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1 From the Shiley Eye Center, University of California, San Diego; 2 Department of Medicine, VA Medical Center and University of California, San Diego, La Jolla, California; and the 3 Navy Hospital, San Diego, California.
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Abstract |
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METHODS. HDP-P-GCV was synthesized by coupling 1-O-hexadecyl-propanediol-3-phosphate to either free hydroxyl of ganciclovir in pyridine with dicyclohexylcarbodiimide as catalyst. The compound was formulated into liposomes. The antiviral activity was assessed by DNA reduction in vitro, and intraocular safety was assessed by ophthalmoscopy, electrophysiology, and histology after intravitreal injections, with resultant intravitreal concentrations of 0.2, 0.632, 1.12, and 2 mM. The treatment efficacy was evaluated by simultaneous intravitreal injection of HDP-P-GCV and herpes simplex virus type 1 (HSV-1) or by intravitreal injection of HDP-P-GCV at various times before HSV-1 intravitreal inoculation. Retinitis was scored with ophthalmoscopy and compared with controls.
RESULTS. In vitro, the IC50 of HDP-P-GCV against HSV-1 and human cytomegalovirus (HCMV)-infected cells was 0.02 and 0.6 µM, respectively. In rabbits in vivo, HDP-P-GCV dispersed evenly and maintained a good vitreous clarity at all doses except 2 mM final intravitreal concentration. Although cataracts were observed in some eyes at the higher doses, they were not observed in eyes with 0.2 mM final intravitreal concentration. No other indications of ocular toxicity were observed. Intravitreal injection of HDP-P-GCV with resultant 0.2 mM intravitreal concentration in the HSV-1 retinitis rabbit model demonstrated a complete protection of the retina with the simultaneous treatment strategy and a 4- (P = 0.03) to 6- (P = 0.058) week significant protection of retina with the pretreatment strategies when compared with ganciclovir or blank liposome controls.
CONCLUSIONS. In the rabbit model of HSV-1 retinitis HDP-P-GCV acts as a long-lasting intravitreal injectable anti-CMV or anti-HSV compound. This self-assembling liposome system could be applicable for many compounds available for intraocular diseases.
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Introduction |
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Ganciclovir (GCV) was the first drug to be approved for CMV infection in AIDS patients. Ganciclovir is effective in treating CMV retinitis by intravenous administration, but the drug does not eliminate the virus from the retina, requiring long-term suppressive maintenance therapy. During maintenance treatment, relapse is almost inevitable in non–immune-reconstituted patients; systemic toxicity such as bone marrow suppression is also a problem. The sustained-release GCV implant is effective treatment for CMV retinitis2 and recurrent CMV retinitis,3 but complications from surgery such as endophthalmitis and retina detachment are sight threatening.3 4
We wished to develop a simple, in-office injectable local therapy that would be effective, minimally toxic, and long-lasting for treatment of CMV retinitis. We have previously reported a self-assembling liposomal foscarnet analog that demonstrated a higher therapeutic index5 6 and longer vitreal half-life than foscarnet, after intravitreal injection.7 In the present study, we evaluated the intravitreal toxicity and experimental treatment efficacy of 1-O-hexadecylpropanediol-3-phospho-ganciclovir (HDP-P-GCV; Fig. 1 ).
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Methods |
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Ganciclovir (4.4 g, 17.2 mmol) was suspended in dry pyridine (100 ml) and cooled to 0°C. Chlorotrimethylsilane (11.2 g, 103 mmol) was added drop-wise over 5 minutes. The mixture was allowed to warm to room temperature.
Monomethoxytrityl chloride (6.18 g, 20 mmol) and
dimethylaminopyridine (0.25 g, 2 mmol) were added, and the mixture
was stirred overnight. The reaction mix was then cooled to 5°C with
ice/H2O, water (20 ml) was added, and the mixture
was stirred for 15 minutes. Concentrated NH4OH
(25 ml) was then added, and the mixture was stirred an additional 15
minutes. The mixture was filtered, the solvent evaporated in vacuo, and
the residue was chromatographed (gradient:
CH2Cl2 
15%
EtOH/CH2Cl2). Fractions
were evaporated, and the residue was crystallized in hot toluene to
give 5.2 g N-monomethoxytrityl-ganciclovir (58% yield).
