February 2002
Volume 43, Issue 2
Free
Retina  |   February 2002
Treatment or Prevention of Herpes Simplex Virus Retinitis with Intravitreally Injectable Crystalline 1-O-Hexadecylpropanediol-3-Phospho-Ganciclovir
Author Affiliations
  • Lingyun Cheng
    From the Shiley Eye Center, University of California San Diego, La Jolla, California; and the
  • Karl Y. Hostetler
    Department of Medicine, Veterans Affairs Medical Center, and University of California San Diego, La Jolla, California.
  • Sunan Chaidhawangul
    From the Shiley Eye Center, University of California San Diego, La Jolla, California; and the
  • Michael F. Gardner
    Department of Medicine, Veterans Affairs Medical Center, and University of California San Diego, La Jolla, California.
  • James R. Beadle
    Department of Medicine, Veterans Affairs Medical Center, and University of California San Diego, La Jolla, California.
  • Mitsuko Toyoguchi
    From the Shiley Eye Center, University of California San Diego, La Jolla, California; and the
  • Germaine Bergeron-Lynn
    From the Shiley Eye Center, University of California San Diego, La Jolla, California; and the
  • William R. Freeman
    From the Shiley Eye Center, University of California San Diego, La Jolla, California; and the
Investigative Ophthalmology & Visual Science February 2002, Vol.43, 515-521. doi:
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Lingyun Cheng, Karl Y. Hostetler, Sunan Chaidhawangul, Michael F. Gardner, James R. Beadle, Mitsuko Toyoguchi, Germaine Bergeron-Lynn, William R. Freeman; Treatment or Prevention of Herpes Simplex Virus Retinitis with Intravitreally Injectable Crystalline 1-O-Hexadecylpropanediol-3-Phospho-Ganciclovir. Invest. Ophthalmol. Vis. Sci. 2002;43(2):515-521.

      Download citation file:


      © 2015 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
 

purpose. To evaluate an intraocular drug delivery system consisting of the crystalline ammonium salt of 1-O-hexadecylpropanediol-3-phospho-ganciclovir (HDP-P-GCV) as a slow-release form of the drug.

 

methods. A dosage of 0.885, 1.57, 2.8, 4.486, or 8.85 μmol of ammonium salt HDP-P-GCV in 0.1 mL was intravitreally injected into rabbit vitreous. The toxicity and safety were evaluated with ophthalmoscopy, electroretinography, and pathology. Drug vitreous levels were determined at various time intervals by means of HPLC. The treatment efficacy and duration of efficacy were tested in a herpes simplex virus (HSV)-1 retinitis rabbit model.

 

results. Intravitreal injections of the compound revealed clear vitreous of optic axis, a desirable drug depot in the inferior vitreous cavity, and no clinical toxicity, except for variable mild local posterior subcapsular cataract and local retinal toxicity with high doses. HPLC analysis showed free ammonium salt of HDP-P-GCV in the upper vitreous at a level of 0.2 μM 12 weeks after the 2.8-μmol initial intravitreal dose. Drug concentration was still 1.95 μM 20 weeks after the 8.85-μmol initial intravitreal dose. These concentrations (0.2 and 1.95 μM) were 10 and 100 times higher, respectively, than the median inhibitory concentration (IC50) of HSV-1 (0.023μ M). Treatment with the highest nontoxic dose (2.8 μmol) and the highest dose (8.85 μmol) showed significant protection from HSV-1 infection (P < 0.05) and provided sustained antiviral effect after a single intravitreal drug injection.

 

conclusions. The crystalline ammonium salt of HDP-P-GCV may be a very useful local therapy for herpes family viral retinitis.

