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Figure 1.
Prevention of stromal disease. Comparison of the least-square means of the severity of stromal keratitis (on a scale of 0-4 [see the "Methods" subsection of the "Materials and Methods" section for an explanation of grading]) in rabbits injected with the RE strain of herpes simplex type 1 in both eyes and treated with 0.2% cidofovir twice a day or 1% trifluridine 7 times a day. Treatment was begun after evaluation on postinjection day 3 (prior to the appearance of stromal disease) and continued through day 21. Drug treatments were coded, and all grading was done in a masked manner. BSS indicates balanced salt solution; bars, least-square mean ± SEs of the least-square mean.

Prevention of stromal disease. Comparison of the least-square means of the severity of stromal keratitis (on a scale of 0-4 [see the "Methods" subsection of the "Materials and Methods" section for an explanation of grading]) in rabbits injected with the RE strain of herpes simplex type 1 in both eyes and treated with 0.2% cidofovir twice a day or 1% trifluridine 7 times a day. Treatment was begun after evaluation on postinjection day 3 (prior to the appearance of stromal disease) and continued through day 21. Drug treatments were coded, and all grading was done in a masked manner. BSS indicates balanced salt solution; bars, least-square mean ± SEs of the least-square mean.

Figure 2.
Prevention of stromal disease. Comparison of the least-square means of the severity of stromal keratitis (on a scale of 0-4 [see the "Methods" subsection of the "Materials and Methods" section for an explanation of grading]) in rabbits injected with the RE strain of herpes simplex type 1 in both eyes and treated with 0.5% cidofovir twice a day or 1% trifluridine 5 times a day. Treatment was begun after evaluation on postinjection day 2 (prior to the appearance of stromal disease) and continued through day 21. Drug treatments were coded, and all grading was done in a masked manner. BSS indicates balanced salt solution; bars, least-square mean ± SE of the least-square mean.

Prevention of stromal disease. Comparison of the least-square means of the severity of stromal keratitis (on a scale of 0-4 [see the "Methods" subsection of the "Materials and Methods" section for an explanation of grading]) in rabbits injected with the RE strain of herpes simplex type 1 in both eyes and treated with 0.5% cidofovir twice a day or 1% trifluridine 5 times a day. Treatment was begun after evaluation on postinjection day 2 (prior to the appearance of stromal disease) and continued through day 21. Drug treatments were coded, and all grading was done in a masked manner. BSS indicates balanced salt solution; bars, least-square mean ± SE of the least-square mean.

Figure 3.
Prevention of stromal disease. Comparison of the least-square means of the severity of stromal keratitis (on a scale of 0-4 [see the "Methods" subsection of the "Materials and Methods" section for an explanation of grading]) in rabbits injected with the RE strain of herpes simplex type 1 in both eyes and treated with 1% cidofovir twice a day, 1% trifluridine 5 times a day, or fluorometholone. Treatment was begun after evaluation on postinjection day 3 (prior to the appearance of stromal disease) and continued through day 17. Drug treatments were coded, and all grading was done in a masked manner. BSS indicates balanced salt solution; bars, least-square mean ± SE of the least-square mean.

Prevention of stromal disease. Comparison of the least-square means of the severity of stromal keratitis (on a scale of 0-4 [see the "Methods" subsection of the "Materials and Methods" section for an explanation of grading]) in rabbits injected with the RE strain of herpes simplex type 1 in both eyes and treated with 1% cidofovir twice a day, 1% trifluridine 5 times a day, or fluorometholone. Treatment was begun after evaluation on postinjection day 3 (prior to the appearance of stromal disease) and continued through day 17. Drug treatments were coded, and all grading was done in a masked manner. BSS indicates balanced salt solution; bars, least-square mean ± SE of the least-square mean.