A mixture of N-monomethoxytrityl ganciclovir (40.6 g, 0.077 mol),
hexadecylpropanediol phosphate (25.0 g, 0.066 mol), and
dicyclohexylcarbodiimide (27.2 g, 0.132 mol) in dry pyridine (600 ml)
was stirred at room temperature for 18 hours, then quenched by the
addition of water (10 ml). The solution was evaporated to dryness and
the residue chromatographed (gradient:
CH2Cl2 15%
EtOH/CH2Cl2) to give the
coupled product as a white solid (36 g, 61% yield). This solid was
suspended in 80% aqueous acetic acid and heated to 55°C for 5 hours
to remove the monomethoxytrityl protecting group. The solution was
concentrated in vacuo and the residue chromatographed (gradient:
CH2Cl2 80:20:1:1
CH2Cl2; MeOH:
NH4OH:H2O) to give
HDP-P-GCV as a white powder that was recrystallized from 2:1
(1,4-dioxane/water) to give analytically pure HDP-P-GCV (9 g, 36%
yield). The material was characterized by 1H-NMR
as follows: (CDCl3 +
DMSO-d6) d 0.852 (t, 3H), 1.249 (br s,
28H), 1.52 (m, 2H), 1.94 (t, 2H), 3.17 (m, 2H), 3.36 (t,2H), 3.45 (t,
2H), 3.76 (s,3H), 3.95 (m, 1H), 4.23 (m, 2H) 4.89 (m, 2H), 6.79 (d,
2H), 7.19 to 7.50 (m, 16H), 7.60 (s, 1H), 10.57 (s, 1H) [s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; br s, broad
singlet].
Preparation of Liposomes Containing HDP-P-GCV for Antiviral Assays
Chloroform solutions of the phospholipids, cholesterol, and drugs
were mixed to provide dioleoylphosphatidylcholine (DOPC),
dioleoylphosphatidylglycerol (DOPG), cholesterol (CH), and HDP-P-GCV at
a molar ratio of 50/10/30/10. The chloroform was removed under a stream
of nitrogen, and the thin lipid film was hydrated by the addition of
360 µl of 250 mM sorbitol/20 mM sodium acetate, pH 5.5. The small
multiple dose vial was sealed under nitrogen with a Teflon-lined cap
and sonicated for 1 hour at 42°C. The clear preparation of sonicated
liposomes, representing a nominal HDP-P-GCV concentration of 5 mM, was
diluted sequentially with Dulbecco’s modified Eagle’s medium
containing 4% fetal bovine serum to provide the indicated range of
drug concentrations.
Human CMV and HSV-1 DNA Reduction Assays
The antiviral activity of GCV and HDP-P-GCV was assessed by DNA
reduction as described previously.8
9
For human
cytomeglovirus (HCMV), subconfluent MRC-5 human lung fibroblasts were
pretreated for 24 hours with the indicated concentrations of drug in
MEM medium containing 2% fetal bovine serum and antibiotics. The
medium was removed, HCMV (AD-169 strain) was added at a dilution that
resulted in 3 to 4+ cytopathic effect in the control wells at 5 days,
and the wells were incubated at 37°C for 1 hour. The supernatant was
removed, replaced with drug dilutions at the indicated concentrations,
and incubated for 5 days. HCMV DNA was quantified by nucleic acid
hybridization using probes supplied by Diagnostic Hybrids (Athens, OH).
The herpes simplex virus type 1 (HSV-1), strain F, assays were
performed similarly but without drug pretreatment. HSV-1–infected
cells were incubated for 24 hours with drug dilutions at the indicated
concentrations. After incubation, the medium was removed, the cells
were lysed, and the HSV-1 DNA present was quantified by nucleic acid
hybridization using HSV-1 DNA probes obtained from Diagnostic Hybrids.
The results are presented as a percentage of the no drug control HCMV
or HSV-1 DNA levels (mean ± SD).
Liposome Preparation for Intravitreal Injection
HDP-P-GCV (5 mg, dry powder) was placed in a vial with DOPC, DOPG,
and cholesterol at a molar ratio of 12:48:10:30
(HDP-P-GCV/DOPC/DOPG/CH). Solvent from the carrier lipids was removed
by evaporation under nitrogen. Trace solvent was eliminated by
lyophilization overnight. 5% dextrose was added to make a final drug
concentration of 28 mM, and the mixture was sonicated for 1 hour at
45°C, producing a opalescent solution of small unilamellar liposomes
(diameter < 220 nanometers).10
This solution was
used directly or diluted with sterile 5% dextrose for injection at
final intravitreal concentrations of approximately 2.0 mM, 1.12 mM,
0.632 mM, and 0.2 mM. For the treatment study, liposomes were made at a
drug concentration of 8.85 mM and diluted to 2.8 mM to give a final
intravitreal concentration of approximately 0.2 mM.