Direct delivery of drug to the posterior segment by intravitreal injection or intravitreal implant has been practiced to treat some acute or chronic vitreoretinal diseases. 1 2 3 4 5 6 Contrasted with systemic drug administration for vitreoretinal diseases, local intravitreal drug administration bypasses the blood–ocular barriers, allowing higher intraocular drug levels and avoiding many of the side effects associated with systemic therapy. Intraocular delivery may also provide relatively constant drug levels in the eye if implants or a slow-release drug are used. Surgical placement and replacement of intravitreal implants can cause significant adverse effects, including vitreous hemorrhage, retinal detachment, and endophthalmitis. 7 Intravitreal injection of a long-acting drug preparation would be less invasive than surgery. Clinically acceptable intervals for repeating injections could be on the order of several weeks or longer. Unfortunately, most intravitreally injected compounds have a short vitreous half-life, which necessitates frequent injections to provide a therapeutic effect. Frequent intravitreal injections are inconvenient and may cause retinal detachment or endophthalmitis. 
We have previously reported a self-assembling liposome system used for delivery of 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA) 8 and 1-O-hexadecylpropanediol-3-phospho-ganciclovir (HDP-P-GCV) 9 into rabbit eyes. These lipid prodrugs in liposome formulation demonstrated longer antiviral activity compared with their parent compounds in herpes simplex virus (HSV)-1 retinitis after a single intravitreal injection in rabbit eyes. 9 10 Considering that these lipid prodrugs bear a long hydrophobic carbon chain, we hypothesize that the compounds may innately possess slow-release properties without formulation into liposomes. In the present study, we report a novel intraocular drug delivery system using the free crystalline lipid prodrug of ganciclovir, HDP-P-GCV, as a prototype. 
Materials and Methods
Preparation for Intravitreal Injection
Solutions for injection were prepared by weighing the powder ammonium salt of HDP-P-GCV directly into a 2-mL vial; 5% dextrose was added to achieve the desired concentration. Stock solutions were made at 14 times the final intravitreal concentrations, assuming that 0.1 mL would be injected into approximately 1.4 mL of vitreous. After the addition of the dextrose, the vials were sonicated briefly in a water-jacketed cup horn at full power. Dispersions were sonicated until they appeared to be homogenous by visual inspection, usually after 2 to 5 minutes. 
Particle Size Evaluation
The ammonium salt of the HDP-P-GCV used in these experiments is a white powder and is sparingly soluble in water. The ammonium salt of HDP-P-GCV was subjected to laser diffraction (HELOS; Sympatec GmbH, Goslar, Germany) particle size analysis by laser light scattering at Cirrus Pharmaceuticals (Research Triangle Park, NC). 
HPLC Analysis of HDP-P-GCV in Vitreous Sample
HDP-P-GCV was extracted by adding 1 mL fresh acetonitrile to 0.1 mL of rabbit vitreous. 1-O-octadecyl-2-methylglycero-3-P-acyclovir (20 μg) was added as an internal standard. Samples were vortexed for 10 seconds and rested for 30 minutes before the precipitated protein was removed by centrifugation. The supernatant was removed and transferred to a clean tube for evaporation, and the residue was resuspended in 25 μL of mobile phase for a 20-μL injection. 
HPLC analysis was performed using a 4.6 × 150-mm column (Xterra RPC8; Waters, Inc., Milford, MA) with a 3.9 × 20-mm guard cartridge in 50 mM pyrrolidine (native pH) with 57% methanol at 0.5 mL/min and with UV detection at 254 nm. The HPLC system was from Beckman Instruments (model 344 HPLC; Beckman Instruments, Fullerton, CA), with a detector (model 165 UV; Beckman) and an injector (model 410A; Rheodyne, Cotati, CA). 
Animal Studies
A total of 119 rabbits with average body weight of 2.7 ± 0.6 kg were used in the study in accordance with the guidelines of the University of California San Diego, Office of Veterinary Affairs and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Toxicity Study.
For the toxicity study, 15 New Zealand White rabbits were used. The anesthesia and intravitreal drug injections were performed as previously described. 8 Attempts were made to place the drug in the inferior and peripheral vitreous cavity. Each of the eyes received an intravitreal injection of 0.885, 1.57, 2.8, 4.486, or 8.85μ mol of the ammonium salt of HDP-P-GCV suspension in the volume of 0.1 mL. Final predicted drug concentrations in the vitreous were 0.632, 1.12, 2.0, 3.2, and 6.32 mM, based on a rabbit vitreous volume of 1.4 mL. Control eyes received 0.1 mL normal saline by injection. Eyes were examined by indirect ophthalmoscopy before injection, on the first postoperative day and during each successive postinjection week. Anterior segment findings, fundus findings, vitreous clarity, and distribution of drug aggregates in the vitreous were documented. A scale of 0 to 4 for vitreous clarity, a scale of 0 to 3 for anterior segment reaction, and a system of cataract grading were used as previously described. 9 Briefly, a clear lens was graded as 0; a local mild lens opacity was graded as 1; a moderately extensive lens opacity with obscuration of fundus detail was graded as 2; a complete lens opacity without view of fundus was graded as 3. All clinical toxicity evaluations were performed and graded by a trained observer without masking. 
At 2 and 8 weeks after injection, the rabbits were killed, and the globes were processed for light and electron microscopic evaluation. 8 Electroretinography (ERG) was performed at baseline and before death. For the ERG procedure, the eyes were dilated with 2.5% phenylephrine-HCl, and 1% tropicamide and dark adapted for 30 minutes. The rabbits were anesthetized with a mixture of ketamine (21 mg/kg) and xylazine (5 mg/kg), and responses were obtained from the cornea, using a Burian-Allen Hansen rabbit bipolar contact lens (Hansen Ophthalmic Development Laboratory, Iowa City, IA). Responses were collected and amplified by a regulated power supply (model RPS21; Grass Instrument Division, West Warwick, RI) and a high-performance AC preamplifier (model P511; Grass Instrument Division). Low- and high-frequency cutoffs were set at 0.1 and 1000 Hz; amplification was set to be 5000. Amplified signals were digitized through an analog-to-digital converter, input-output board (model PCI-1200; National Instruments, Austin, TX) in a desktop computer. 11 The illuminance on the surface of the eye was approximately 2.8 lux. The duration of the flash stimulus was 10 μsec. The interstimulus interval was 30 seconds. ERGs elicited by five stimuli were averaged using custom software over a response window of 1000 msec. The b-wave amplitude (measured from the trough of the a-wave to the peak of the b-wave) and b-wave implicit times (measured from the flash onset to the peak of the b-wave) were compared between the eyes with drug injection and the pooled normal control eyes, which included baseline and concurrent control eyes. 12  
Pharmacokinetic Study.
For the pharmacokinetic study, 14 rabbits were used. Nine rabbits (18 eyes) were used for an in vivo pharmacokinetic study of drug levels in vitreous aspirates at different time points, and five (5 eyes) were used for an in vivo pharmacokinetic study of whole vitreous at postinjection week 8. 
In the vitreous aspirate study, 18 eyes, which received one of the five doses, were tapped after initial intravitreal injections. Three eyes were injected with 0.885 μmol, three with 1.57, three with 2.8, four with 4.486, four with 8.85, and one with normal saline. At postinjection day 1 and at 1, 2, 3, 5, 8, 12, and 18 weeks, a vitreous sample was removed through the pars plana with a 23-gauge needle attached to a 0.5-mL syringe. Vitreous fluid (0.05 mL) was obtained from a location deliberately away from the drug depot. Vitreous samples were collected in a preweighed vial and stored at −70°C until analysis. 
For the whole vitreous study, five eyes of five rabbits injected with the different doses were enucleated 8 weeks after drug injection. Eyes were dissected, and vitreous was sampled as previously described. 13 The vitreous samples were analyzed for HDP-P-GCV, by using HPLC as described earlier. 
Treatment Study.