Figure 4.
Treatment of established stromal disease. Comparison of the least-square means of the severity of stromal keratitis (on a scale of 0-4 [see the "Methods" subsection of the "Materials and Methods" section for an explanation of grading]) in rabbits injected with the RE strain of herpes simplex type 1 in both eyes and treated with 1% cidofovir twice a day, 1% trifluridine 5 times a day, or fluorometholone. Treatment was begun after evaluation on postinjection day 7 (after the appearance of stromal disease) and continued through day 20. Drug treatments were coded, and all grading was done in a masked manner. BSS indicates balanced salt solution; bars, least-square mean ± SE of the least-square mean.

Treatment of established stromal disease. Comparison of the least-square means of the severity of stromal keratitis (on a scale of 0-4 [see the "Methods" subsection of the "Materials and Methods" section for an explanation of grading]) in rabbits injected with the RE strain of herpes simplex type 1 in both eyes and treated with 1% cidofovir twice a day, 1% trifluridine 5 times a day, or fluorometholone. Treatment was begun after evaluation on postinjection day 7 (after the appearance of stromal disease) and continued through day 20. Drug treatments were coded, and all grading was done in a masked manner. BSS indicates balanced salt solution; bars, least-square mean ± SE of the least-square mean.

1.
McNeill  JIKaufman  HE Local antivirals in a herpes simplex stromal keratitis model. Arch Ophthalmol. 1979;97727- 729Article
2.
De Clercq  ESakuma  TBaba  M  et al.  Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and pyrimidines. Antiviral Res. 1987;8261- 272Article
3.
Gordon  YJRomanowski  EGAraullo-Cruz  T Topical HPMPC inhibits adenovirus type 5 in the New Zealand rabbit ocular replication model. Invest Ophthalmol Vis Sci. 1994;354135- 4143
4.
Gordon  YJNaesens  LDe Clercq  EMaudgal  PBVeckeneer  M Treatment of adenoviral conjunctivitis with topical cidofovir [letter]. Cornea. 1996;15546Article
5.
Gordon  YJRomanowski  EGAraullo-Cruz  T HPMPC, a broad-spectrum topical antiviral agent, inhibits herpes simplex virus type 1 replication and promotes healing of dendritic keratitis in the New Zealand rabbit ocular model. Cornea. 1994;13516- 520Article
6.
Kaufman  HEVarnell  EDThompson  HW Trifluridine, cidofovir, and penciclovir in the treatment of experimental herpetic keratitis. Arch Ophthalmol. 1998;116777- 780Article
7.
Davis  JLTaskintuna  IFreeman  WRWeinberg  DVFeuer  WJLeonard  RE Iritis and hypotony after treatment with intravenous cidofovir for cytomegalovirus retinitis. Arch Ophthalmol. 1997;115733- 737Article
8.
Barrier  JHBani-Sadr  FGaillard  FRaffi  F Recurrent iritis after intravenous administration of cidofovir. Clin Infect Dis. 1997;25337- 338Article
9.
Friedberg  DN Hypotony and visual loss with intravenous cidofovir treatment of cytomegalovirus retinitis. Arch Ophthalmol. 1997;115801- 802Article
10.
Banker  ASArevalo  JFMunguia  D  et al.  Intraocular pressure and aqueous humor dynamics in patients with AIDS treated with intravitreal cidofovir (HPMPC) for cytomegalovirus retinitis. Am J Ophthalmol. 1997;124168- 180
11.
Chavez-de la Paz  EArevalo  JFKirsch  LS  et al.  Anterior nongranulomatous uveitis after intravitreal HPMPC (cidofovir) for the treatment of cytomegalovirus retinitis. Ophthalmology. 1997;104539- 544Article
12.
Taskintuna  IRahhal  FMArevalo  JF  et al.  Low-dose intravitreal cidofovir (HPMPC) therapy of cytomegalovirus retinitis in patients with acquired immune deficiency syndrome. Ophthalmology. 1997;1041049- 1057Article
13.
Taskintuna  IRahhal  FMRao  NA  et al.  Adverse events and autopsy findings after intravitreous cidofovir (HPMPC) therapy in patients with acquired immune deficiency syndrome (AIDS). Ophthalmology. 1997;1041827- 1837Article
14.
Akler  MEJohnson  DWBurman  WJJohnson  SC Anterior uveitis and hypotony after intravenous cidofovir for the treatment of cytomegalovirus retinitis. Ophthalmology. 1998;105651- 657Article
15.
Taskintuna  IBanker  ASRao  NA  et al.  An animal model for cidofovir (HPMPC) toxicity: intraocular pressure and histopathologic effects. Exp Eye Res. 1997;64795- 806Article
16.
Banker  ASBergeron-Lynn  GKeefe  KSDe Clercq  E Effects of topical and subconjunctival cidofovir (HPMPC) in an animal model. Curr Eye Res. 1998;17560- 566Article
17.
Otova  BVotruba  IHoly  A Pretreatment of the host cell with 1-(S)-(3-hydroxy-2-phosphonomethoxypropyl)cytosine (HPMPC) is sufficient for its antiviral effect. Acta Virol. 1992;36313- 319
18.
Soike  KFHuang  J-LZhang  J-YBohm  RHitchcock  MJMMartin  JC Evaluation of infrequent dosing regimens with (S)-1-[3-hydroxy-2-phosphonomethoxy)propyl]-cytosine (S-HPMPC) on simian varicella infection in monkeys. Antiviral Res. 1991;1617- 28Article
19.
SAS Institute Inc, SAS Language Reference, Version 6.  Cary, NC SAS Institute Inc1990;
Laboratory Sciences
July 1999

