were gently rubbed over the cornea for 15 seconds.
Three days after virus inoculation, the corneas were stained with fluorescein, and the severity of keratitis was graded on a scale of 0 to 4 as follows: 0, normal cornea; 1, epithelial ulceration involving a quarter of the epithelial area; 2, epithelial ulceration involving half of the epithelial area; 3, epithelial ulceration involving three quarters of the epithelial area; and 4, total epithelial involvement. Rabbits were randomly assigned to groups of comparable severity, and treatment was administered in a coded manner. Both eyes of each rabbit were treated identically.
In 1 study designed to compare a variety of treatment regimens, there were 6 drug treatment groups consisting of 8 rabbits (16 eyes) each and 1 control group of 7 rabbits (14 eyes). Eyes were graded through day 14 after inoculation. Treatment regimens were as follows: trifluridine, 1% drops twice a day (8 AM and 8 PM); trifluridine, 1% drops 4 times a day (8 AM, noon, 4 PM, and 8 PM); trifluridine, 1% drops 7 times a day (8 AM, 10 AM, noon, and 2, 4, 6, and 8 PM); cidofovir, 0.2% drops twice a day (8 AM and 8 PM); penciclovir, 3% ointment twice a day (8 AM and 8 PM); penciclovir, 3% ointment 4 times a day (8 AM, noon, 4 PM, and 8 PM); and PBS (control), twice a day (8 AM and 8 PM).
In a second study designed to compare the efficacy of a single daily treatment, there were 3 drug treatment groups and 1 control group consisting of 10 rabbits (20 eyes) each. Eyes were graded through day 10 after inoculation. Treatment regimens were as follows: trifluridine, 1% drops; cidofovir, 0.2% drops; penciclovir, 3% ointment; and PBS (control).
In both studies, treatment was given beginning after examination on postinfection day 3 through postinfection day 9. Corneas were stained with fluorescein and examined with the slitlamp daily, and the severity of keratitis was graded in a masked manner.
A third study was undertaken to test the efficacy of a higher concentration of cidofovir eyedrops using 10 rabbits (20 eyes) in each group. Eyes were graded through day 7 after inoculation. Treatment regimens were as follows: trifluridine, 1% drops 5 times a day (8:00 and 10:30 AM and 1:00, 3:30, and 6:00 PM); cidofovir, 1% drops twice a day (8 AM and 6 PM); and PBS (control), twice a day (8 AM and 6 PM).
STATISTICAL ANALYSIS
The outcome variable was the severity of the keratitis grade. The drug treatment regimens were the dependent variable. Variability due to differences between rabbits (each with 2 identically treated eyes) within treatments was controlled for by the application of a repeated-measures analysis of variance. Comparisons between the keratitis severity score means for each drug regimen were conducted by protected t tests on least squares means derived from the analysis of variance.18 All P values were derived from these tests. The analysis of variance and all subsequent comparisons of treatment means were conducted using programs and procedures from statistical software.19
RESULTS
In the initial study, all treatments with trifluridine, cidofovir, or penciclovir resulted in statistically less severe herpetic keratitis than treatment with PBS (P<.001 for all comparisons) (Figure 1). Twice-a-day and 4-times-a-day regimens of penciclovir were equally effective (P=.62). There was no difference in the effectiveness of trifluridine given 2, 4, or 7 times a day (2 vs 4 times a day, P=.26; 2 vs 7 times a day, P =.55; and 4 vs 7 times a day, P =.60), and there was no difference in efficacy between cidofovir given twice a day and trifluridine given twice a day (P=.06). Although all treatments were effective in promoting the healing of herpetic keratitis, none prevented spontaneous recurrences, even while treatment was continuing. All groups showed spontaneous recurrences after treatment was stopped.
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Figure 1. Comparison of the severity of keratitis (on a scale of 0-4 [see the "Methods" subsection for an explanation of the grades on the scale]) in rabbits infected with the McKrae strain of herpes simplex virus type 1 in both eyes and treated with 1 of the 3 drugs on various dosage schedules. Treatment was begun after the evaluation on postinfection day 3 and continued through day 9. Treatments with 1% trifluridine drops, 0.2% cidofovir solution, or 3% penciclovir ointment were administered in a coded manner, and all grading was done in a masked manner.