HSV-1 for In Vivo Study
HSV-1 PH strain,11
provided courtesy of Jang Oh
(Proctor Foundation, University of California, San Francisco), was
cultured as described previously.6
Immediately before
intraocular viral inoculations, an aliquot of the virus was thawed and
further diluted 10,000-fold.
Animal Studies
For the safety and toxicity study, 10 New Zealand white rabbits
were used according to the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research. Rabbits, weighing from 2.3 to 2.8 kg
each, were divided into two groups, 2 and 8 weeks, with 5 rabbits each.
A total of 20 eyes were evaluated including controls (Tables 1
2)
. The animal anesthesia and intravitreal drug injections were
conducted as previously described.5
The rabbits received
intravitreal injections of 0.1 ml of HDP-P-GCV in liposome formulation
at concentrations of either 2.8, 8.85, 15.7, or 28 mM. Final calculated
drug concentrations in the eye were 0.2, 0.632, 1.12, and 2 mM,
assuming a vitreous volume of 1.4 ml.7
12
The same volume
of saline, 0.9%, was injected into 4 eyes as controls.
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At 2 or 8 weeks after injection, the rabbits were euthanatized. Enucleated globes were fixed, dissected, and processed as previously described5 for light and electron microscopic examination.
Electroretinography (ERG) was performed on all drug-injected rabbits and control rabbits postoperatively before they were killed as described previously by our group.5
For the treatment study, a total of 72 pigmented rabbits (weighing 1.7–3.6 kg) were used in accordance with the guidelines of the University of California, San Diego, Office of Campus Veterinary Services. Only the right eye of each rabbit was used for the study. The rabbits were divided into five groups: simultaneous treatment (22 rabbits), 1-week pretreatment (14 rabbits), 2-week pretreatment (13 rabbits), 4-week pretreatment (15 rabbits), and 6-week pretreatment (8 rabbits). For the simultaneous treatment study, 7 rabbits received intravitreal injections of 0.1 ml saline and 0.06 ml of a 1 x 10-4 dilution of 10-7.6 TCID50/ml titered HSV-1 (TCID50: the 50% tissue culture infective dose); 6 rabbits received intravitreal injections of 0.1 ml blank liposomes (equivalent to the amount of liposomes in 0.2 mM final intravitreal concentration of HDP-P-GCV) and 0.06 ml of a 1 x 10-4 dilution of 10-7.6 TCID50/ml titered HSV-1; 5 rabbits received intravitreal injections of 71.4 µg GCV in 0.1 ml (equivalent vitreous concentration of 0.2 mM, i.e., 0.1 ml of 2.8 mM HDP-P-GCV) and 0.06 ml of a 1 x 10-4 dilution of 10-7.6 TCID50/ml titered HSV-1; and four rabbits were intravitreally injected with 0.1 ml of 2.8 mM HDP-P-GCV and 0.06 ml of a 1 x 10-4 dilution of 10-7.6 TCID50/ml titered HSV-1. For the 1-week pretreatment study, 6 rabbits were injected with 0.1 ml of 2.8 mM HDP-P-GCV, whereas the other 7 rabbits were injected with 700 µg GCV in 0.1 ml as control. One week later those 13 eyes were inoculated with 0.06 ml of a 1 x 10-4 dilution of 10-7.6 TCID50/ml titered HSV-1. For the 2-week pretreatment study, 8 rabbits were intravitreally injected with HDP-P-GCV, and 4 rabbits were injected with 700 µg GCV as control. Two weeks later those 12 eyes were inoculated with 0.06 ml of a 1 x 10-4 dilution of 10-7.6 TCID50/ml titered HSV-1. For the 4-week pretreatment study, 7 rabbits were intravitreally injected with 0.1 ml of 2.8 mM HDP-P-GCV, and 7 rabbits were injected with 700 µg GCV in 0.1 ml as control. Four weeks later those 14 rabbits were inoculated with 0.06 ml of a 1 x 10-4 dilution of 10-7.6 TCID50/ml titered HSV-1. For the 6-week pretreatment, 5 eyes were injected with 0.1 ml of 2.8 mM HDP-P-GCV, and 3 eyes were injected with saline as control to confirm the virus viability. In addition, 1 rabbit each was used to confirm the virus viability with intravitreal virus inoculation in the 1-, 2-, and 4-week pretreatment groups.