Cidofovir and Experimental Herpetic Stromal Disease

Author Affiliations

From the LSU Eye Center, Louisiana State University Medical Center School of Medicine, New Orleans. None of the authors has any financial interest in or receive payment as a consultant, reviewer, or evaluator from Gilead Sciences Inc.

Arch Ophthalmol. 1999;117(7):925-928. doi:10.1001/archopht.117.7.925
Abstract

Objective  To compare topical cidofovir with topical trifluridine for the prevention and treatment of herpes simplex type 1 stromal keratitis in rabbits.

Methods  The RE strain of herpes simplex virus 1 was injected into the central stroma of both eyes of New Zealand white rabbits. Two to 3 days after virus inoculation, the rabbits were randomized to treatment groups of 10 each and treated with 1% trifluridine administered 5 or 7 times a day, 1%, 0.5%, or 0.2% cidofovir administered twice a day, fluorometholone administered twice a day, or balanced salt solution (BSS) administered twice a day (control) until day 21 after injection. The treated corneas were examined 3 times a week and the severity of stromal keratitis was graded in a masked fashion. To evaluate the ability of cidofovir to treat established stromal disease, groups of 10 rabbits each were inoculated with herpes simplex virus and treated with 1% cidofovir twice a day, 1% trifluridine 5 times a day, fluorometholone twice a day, or BSS twice a day beginning on day 7 after virus inoculation through day 21.

Results  Treatment with 0.2% cidofovir twice a day was not effective in preventing the appearance of stromal disease (P=.89), whereas treatment with 0.5% (P<.001) or 1% (P<.001) cidofovir twice a day or 1% trifluridine 5 times a day (P<.001) or 7 times a day (P=.006) significantly reduced the appearance of stromal keratitis on the 8 evaluation days, compared with BSS treatment (F test analysis of variance). There was no difference between the eyes treated with 0.5% cidofovir twice a day and those treated with 1% trifluridine 5 times a day. Treatment with 1% cidofovir was not effective in treating established stromal disease.

Conclusions  Cidofovir and trifluridine are highly effective in preventing the appearance of herpetic stromal disease. Cidofovir is as effective as, but no more effective than, trifluridine in this model. Neither cidofovir nor trifluridine benefits established stromal disease, however.