For the retinitis treatment study, 90 rabbits were used, including 11 rabbits for treatment study and 79 for prophylaxis study. Only the right eye of each rabbit was used. Ophthalmoscopic retinitis grading was performed by an unmasked observer, who used a previously reported method with a standardized grading scheme 10 : 0, a normal optic nerve head and retina compared with the fellow eye; 0.5, significant optic nerve head swelling and hyperemia without hemorrhage; 1, optic nerve head and medullary ray flame hemorrhage; 2, scattered infectious foci on the inferior retina in addition to the grade 1 changes; 3, confluent white retinitis lesions over the inferior retina without involvement of the superior retina; and 4, whole retina involvement with retinal detachment and severe vitreous clouding. 
For the treatment study, the right eyes of 14 rabbits were intravitreally injected with 0.06 mL of a 5 × 10−5 dilution of 10−7.6 mean tissue culture infective dose (TCID50)/mL titered HSV-1. When retinitis developed and reached grade 1 (earliest detected retinitis grade: 1 or 2), the infected eyes received 2.8 μmol of ammonium salt of HDP-P-GCV in 0.1 mL (final predicted intravitreal concentration of 2 mM) 5% dextrose for treatment or 0.1 mL 5% dextrose only for a control. For the prophylaxis study, two doses, 2.8 and 8.85 μmol (final predicted intravitreal concentrations of 2.0 mM and 6.32 mM), were tested separately. In this strategy, HSV-1 was intravitreally inoculated into right eyes at given time periods after drug (prophylaxis group) or saline (control group) intravitreal injections. Each group of control eyes were injected concurrently with each corresponding group of experimental eyes. For the 2.8-μmol dose, 39 rabbits were divided into four groups: the 4-, 8-, 12-, and 20-week prophylaxis groups. For the 8.85-μmol dose, 37 rabbits were divided into four groups: the 2-, 6-, 12-, and 20-week prophylaxis groups. The HSV-1 virus dose, injection method, and clinical retinitis grading were as previously reported. 9 10 Rabbits were killed 2 weeks after development of retinitis. Rabbits without retinitis 3 or 4 weeks after HSV-1 inoculation were killed. After death, enucleated globes were fixed with 10% formalin, and gross dissection was performed before routine histologic processing. 
Statistical Analysis
Nonparametric analysis of variance was performed by using the Kruskal-Wallis test on computer (JMP software, ver. 3.1; SAS Inc., Cary, NC). Differences in retinitis scores in treated versus control groups were compared. For the incidence of local retinal toxicity, comparison between lower doses and higher doses was performed by the Fisher exact test. ERG amplitudes and implicit times were compared between treated and control eyes by Student’s t-test. 
Results
Properties of HDP-P-GCV
The ammonium salt of HDP-P-GCV is a white crystalline powder. Analysis of the particle size of HDP-P-GCV by laser light scattering showed a bimodal particle size distribution with mean diameters of 8 to 43 μm. The compound is slightly soluble in water, and a stable slurry can be prepared in 5% dextrose by sonication. This preparation was readily injectable directly into the vitreous through a small-gauge needle. 
Evaluation of Toxicity
After intravitreal injection, the drug aggregated and formed a drug depot at the injection site, with minimal initial visible dispersion into the surrounding vitreous. After 24 hours, drug remained visible at the injection site, with slight dispersion into the vitreous near the injection site. The central vitreous was completely clear (Fig. 1) . Over time, the drug depot became smaller but was still visible at the end of the experiment (8 weeks), even with the lowest dose (0.885μ mol; 0.632 mM final predicted intravitreal concentration). The size of the visible drug depot was dose dependent. No clinical toxicity was noted during the entire experiment, except for mild posterior subcapsular cataract in two eyes at week 3 after intravitreal drug injection: one eye with a dose of 4.486 μmol and the other with 8.85μ mol (Table 1)
The full-field ERGs from normal rabbit eyes showed a mean b-wave amplitude of 132.5 ± 38.4 μV and b-wave implicit time of 66.5 ± 17.4 msec. ERGs from the eyes with different doses of HDP-P-GCV demonstrated normal ERG waveforms. ERG amplitudes and implicit times were in the mean ± 2 SD range of the normal rabbit ERGs. 12 For the statistical assessment, the eyes with the 2.8-μmol or lower doses were classified as the low-dose group, and the eyes with 4.486-μmol and 8.85-μmol doses were classified as the high dose group. At the 2-week time point, the low-dose group had a mean b-wave amplitude of 155 ± 18 μV and a mean b-wave implicit time of 77.3 ± 13 msec; the high-dose group showed a mean b-wave amplitude of 145.4 ± 27.6 μV and a mean b-wave implicit time of 73.1 ± 18.1 msec. At the 8-week time point, the low-dose group exhibited a mean b-wave amplitude of 175.8 ± 21.9 μV and a mean b-wave implicit time of 74 ± 9.8 msec; the high-dose group demonstrated a mean b-wave amplitude of 137.4 ± 34 μV and a mean b-wave implicit time of 64.7 ± 9.7 msec. All these parameters were not statistically different from those in the normal control eyes (P > 0.05) except for the 8-week b-wave amplitude of the low-dose group, which was higher than the control b-wave amplitude. 
In the pathologic evaluation, eyes with 2.8-μmol and lower doses showed normal retinal structures, including the retina adjacent to the drug depot (Fig. 2) . Local retinal toxicity was noted in 56% of the eyes injected with the 4.486- and 8.85-μmol doses (Table 1) . The local toxicity was present only in the area of retina that was in contact with the drug depot. The toxicity was characterized by loss of the outer nuclear layer, or even part of the inner nuclear layer in advanced cases, and by minimal responsive inflammatory infiltration at the contacting site (Fig. 3 ; Table 1 ). 
Pharmacokinetic Study
At day 1 after intravitreal drug injection, free intravitreal drug levels ranged from 2.1 ± 1.47 to 10.1 ± 4.84 μM. The free intravitreal HDP-P-GCV level in vitreous aspirates increased over time with dose dependence and with peak concentrations at 1 week for the 1.57- and 2.8-μmol doses and at 3 weeks for the 4.486- and 8.85-μmol doses. Free drug levels in the vitreous with the 0.885-μmol dose were low and without obvious peak. After the peak concentration was reached, free drug levels in the vitreous gradually decreased over time. The free drug level for the 2.8-μmol dose was 0.2 ± 0 μM at week 12 after a single injection and 1.95 ± 2.03 μM at week 18 for the 8.85-μmol dose after a single injection (Fig. 4) . The whole vitreous samples at week 8 after a single intravitreal injection showed that the HDP-P-GCV vitreous levels were 0.1 mM for the 0.885-μmol dose, 0.15 mM for 1.57 μmol, 0.43 mM for 2.8 μmol, 0.71 mM for 4.486 μmol, and 1.03 mM for 8.85 μmol. 
Treatment Studies
Treatment of Established Retinitis.
Eleven eyes that were inoculated with HSV-1 virus showed development of retinitis between 4 and 6 days after viral inoculation. After retinitis reached grade 1 (documented by indirect ophthalmoscopy), these eyes were treated with drug or 5% dextrose. The baseline retinitis scores before intervention were similar between drug-treated and control groups (Table 2) . At days 4 and 10 after intervention, drug-treated eyes showed a median retinitis score of 3 and 3.5, respectively, versus 4 and 4 in the control group (P = 0.011, P = 0.077; Table 2 ). 
Prophylaxis Results.
In the prophylaxis study, the 2.8- and 8.85-μmol doses (2.0 and 6.32 mM final predicted intravitreal concentrations, respectively) were tested. In the 2.8-μmol 4-week prophylaxis study, five of six eyes were completely protected from HSV-1 infection, and delayed retinitis of less severity developed in one eye, when compared with the control eyes, all of which showed development of typical HSV-1 retinitis, as we have described previously. 10 In the 8-, 12-, and 20-week prophylaxis studies, retinitis eventually developed in all treated eyes. However, retinitis was delayed and was significantly less severe in the drug-treated eyes than in the control eyes (Table 3)
In the 8.85-μmol prophylaxis study, four of five treated eyes in the 2-week prophylaxis group did not have retinitis; five of six in the 6-week prophylaxis group showed complete protection; four of five treated eyes showed complete protection in the 12-week prophylaxis group; and all three eyes showed complete protection in the 20-week prophylaxis group (Table 4) . Rabbits in the 20-week prophylaxis group were observed for 25 weeks after drug injection and 5 weeks after HSV-1 virus intravitreal inoculation before death. 