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Trifluridine and cidofovir treatment resulted in significantly less severe keratitis than treatment with penciclovir at all tested dosage schedules (trifluridine twice a day vs penciclovir twice a day, P <.001; trifluridine 4 times a day vs penciclovir 4 times a day, P=.02; and cidofovir twice a day vs penciclovir twice a day, P<.001).
Once-a-day treatment with trifluridine, cidofovir, or penciclovir resulted in significantly less severe keratitis than treatment with PBS (P<.001 for all 3 comparisons) (Figure 2).
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Figure 2. Comparison of the severity of keratitis (on a scale of 0-4 [see the "Methods subsection for an explanation of the grades on the scale]) in rabbits infected with the McKrae strain of herpes simplex virus type 1 in both eyes and treated with 1 of the 3 drugs on a once-a-day schedule. Treatment was begun after the evaluation on postinfection day 3 and continued through day 9. Treatments with 1% trifluridine drops, 0.2% cidofovir solution, or 3% penciclovir ointment were administered once a day, in a coded manner, and all grading was done in a masked manner.
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Treatment with 1% cidofovir twice a day and 1% trifluridine 5 times a day resulted in the rapid healing of the keratitis soon after treatment was initiated. On each study day, treatment with either drug resulted in significantly less severe keratitis than treatment with PBS (P<.001); however, there was no significant difference in the severity of keratitis in eyes treated with either trifluridine or cidofovir (Figure 3).
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Figure 3. Comparison of the severity of keratitis (on a scale of 0-4 [see the "Methods subsection for an explanation of the grades on the scale]) in rabbits infected with the McKrae strain of herpes simplex virus type 1 in both eyes and treated with 1% cidofovir twice a day or 1% trifluridine 5 times a day. Treatment was begun on postinfection day 3 and continued through day 7. Drug treatments were coded, and all grading was done in a masked manner.
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Mild to moderate punctate keratitis was noted with trifluridine treatment given 7 times a day, with all penciclovir ointment treatments, and with the 1% cidofovir treatment. The punctate keratitis appeared 4 to 5 days after the start of treatment.
COMMENT
In this model, it was possible to show that both trifluridine and cidofovir given as infrequently as once a day are effective for treating herpes simplex virus keratitis, even with treatment beginning 72 hours after infection when the lesions are well established.20-21 These findings suggest the possibility that trifluridine is being used clinically more frequently than is necessary. When corticosteroids are being administered, frequent dosage of trifluridine may be needed because corticosteroids reduce the antiviral effect of all these drugs. In general, however, it may be that the antiviral drugs are being administered more frequently in clinical practice than is required.
The comparison studies showed that topical penciclovir is slightly less effective than trifluridine or cidofovir and seems to offer no advantages. Cidofovir and trifluridine were equally effective against herpetic keratitis and have been reported to be effective against ocular adenovirus infection.15, 22-23
AUTHOR INFORMATION
Accepted for publication January 9, 1998.
This study was supported in part by US Public Health Service grants EY02672 and EY02377 from the National Eye Institute, National Institutes of Health, Bethesda, Md; Department of the Army, Cooperative Agreement DAMD17-93-V-3013 (this does not necessarily reflect the position or the policy of the government, and no official endorsement should be inferred); and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc, New York, NY.
Cidofovir powder was supplied by Gilead Sciences Inc, Foster City, Calif. Penciclovir powder was supplied by SmithKline Beecham Pharmaceuticals, King of Prussia, Pa. None of the authors have any financial interest in or receive payment as a consultant, reviewer, or evaluator from either company.
Presented in part as a poster at the Association for Research in Vision and Ophthalmology Annual Meeting, Fort Lauderdale, Fla, May 11, 1997.
Reprints: Emily D. Varnell, BS, LSU Eye Center, 2020 Gravier St, Suite B, New Orleans, LA 70112-2234 (e-mail: evarne{at}lsumc.edu)
From the LSU Eye Center, Louisiana State University Medical Center School of Medicine, New Orleans.
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