Clinical Relevance  Cidofovir is a new, potent antiviral that seems similar in efficacy to trifluridine and is effective in the prevention of the development of stromal herpes, but is not effective in the treatment of established stromal disease in which hypersensitivity predominates.

IN 1979, TRIFLURIDINE eye drops were shown to prevent the development of herpes simplex virus (HSV) stromal keratitis in rabbit eyes injected intrastromally with the RE strain of herpes simplex virus type 1 (HSV-1).1 Since that time, there have been, to our knowledge, no published reports of another antiviral with this capability.

Cidofovir (1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) is a phosphonate with a broad antiviral spectrum.24 It is highly effective against HSV epithelial keratitis when administered twice a day as 0.2% to 1% eye drops.36 Cidofovir can produce adverse events when administered systemically to humans,714 and topical application can cause decreased intraocular pressure and morphologic changes in the corneas of guinea pigs15,16; however, the eye drops do not affect intraocular pressure or appear to be toxic when applied to the rabbit cornea.5,6 Cidofovir has been reported to persist in tissues for a long time.17,18 We evaluated the ability of this compound to prevent the appearance of herpetic stromal disease in the New Zealand white (NZW) rabbit and compared its efficacy with that of 1% trifluridine. We also tested cidofovir for the ability to treat established stromal disease in the rabbit.

MATERIALS AND METHODS
MATERIALS

New Zealand white rabbits (weighing 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) and 0.2%, 0.5%, and 1% cidofovir (Gilead Sciences, Foster City, Calif). Cidofovir drops were formulated from cidofovir powder in a 1% carboxymethyl cellulose solution in our laboratory. Trifluridine at a concentration of 1% was chosen because our experience shows that 1% trifluridine administered 2, 4, or 7 times a day in the herpetic epithelial disease model provides an effect that is similar to that of cidofovir given 2 times a day.6 There are, to our knowledge, no reports that twice-a-day treatment with trifluridine is effective against herpetic stromal disease; thus, we chose to administer 1% trifluridine 5 to 7 times a day, a dosage schedule that has been shown to have such an effect.1

Eyes were also treated with fluorometholone (FML ophthalmic suspension; Allergan Pharmaceuticals, Hormigueros, PR) or balanced salt solution (BSS; Alcon Surgical Inc, Fort Worth, Tex). Although dexamethasone sodium phosphate is very effective in this model, it can be administered only a few times (≤6) before the rabbits develop gastrointestinal ulcers. Therefore, fluorometholone was used to provide a mild steroid effect while avoiding the adverse effects of dexamethasone.

METHODS

Both corneas of anesthetized NZW rabbits were injected with 10 µL of the RE strain of HSV-1 (1 × 107 plaque-forming units per milliliter). To determine the efficacy of cidofovir in preventing stromal disease, the rabbits were randomized to groups of 10 each on day 2 or 3 after virus injection; treatment was begun on that day and continued through day 21. Both eyes of each rabbit received the same treatment. In 3 separate studies, groups of eyes were treated with (1) 0.2% cidofovir twice a day, 1% trifluridine 7 times a day, or BSS twice a day; (2) 0.5% cidofovir twice a day, 1% trifluridine 5 times a day, or BSS twice a day; and (3) 1% cidofovir twice a day, 1% trifluridine 5 times a day, fluorometholone twice a day, or BSS twice a day.

In another study undertaken to test the efficacy of cidofovir in treating established stromal disease, both eyes of 4 groups of 10 rabbits each were inoculated as described above. The severity of stromal keratitis was evaluated 7 days after virus injection and the rabbits were randomized to 4 groups of comparable severity prior to the start of treatment. Treatment was begun 7 days after virus injection and continued through day 21. Treatments consisted of 1% cidofovir twice a day, 1% trifluridine 5 times a day, fluorometholone twice a day, or BSS twice a day. Because of variability in the severity of stromal keratitis in individual experiments, a concurrent control group was included with each study.