HSV-1 retinitis, with a typical course of evolution, developed in all control eyes. 
Discussion
We report a new intraocular drug delivery system using HDP-P-GCV as the prototype compound. This compound is an example of an engineered biologically active compound designed to be slow releasing. We achieved this by conjugating an alkylpropanediol of an appropriate carbon chain length to the phosphate of GCV-MP, yielding an amphiphilic compound with hydrophobic and hydrophilic moieties. This compound is a crystalline white powder with a mean particle size ranging from 8 to 43μ m. The preparation can be directly injected into vitreous through a small-gauge needle to form a drug depot that appears to release the drug slowly and to provide a long-lasting level of antiviral drug in the retina. 
The powdered ammonium salt of HDP-P-GCV was added to 5% dextrose and mixed to form the intravitreally injectable suspension. The suspension was visually similar to the triamcinolone acetonide used clinically. 14 After intravitreal injection, the drug formed a bound drug depot in the peripheral vitreous near the injection site, with completely clear vitreous elsewhere. The drug depot remained in a relatively stable position in the vitreous outside the visual axis in all cases. No clinical or pathologic vitritis was observed. We assume that the drug depot (bound drug) continuously released free HDP-P-GCV into the vitreous and then into the retina and choroid, where it metabolized to GCV triphosphate. 
Variable local retinal toxicity and local posterior subcapsular cataract were observed with the 4.486- and 8.85-μmol doses in the present study. The local toxicity appeared to be caused by direct contact between the drug depot and intraocular tissues. It was apparent that the size of the drug depot was the cause of local retinal or lens toxicity, because the local toxicity was observed only in the eyes with the 4.486-μmol and higher doses, which formed a larger visible drug depot. However, ERGs in those eyes with the high drug doses were normal. No local retinal or lens toxicity was found in eyes with the 2.8-μmol and lower doses, and ERGs in these eyes were normal. The 8-week ERG of the low-dose group showed a higher b-wave amplitude, which we think was a deviation due to the small number of samples (n = 5). In our previous study, the 2.8-μmol dose in liposome formulation caused vitreous opacification, cataract, and anterior segment congestion. 9 The highest nontoxic dose for HDP-P-GCV in liposome formulation was 0.28 μmol (0.2 mM final predicted intravitreal concentration). 9 Compared with HDP-P-GCV in liposome formulation, crystalline free HDP-P-GCV provided a much higher nontoxic dose (2.8 μmol; 2 mM final predicted intravitreal concentration) which also demonstrated a clear vitreous and lens. The reason is that more HDP-P-GCV in liposome formulation is available to intraocular tissues because of the complete liposome water solubility, whereas only the dissolved amount of hydrophobic crystalline ammonium salt of HDP-P-GCV in vitreous is available to the tissues after intravitreal injections. 
The pharmacokinetic study of vitreous aspirates (upper vitreous) showed active free ammonium salt of HDP-P-GCV in vitreous at a concentration of 0.2 μM 12 weeks after the 2.8-μmol initial intravitreal dose. It was still at 1.95 μM 18 weeks after the 8.85-μmol initial intravitreal dose. These concentrations (0.2 and 1.95 μM) were much higher than the mean inhibitory concentration (IC50) for HSV-1 (0.02 μM). 9 For human cytomegalovirus (HCMV), the HDP-P-GCV IC50 is 0.6 μM. For the highest nontoxic dose, 2.8 μmol, in the present study, the free drug concentration (5.79 μM) at week 5 after a single intravitreal injection was 10 times higher than the IC50 for HCMV. For the highest dose tested in this study (8.85 μmol with 6.32 mM final predicted intravitreal concentration), the active free drug level at week 18 (1.95 μM) was still above the IC50 for HCMV. In a recent report, 15 the GCV level in rabbit eyes at day 70 (10 weeks) after a standard GCV implant was 1.3 μg/mL. This is equivalent to an intravitreal concentration of 4.3 μM. The 2.8-μmol injection yielded an HDP-P-GCV level of 0.2 μM at week 12, which was lower than the GCV concentration achieved by GCV implant at week 10. However, the 8.85-μmol dose in this study demonstrated a HDP-P-GCV level of 3.8μ M at week 12 after a single intravitreal injection, which is similar to the GCV level achieved by GCV implant at week 10. In addition, HDP-P-GCV is three times as potent for HCMV compared with GCV (IC50 0.6 μM for HDP-P-GCV versus 1.6 μM for GCV, P < 0.05). 9 Therefore, free crystalline ammonium salt of HDP-P-GCV may be as effective as the GCV implant in the prevention and treatment of HCMV in immunocompromised patients. 
In our vitreous pharmacokinetic study, whole vitreous HDP-P-GCV concentration was 1000-fold (millimolar versus micromolar) higher than the HDP-P-GCV concentration in the upper vitreous at week 8, when the bound drug depot was in the whole vitreous sample. This finding suggests that this compound may provide a higher free drug level without diminishing the intraocular sustained release course by minimizing the drug particle size, thus increasing the particle surface area and its water solubility. 
In the prophylaxis study, both the 2.8- and 8.85-μmol doses demonstrated a 20-week antiviral duration of action, with the 8.85-μmol dose providing 20 weeks of complete retinal protection against HSV-1 infection. The 2.8-μmol dose, after showing a 4-week complete retinal protection against HSV-1 infection, significantly delayed retinitis occurrence and inhibited severity in the subsequent weeks, compared with the control eyes. The retinitis model used in this study is a much more severe and rapidly progressive retinitis than that in humans. We hypothesize that the 2.8-μmol dose may provide anti-HSV or anti-HCMV action in human retinitis beyond the period observed in the present study. In the current experimental setting, the duration of treatment efficacy was longer than the duration of vitreous therapeutic drug concentration determined by the pharmacokinetic study. This may be due to the incorporation of HDP-P-GCV into the membrane lipids of the retinal cells and the slow release into the cytoplasm by cellular phosphodiesterases or phospholipase C. The antiviral protection provided by a single intravitreal injection of the crystalline ammonium salt of HDP-P-GCV is at least 20 times longer than that provided by a single intravitreal GCV injection and at least 4 times longer than that provided by a single self-assembling liposomal HDP-P-GCV intravitreal injection, in the similar experimental setting. 9 The crystalline ammonium salt of HDP-P-GCV intravitreal injection may provide a long sustained intraocular maintenance treatment for HCMV or other forms of the herpes family virus retinitis. 
In the retinitis treatment study, we used only the highest nontoxic dose (2.8 μmol; 2.0 mM final predicted intravitreal concentration) to treat already-established HSV-1 retinitis. Treated eyes demonstrated significantly slower progression and less severity of retinitis than did the control eyes. However, in all treated eyes, the therapy failed to prevent progression of retinitis. The failure to completely control the progression of experimental HSV-1 retinitis is probably related to the fulminant nature of this retinitis model, which completely destroys the entire retina within 2 weeks of viral inoculation, if left untreated. 10 16 However, the slow-release nature of this delivery system may not provide the immediate therapeutic levels that are needed for an induction treatment. To overcome this limitation, GCV could be used combined with the crystalline ammonium salt of HDP-P-GCV to initiate an immediate therapeutic effect. 
In summary, we have shown that crystalline HDP-P-GCV in the form of 8- to 43-μm particles may have utility in treating or preventing HSV retinitis when injected intravitreally as infrequently as once a month or less frequently. The local retinal or lens toxicity observed with high doses may be eliminated, and antiviral duration could even be prolonged by using smaller drug particles, which may provide a better release rate and require less drug to maintain a therapeutic vitreous level with the advantage of a smaller drug depot. 
 