The HSV-1 was intravitreally inoculated under direct observation using a surgical microscope as described before by us.6 After the intravitreal HSV-1 inoculation, both eyes were examined on days 2, 4, 6, 9, 11, and 14. After careful fundus examination with an indirect ophthalmoscope, diagrams were made and the retinitis was graded according to the previously published score criteria.6 In selected cases the fundus was photographed. Most rabbits were killed 2 weeks after receiving HSV-1; however, a few of them were killed before the 2-week point because HSV-1 encephalitis developed or the fellow eye became infected. After eye enucleation, the globes were fixed in 10% formalin for 2 to 5 days before processing for routine histopathologic examination.
Immunohistochemical Assay for HSV-1 Antigen
Paraffin sections (5-µm-thick) were deparaffinized, then placed
in 1% hydrogen peroxide in absolute methanol solution to neutralize
endogenous peroxidase activity. Tissue sections were hydrated in a
series of graded alcohols to water and then to a Tris buffer (0.05 M
Tris, 1.5% NaCl, 2 mM CaCl2, pH 7.6) and placed
in proteinase K in Tris buffer (42 µg/ml) at 37°C for 20 minutes.
Sections were then transferred into 2% glycine in phosphate-buffered
saline (PBS) for 5 minutes and subsequently placed in PBS (pH 7.3) for
5 minutes. The sections were then covered with a 1:40 dilution of
antibody (horseradish peroxidase–conjugated rabbit anti-human HSV-1
antibody, No. P0175; DAKO, Carpinteria, CA) in a fish gelatin block,
coverslipped, and incubated at 4°C overnight in a humidity chamber.
The next day the coverslips were removed in a beaker of
dH2O. The slides were washed with PBS, followed
by 0.1 M acetate buffer (pH 5.2), and then incubated with
aminoethylcarbazole at 37°C in a humidity chamber for 20 to 30
minutes. The reaction was killed in dH2O, and the
sections were counterstained with Mayer’s hematoxylin.
Statistical Analysis
Statistical analysis was done using the Kruskal-Wallis test and
JMP software, version 3.1 (SAS, Cary, NC). The scores from evaluation
of vitreous clarity, anterior segment inflammation, cataract, and
vitritis were computerized across the doses to reveal the meaningful
differences. Differences in retinitis scores in HDP-P-GCV–treated
versus control groups were also compared with the Kruskal-Wallis test.
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Results |
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Clinical Evaluation of Safety and Toxicity
After injection, HDP-P-GCV dispersed evenly in the vitreous in all
eyes except the eyes that received the highest dose (0.1 ml of 28 mM).
The vitreous was totally clear at 0.2 mM final intravitreal
concentration. Vitreous clarity decreased with increasing dose. Both
0.632 and 1.12 mM final intravitreal concentrations demonstrated a
vitreous clarity that permitted a clear view of small vessels on the
medullary ray. The 2 mM final intravitreal dose resulted in a cloudy
vitreous with the obscuration of the optic nerve head and medullary ray
(Table 1)
. Variable cataract and anterior segment injection were
observed in these doses, except for 0.2 mM final intravitreal
concentration (Table 1)
. The frequency and severity of cataract and
anterior segment injection were associated with drug concentration
(Table 1)
. Indirect ophthalmoscopy of fundus did not reveal any
abnormality in any eye. All eyes at all concentrations of HDP-P-GCV
exhibited normal ERGs when compared with the saline controls (amplitude
and latency; Fig. 2
).
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In the 2-week pretreatment study, of 8 rabbits with HDP-P-GCV, 2 did not develop retinitis and 6 developed delayed retinitis. All 4 rabbits treated with 700 µg of GCV developed typical retinitis, as did the saline controls. There were significant differences between HDP-P-GCV– and the GCV-treated eyes (Table 3) .
In the 4-week pretreatment study, the retinitis scores between HDP-P-GCV– and the maximum (700 µg) GCV–treated eyes on day 4 and day 6 still demonstrated significant differences (P = 0.0044 and P = 0.03). The eyes treated with HDP-P-GCV showed a delay in time of retinitis onset and less severity in clinical appearance.