Corneas were evaluated 3 times a week in a masked fashion. Stromal disease was graded as follows: 0=normal, thin cornea; 1=detectable edema, iris details clearly visible; 2=gross edema with stromal swelling, iris details still distinct; 3=pupillary border no longer distinct; 4=opaque cornea, anterior chamber structures not visible.

STATISTICAL ANALYSES

The outcome variable was the grade of severity of stromal disease. The drug treatment regimens were the dependent variable. Variability due to rabbit differences observed over time within treatments was controlled for by application of a repeated-measures design in the analysis of variance (ANOVA). Comparisons between the stromal disease severity score means for each drug regimen were conducted by protected t tests on least-square means derived from the ANOVA. All P values were derived from these tests; the ANOVA and all subsequent comparisons of treatment means were conducted using programs and procedures from the Statistical Analysis System19 language.

RESULTS

Treatment of rabbit corneas with 0.2% cidofovir eye drops twice a day was not effective in preventing the development of HSV stromal disease; stromal disease scores with this treatment were similar to the scores of control eyes treated with BSS (cidofovir vs BSS; P=.89), whereas the scores of eyes treated with 1% trifluridine eye drops 7 times a day were significantly better than those of the controls (trifluridine vs BSS; P=.006) (Figure 1). However, treatment with 0.5% cidofovir twice a day was effective in preventing HSV stromal disease (cidofovir vs BSS; P<.001), as was treatment with 1% trifluridine 5 times a day (trifluridine vs BSS; P<.001) (Figure 2); there was no difference in efficacy between these 2 drugs (cidofovir vs trifluridine; P=.87). Similarly, 1% cidofovir twice a day was as effective as 1% trifluridine 5 times a day in preventing the development of stromal disease (cidofovir vs trifluridine; P=.54) (Figure 3). Predictably, treatment with fluorometholone beginning 3 days after virus injection led to very severe stromal disease.

Treatment of established HSV stromal disease with either cidofovir or trifluridine had no beneficial effect; scores in both the trifluridine- and cidofovir-treated groups were significantly worse than scores in the BSS-treated group (P=.02 and P=.003, respectively) (Figure 4). In the calculation of overall effect, fluorometholone treatment twice a day was not significantly different from BSS treatment (P=.58) (Figure 4).

Punctate keratitis was noted in the eyes of animals that received trifluridine 7 times a day and in those that received 1% cidofovir. The punctate keratitis appeared 4 to 5 days after the start of treatment and remained mild to moderate.

COMMENT

Cidofovir at concentrations of 0.5% and 1% and 1% trifluridine are highly and equally effective in preventing the development of herpetic stromal disease in the NZW rabbit injected with the RE strain of HSV-1. Neither cidofovir nor trifluridine is effective for treating established stromal disease in this model. Although corticosteroids are effective in treating established herpetic stromal disease, in this study treatment with the mild corticosteroid fluorometholone was significantly better than BSS treatment only at 9 days after virus injection.

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Article Information

Accepted for publication March 22, 1999.

This work was supported in part by Public Health Service grants EY02672 (Dr Kaufman) and EY02377 (departmental core grant) from the National Eye Institute, National Institutes of Health, Bethesda, Md, 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.

Presented in part as a poster at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Fla, March 15, 1998.

Reprints: Emily D. Varnell, LSU Eye Center, 2020 Gravier St, Suite B, New Orleans, LA 70112-2234.