Figure 1.
 
Fundus photograph showing aggregated drug depot in peripheral vitreous cavity. Vitreous is clear around the drug depot, and vortex veins can be seen clearly.
Figure 1.
 
Fundus photograph showing aggregated drug depot in peripheral vitreous cavity. Vitreous is clear around the drug depot, and vortex veins can be seen clearly.
Table 1.
 
Safety and Toxicity with Intravitreal Injection of Crystaline HDP-P-GCV
Table 1.
 
Safety and Toxicity with Intravitreal Injection of Crystaline HDP-P-GCV
Intravitreal Doses 2 Weeks after Injection 8 Weeks after Injection
Eyes (n) VCS (+)* Visible Drug Depot Cataract, † ERG General Pathology Local Retinal Toxicity Eyes (n) VCS (+)* Visible Drug Depot Cataract, † ERG General Pathology Local Retinal Toxicity
Saline (0.1 mL) 2 0, 0 No 0, 0 NL NL N, N 1 0 No 0 NL NL N, N
Crystaline HDP-P-GCV (μmol)
0.885 2 0, 0 Small 0, 0 NL NL N, N 2 0, 0 Small 0, 0 NL NL N, N
1.57 2 0, 0 Medium 0, 0 NL NL N, N 2 0, 0 Medium 0, 0 NL NL N, N
2.8 2 0, 0 Medium 0, 0 NL NL N, N 1 0 Medium 0 NL NL N
4.486 6 0, 0, 0, 0, 0, 0 Larger 0, 0, 0, 0, 0, 0 NL NL N, Y, N, Y, Y, N 3 0, 0, 0 Larger 0, 0, 1 NL NL N, Y, Y
8.85 6 0, 0, 0, 0, 0, 0 Larger 0, 0, 0, 0, 0, 0 NL NL Y, N, Y, N, Y, Y 3 0, 0, 0 Larger 0, 0, 1 NL NL N, Y, N
P , ‡ P = 0.04, ‡
Figure 2.
 
