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1 From the Shiley Eye Center, University of California San Diego, La Jolla, California; and the 2 Department of Medicine, Veterans Affairs Medical Center, and University of California San Diego, La Jolla, California.
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Abstract |
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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 (0.023 µM). 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.
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Introduction |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 150-mm column (Xterra RPC8; Waters, Inc., Milford, MA) with a 3.9 x 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 8.85
µmol 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 scheme10
: 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 x 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (0.885
µmol; 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 8.85
µmol (Table 1)
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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 ).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 3.8 µM 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.
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Footnotes |
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Submitted for publication June 1, 2001; revised September 6, 2001; accepted September 28, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: William R. Freeman, Department of Ophthalmology, Shiley Eye Center, University of California San Diego, 9415 Campus Point Drive, La Jolla, CA 92093-0946; freeman{at}eyecenter.ucsd.edu
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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