References
1.
McNeill  JIKaufman  HE Local antivirals in a herpes simplex stromal keratitis model. Arch Ophthalmol. 1979;97727- 729Article
2.
De Clercq  ESakuma  TBaba  M  et al.  Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and pyrimidines. Antiviral Res. 1987;8261- 272Article
3.
Gordon  YJRomanowski  EGAraullo-Cruz  T Topical HPMPC inhibits adenovirus type 5 in the New Zealand rabbit ocular replication model. Invest Ophthalmol Vis Sci. 1994;354135- 4143
4.
Gordon  YJNaesens  LDe Clercq  EMaudgal  PBVeckeneer  M Treatment of adenoviral conjunctivitis with topical cidofovir [letter]. Cornea. 1996;15546Article
5.
Gordon  YJRomanowski  EGAraullo-Cruz  T HPMPC, a broad-spectrum topical antiviral agent, inhibits herpes simplex virus type 1 replication and promotes healing of dendritic keratitis in the New Zealand rabbit ocular model. Cornea. 1994;13516- 520Article
6.
Kaufman  HEVarnell  EDThompson  HW Trifluridine, cidofovir, and penciclovir in the treatment of experimental herpetic keratitis. Arch Ophthalmol. 1998;116777- 780Article
7.
Davis  JLTaskintuna  IFreeman  WRWeinberg  DVFeuer  WJLeonard  RE Iritis and hypotony after treatment with intravenous cidofovir for cytomegalovirus retinitis. Arch Ophthalmol. 1997;115733- 737Article
8.
Barrier  JHBani-Sadr  FGaillard  FRaffi  F Recurrent iritis after intravenous administration of cidofovir. Clin Infect Dis. 1997;25337- 338Article
9.
Friedberg  DN Hypotony and visual loss with intravenous cidofovir treatment of cytomegalovirus retinitis. Arch Ophthalmol. 1997;115801- 802Article
10.
Banker  ASArevalo  JFMunguia  D  et al.  Intraocular pressure and aqueous humor dynamics in patients with AIDS treated with intravitreal cidofovir (HPMPC) for cytomegalovirus retinitis. Am J Ophthalmol. 1997;124168- 180
11.
Chavez-de la Paz  EArevalo  JFKirsch  LS  et al.  Anterior nongranulomatous uveitis after intravitreal HPMPC (cidofovir) for the treatment of cytomegalovirus retinitis. Ophthalmology. 1997;104539- 544Article
12.
Taskintuna  IRahhal  FMArevalo  JF  et al.  Low-dose intravitreal cidofovir (HPMPC) therapy of cytomegalovirus retinitis in patients with acquired immune deficiency syndrome. Ophthalmology. 1997;1041049- 1057Article
13.
Taskintuna  IRahhal  FMRao  NA  et al.  Adverse events and autopsy findings after intravitreous cidofovir (HPMPC) therapy in patients with acquired immune deficiency syndrome (AIDS). Ophthalmology. 1997;1041827- 1837Article
14.
Akler  MEJohnson  DWBurman  WJJohnson  SC Anterior uveitis and hypotony after intravenous cidofovir for the treatment of cytomegalovirus retinitis. Ophthalmology. 1998;105651- 657Article
15.
Taskintuna  IBanker  ASRao  NA  et al.  An animal model for cidofovir (HPMPC) toxicity: intraocular pressure and histopathologic effects. Exp Eye Res. 1997;64795- 806Article
16.
Banker  ASBergeron-Lynn  GKeefe  KSDe Clercq  E Effects of topical and subconjunctival cidofovir (HPMPC) in an animal model. Curr Eye Res. 1998;17560- 566Article
17.
Otova  BVotruba  IHoly  A Pretreatment of the host cell with 1-(S)-(3-hydroxy-2-phosphonomethoxypropyl)cytosine (HPMPC) is sufficient for its antiviral effect. Acta Virol. 1992;36313- 319
18.
Soike  KFHuang  J-LZhang  J-YBohm  RHitchcock  MJMMartin  JC Evaluation of infrequent dosing regimens with (S)-1-[3-hydroxy-2-phosphonomethoxy)propyl]-cytosine (S-HPMPC) on simian varicella infection in monkeys. Antiviral Res. 1991;1617- 28Article
19.
SAS Institute Inc, SAS Language Reference, Version 6.  Cary, NC SAS Institute Inc1990;
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