Light microphotograph of an eye that received the 2.8-μmol dose (2.0 mM final predicted intravitreal concentration) showing drug depot (arrow) in vitreous and normal retina (mild artificial retinal detachment present). Magnification, ×10.
Figure 2.
 
Light microphotograph of an eye that received the 2.8-μmol dose (2.0 mM final predicted intravitreal concentration) showing drug depot (arrow) in vitreous and normal retina (mild artificial retinal detachment present). Magnification, ×10.
Figure 3.
 
Light microphotograph from an eye with 8.85-μmol dose (6.32 mM final predicted intravitreal concentration), showing the contact between drug depot and retina (arrow). The retina in contact with the drug depot demonstrated toxic changes of retinal gliosis and diminished inner and outer nuclear layer thickness. The retinal detachment is artificial. Magnification, ×25.
Figure 3.
 
Light microphotograph from an eye with 8.85-μmol dose (6.32 mM final predicted intravitreal concentration), showing the contact between drug depot and retina (arrow). The retina in contact with the drug depot demonstrated toxic changes of retinal gliosis and diminished inner and outer nuclear layer thickness. The retinal detachment is artificial. Magnification, ×25.
Figure 4.
 
Noncrystalline HDP-P-GCV concentrations in vitreous aspirates at time points (0.14 indicates day 1) after the initial injection of different doses. The free HDP-P-GCV concentration was dose dependent. The concentrations are expressed as the mean ± SD.
Figure 4.
 
Noncrystalline HDP-P-GCV concentrations in vitreous aspirates at time points (0.14 indicates day 1) after the initial injection of different doses. The free HDP-P-GCV concentration was dose dependent. The concentrations are expressed as the mean ± SD.
Table 2.
 
Progression of Retinitis in HDP-P-GCV–Treated and Control Eyes
Table 2.
 
Progression of Retinitis in HDP-P-GCV–Treated and Control Eyes
Group Eyes (n) Preinjection Retinitis Score (median) Intravitreal Injection Retinitis Score after Treatment (Median)
Day 4 Day 10
Treatment 6 1.5 HDP-P-GCV (2.8 μmol) 3 3.5
Control 5 1 5% Dextrose (0.1 mL) 4 4
P* 0.33 0.011 0.077
Table 3.
 
Time Course and Median Retinitis Scores in Crystalline HDP-P-GCV (2.8μ mol)–Treated and –Untreated Control Rabbits
Table 3.
 
Time Course and Median Retinitis Scores in Crystalline HDP-P-GCV (2.8μ mol)–Treated and –Untreated Control Rabbits
Group Rabbits (n) Time Course and Median Retinitis Score (Infection %)*
Day 4 Day 6 Day 9 Day 14
4-wk prophylaxis
HDP-P-GCV 6 0 (0/6) 0 (1/6) 0 (1/6) 0 (1/6)
Saline control 4 1.5 (4/4) 4 (4/4) 4 (4/4) 4 (4/4)
P, † 0.0038 0.0047 0.0143 0.0143
8-wk prophylaxis
HDP-P-GCV 4 0 (0/4) 1 (3/4) 3 (4/4) 3.5 (4/4)
Saline control 5 1 (5/5) 3 (5/5) 4 (5/5) 4 (5/5)
P, † 0.0047 0.0123 0.0281 0.0935
12-wk prophylaxis
HDP-P-GCV 5 0 (0/5) 0.5 (4/5) 3 (5/5) 4 (5/5)
Saline control 5 0.5 (5/5) 4 (5/5) 4 (5/5) 4 (5/5)
P, † 0.0047 0.008 0.05 NS
20-wk prophylaxis
HDP-P-GCV 5 0 (1/5) 2 (4/5) 4 (5/5)
Saline control 5 1 (4/5) 3 (5/5) 4 (5/5)
P, † 0.0282 0.0827 NS
Table 4.
 
Time Course and Median Retinitis Scores in Crystalline HDP-P-GCV (8.85μ mol)–Treated and –Untreated Control Rabbits
Table 4.
 
Time Course and Median Retinitis Scores in Crystalline HDP-P-GCV (8.85μ mol)–Treated and –Untreated Control Rabbits
Group Rabbits (n) Time Course and Median Retinitis Score (Infection %)*
Day 4 Day 6 Day 9 Day 14
2-wk prophylaxis
HDP-GCV 5 0 (0/5) 0 (0/5) 0 (0/5) 0 (1/5)
Saline control 8 2.5 (8/8) 4 (8/8) 4 (8/8) 4 (8/8)
P, † 0.0025 0.001 0.0005 0.0007
6-wk prophylaxis
HDP-P-GCV 6 0 (0/6) 0 (0/6) 0 (0/6) 0 (1/6)
Saline control 4 1.5 (4/4) 3.5 (4/4) 4 (4/4) 4 (4/4)
P, † 0.0038 0.0038 0.0027 0.0047
12-wk prophylaxis
HDP-P-GCV 5 0 (0/5) 0 (0/5) 0 (1/5) 0 (1/5)
Saline control 5 1 (5/5) 4 (5/5) 4 (5/5) 4 (5/5)
P, † 0.0047 0.0039 0.0039 0.0143
20-wk prophylaxis
HDP-P-GCV 3 0 (0/3) 0 (0/3) 0 (0/3) 0 (0/3)
Saline control 4 1 (4/4) 3 (4/4) 4 (4/4) 4 (4/4)
P, † 0.0143 0.0219 0.0143 0.0143
Diaz-Llopis M, Espana E, Munoz G, et al. High dose intravitreal foscarnet in the treatment of cytomegalovirus retinitis in AIDS. Br J Ophthalmol. 1994;78:120–124. [CrossRef] [PubMed]
Cochereau-Massin I, Lehoang P, Lautier-Frau M, et al. Efficacy and tolerance of intravitreal ganciclovir in cytomegalovirus retinitis in acquired immune deficiency syndrome. Ophthalmology. 1991;98:1348–1353. [CrossRef] [PubMed]
Young S, Morlet N, Besen G, et al. High-dose (2000-microgram) intravitreous ganciclovir in the treatment of cytomegalovirus retinitis. Ophthalmology. 1998;105:1404–1410. [CrossRef] [PubMed]
Rahhal FM, Arevalo JF, Chavez de la Paz E, Munguia D, Azen SP, Freeman WR. Treatment of cytomegalovirus retinitis with intravitreous cidofovir in patients with AIDS: a preliminary report (see comments). Ann Intern Med. 1996;125:98–103. [CrossRef] [PubMed]
Martin DF, Parks DJ, Mellow SD, et al. Treatment of cytomegalovirus retinitis with an intraocular sustained-release ganciclovir implant: a randomized controlled clinical trial (see comments). Arch Ophthalmol. 1994;112:1531–1539. [CrossRef] [PubMed]
Roth DB, Flynn HW, Jr. Antibiotic selection in the treatment of endophthalmitis: the significance of drug combinations and synergy. Surv Ophthalmol. 1997;41:395–401. [CrossRef] [PubMed]
Martin DF, Ferris FL, Parks DJ, et al. Ganciclovir implant exchange: timing, surgical procedure, and complications. Arch Ophthalmol. 1997;115:1389–1394. [CrossRef] [PubMed]
Cheng L, Hostetler KY, Gardner MF, et al. Intravitreal toxicology in rabbits of two preparations of 1-O-octadecyl-sn-glycerol-3-phosphonoformate, a sustained-delivery anti-CMV drug. Invest Ophthalmol Vis Sci. 1999;40:1487–1495. [PubMed]
Cheng L, Hostetler KY, Chaidhawangul S, et al. Intravitreal toxicology and duration of efficacy of a novel antiviral lipid prodrug of ganciclovir in liposome formulation. Invest Ophthalmol Vis Sci. 2000;41:1523–1532. [PubMed]
Cheng L, Hostetler KY, Chaidhawangul S, et al. Treatment of herpes retinitis in an animal model with a sustained delivery antiviral drug, liposomal 1-O-octadecyl-SN-glycerol-3-phosphonoformate. Retina. 1999;19:325–331. [CrossRef] [PubMed]
Nusinowitz S, Birch DG, Birch EE. Rod photoresponses in 6-week and 4-month-old human infants. Vision Res. 1998;38:627–635. [CrossRef] [PubMed]
Iijima H. Distribution of ERG amplitudes, latencies, and implicit times. Heckenlively JR eds. Principles and Practice of Clinical Electrophysiology of Vision. 1991;289–290. Mosby-Year Book, Inc St. Louis.
Cheng L, Hostetler KY, Gardner MF, et al. Intravitreal pharmacokinetics in rabbits of the foscarnet lipid prodrug: 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA). Curr Eye Res. 1999;18:161–167. [CrossRef] [PubMed]
Danis RP, Ciulla TA, Pratt LM, Anliker W. Intravitreal triamcinolone acetonide in exudative age-related macular degeneration. Retina. 2000;20:244–250. [CrossRef] [PubMed]
Perkins SL, Yang CH, Ashton PA, Jaffe GJ. Pharmacokinetics of the ganciclovir implant in the silicone-filled eye. Retina. 2001;21:10–14. [CrossRef] [PubMed]
Flores-Aguilar M, Huang JS, Wiley CA, et al. Long-acting therapy of viral retinitis with (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine. J Infect Dis. 1994;169:642–647. [CrossRef] [PubMed]
Figure 1.
 
Fundus photograph showing aggregated drug depot in peripheral vitreous cavity. Vitreous is clear around the drug depot, and vortex veins can be seen clearly.
Figure 1.
 
Fundus photograph showing aggregated drug depot in peripheral vitreous cavity. Vitreous is clear around the drug depot, and vortex veins can be seen clearly.
Figure 2.
 
Light microphotograph of an eye that received the 2.8-μmol dose (2.0 mM final predicted intravitreal concentration) showing drug depot (arrow) in vitreous and normal retina (mild artificial retinal detachment present). Magnification, ×10.
Figure 2.
 
Light microphotograph of an eye that received the 2.8-μmol dose (2.0 mM final predicted intravitreal concentration) showing drug depot (arrow) in vitreous and normal retina (mild artificial retinal detachment present). Magnification, ×10.
Figure 3.
 
Light microphotograph from an eye with 8.85-μmol dose (6.32 mM final predicted intravitreal concentration), showing the contact between drug depot and retina (arrow). The retina in contact with the drug depot demonstrated toxic changes of retinal gliosis and diminished inner and outer nuclear layer thickness. The retinal detachment is artificial. Magnification, ×25.
Figure 3.
 
Light microphotograph from an eye with 8.85-μmol dose (6.32 mM final predicted intravitreal concentration), showing the contact between drug depot and retina (arrow). The retina in contact with the drug depot demonstrated toxic changes of retinal gliosis and diminished inner and outer nuclear layer thickness. The retinal detachment is artificial. Magnification, ×25.
Figure 4.
 
Noncrystalline HDP-P-GCV concentrations in vitreous aspirates at time points (0.14 indicates day 1) after the initial injection of different doses. The free HDP-P-GCV concentration was dose dependent. The concentrations are expressed as the mean ± SD.
Figure 4.
 
Noncrystalline HDP-P-GCV concentrations in vitreous aspirates at time points (0.14 indicates day 1) after the initial injection of different doses. The free HDP-P-GCV concentration was dose dependent. The concentrations are expressed as the mean ± SD.
Table 1.
 
Safety and Toxicity with Intravitreal Injection of Crystaline HDP-P-GCV
Table 1.
 
Safety and Toxicity with Intravitreal Injection of Crystaline HDP-P-GCV
Intravitreal Doses 2 Weeks after Injection 8 Weeks after Injection
Eyes (n) VCS (+)* Visible Drug Depot Cataract, † ERG General Pathology Local Retinal Toxicity Eyes (n) VCS (+)* Visible Drug Depot Cataract, † ERG General Pathology Local Retinal Toxicity
Saline (0.1 mL) 2 0, 0 No 0, 0 NL NL N, N 1 0 No 0 NL NL N, N
Crystaline HDP-P-GCV (μmol)
0.885 2 0, 0 Small 0, 0 NL NL N, N 2 0, 0 Small 0, 0 NL NL N, N
1.57 2 0, 0 Medium 0, 0 NL NL N, N 2 0, 0 Medium 0, 0 NL NL N, N
2.8 2 0, 0 Medium 0, 0 NL NL N, N 1 0 Medium 0 NL NL N
4.486 6 0, 0, 0, 0, 0, 0 Larger 0, 0, 0, 0, 0, 0 NL NL N, Y, N, Y, Y, N 3 0, 0, 0 Larger 0, 0, 1 NL NL N, Y, Y
8.85 6 0, 0, 0, 0, 0, 0 Larger 0, 0, 0, 0, 0, 0 NL NL Y, N, Y, N, Y, Y 3 0, 0, 0 Larger 0, 0, 1 NL NL N, Y, N
P , ‡ P = 0.04, ‡
Table 2.
 
Progression of Retinitis in HDP-P-GCV–Treated and Control Eyes
Table 2.
 
Progression of Retinitis in HDP-P-GCV–Treated and Control Eyes
Group Eyes (n) Preinjection Retinitis Score (median) Intravitreal Injection Retinitis Score after Treatment (Median)
Day 4 Day 10
Treatment 6 1.5 HDP-P-GCV (2.8 μmol) 3 3.5
Control 5 1 5% Dextrose (0.1 mL) 4 4
P* 0.33 0.011 0.077
Table 3.
 
Time Course and Median Retinitis Scores in Crystalline HDP-P-GCV (2.8μ mol)–Treated and –Untreated Control Rabbits
Table 3.
 
Time Course and Median Retinitis Scores in Crystalline HDP-P-GCV (2.8μ mol)–Treated and –Untreated Control Rabbits
Group Rabbits (n) Time Course and Median Retinitis Score (Infection %)*
Day 4 Day 6 Day 9 Day 14
4-wk prophylaxis
HDP-P-GCV 6 0 (0/6) 0 (1/6) 0 (1/6) 0 (1/6)
Saline control 4 1.5 (4/4) 4 (4/4) 4 (4/4) 4 (4/4)
P, † 0.0038 0.0047 0.0143 0.0143
8-wk prophylaxis
HDP-P-GCV 4 0 (0/4) 1 (3/4) 3 (4/4) 3.5 (4/4)
Saline control 5 1 (5/5) 3 (5/5) 4 (5/5) 4 (5/5)
P, † 0.0047 0.0123 0.0281 0.0935
12-wk prophylaxis
HDP-P-GCV 5 0 (0/5) 0.5 (4/5) 3 (5/5) 4 (5/5)
Saline control 5 0.5 (5/5) 4 (5/5) 4 (5/5) 4 (5/5)
P, † 0.0047 0.008 0.05 NS
20-wk prophylaxis
HDP-P-GCV 5 0 (1/5) 2 (4/5) 4 (5/5)
Saline control 5 1 (4/5) 3 (5/5) 4 (5/5)
P, † 0.0282 0.0827 NS
Table 4.
 
Time Course and Median Retinitis Scores in Crystalline HDP-P-GCV (8.85μ mol)–Treated and –Untreated Control Rabbits
Table 4.
 
Time Course and Median Retinitis Scores in Crystalline HDP-P-GCV (8.85μ mol)–Treated and –Untreated Control Rabbits
Group Rabbits (n) Time Course and Median Retinitis Score (Infection %)*
Day 4 Day 6 Day 9 Day 14
2-wk prophylaxis
HDP-GCV 5 0 (0/5) 0 (0/5) 0 (0/5) 0 (1/5)
Saline control 8 2.5 (8/8) 4 (8/8) 4 (8/8) 4 (8/8)
P, † 0.0025 0.001 0.0005 0.0007
6-wk prophylaxis
HDP-P-GCV 6 0 (0/6) 0 (0/6) 0 (0/6) 0 (1/6)
Saline control 4 1.5 (4/4) 3.5 (4/4) 4 (4/4) 4 (4/4)
P, † 0.0038 0.0038 0.0027 0.0047
12-wk prophylaxis
HDP-P-GCV 5 0 (0/5) 0 (0/5) 0 (1/5) 0 (1/5)
Saline control 5 1 (5/5) 4 (5/5) 4 (5/5) 4 (5/5)
P, † 0.0047 0.0039 0.0039 0.0143
20-wk prophylaxis
HDP-P-GCV 3 0 (0/3) 0 (0/3) 0 (0/3) 0 (0/3)
Saline control 4 1 (4/4) 3 (4/4) 4 (4/4) 4 (4/4)
P, † 0.0143 0.0219 0.0143 0.0143
Copyright 2002 The Association for Research in Vision and Ophthalmology, Inc.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×