All 3 control eyes used for confirmation of virus viability in the 1-, 2-, and 4-week pretreatment groups developed typical HSV-1 retinitis.
With the 6-week pretreatment, of 5 eyes pretreated with HDP-P-GCV, 3 developed similar retinitis to the saline control eyes, and 2 eyes were completely protected from infection (P = 0.058).
Pathologic Evaluation of the Retinitis
Pathologic examination of eyes with retinitis showed destruction
of whole layers of retina with retinal cell necrosis accompanied by
severe choroiditis, optic nerve inflammation, vitritis, and retinal
detachment (Fig. 6)
. In less severe cases, a sharp border between infected retina and
normal retina was observed, reflecting a leading edge of spreading of
viral retinitis (Fig. 7) . HSV-1 infection of retina was confirmed by immunohistochemical assay
using anti–HSV-1 antibody. The treated eyes with normal clinical
appearance of the fundus also showed normal retinal architecture
histologically.
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Discussion |
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To evaluate the in vivo duration of efficacy, we conducted a treatment study in our HSV-1 retinitis animal model. In this model, virus is inoculated onto the retina, drug efficacy can be assessed, and the duration of action of anti-HSV drugs can be determined.6 14 A direct measurement of the intravitreal concentration of HDP-P-GCV would be preferable. However, based on our previous studies,6 7 we have found that with the IC50 or IC90 obtained by the in vitro study and the drug level in living tissues determined by pharmacokinetics, the duration of protection provided by the antiviral compound against the virus cannot be precisely assessed. The pretreatment strategy used by us provides valuable information and allows comparison of relative duration of antiviral action in the eye of various compounds and formulations of antiviral drugs. We use this model because a natural rodent model of HCMV retinitis does not exist.15 Recently, attempts have been made to develop models of HCMV infection in which human retina was implanted in the anterior chambers of laboratory animals to support human cytomegalovirus infection.16 17 These models allow HCMV infection of human retina to be established in an experimental setting, but these are not natural retinitis models. It is difficult to clinically evaluate the antiviral effects of anti-CMV compounds in these settings. The murine CMV retinitis model is available,18 but there are essential differences between murine CMV and HCMV. For instance, acyclovir is highly effective against murine CMV but relatively ineffective against HCMV.19 In our model, drug treatment at the time of virus inoculation (simultaneous treatment) can be used to assess drug efficacy in treating retinitis. Pretreatment strategies with this model (drug injections days or weeks before virus inoculation) can assess duration of the antiviral effect of different intravitreally injected compounds. In the present study, the HSV-1 retinitis model produces a focal, nonlethal, and expanding infection that is highly consistent and reproducible as seen in the control eyes and a previous report.6
Both HDP-P-GCV and equivalent GCV in the simultaneous treatment study protected retinas from viral infection, except for one eye with GCV. In contrast, all saline and blank liposome control eyes developed typical HSV-1 retinitis. We wished to compare the potency of HDP-P-GCV and GCV with injected equivalent doses of GCV (yielding 0.2 mM final intravitreal concentration). The results did not show a difference between HDP-P-GCV and GCV with the dose tested (0.2 mM of final intravitreal concentration), although there was one eye in the GCV group that developed delayed retinitis. It is possible that doses lower than 0.2 mM final intravitreal concentration might reveal a difference between the two drugs if tested.
In the pretreatment study, we used 0.2 mM final intravitreal concentration of HDP-P-GCV, whereas the maximum tolerated dose of GCV was used. Based on a recent report,20 2000 µg of GCV can safely be intravitreally injected in human eyes. Assuming 1.4 ml of rabbit vitreous volume,12 700 µg was equivalent to 2000 µg for the human eye. We used 700 µg GCV intravitreal injection in all GCV pretreated eyes. The pretreatment was designed to measure duration of antiviral effect after a single intravitreal injection. The pretreatment studies revealed that the antiviral effect of HDP-P-GCV lasted more than 4 weeks after a single intravitreal injection, whereas the maximum tolerated dose of GCV did not show antiviral effect at 1 week after intravitreal injection when compared with simultaneous saline control (P = 0.1334).
GCV can be intravitreally injected to treat viral retinitis with no systemic toxicity.21 22 However, this local treatment often requires frequent intravitreal injections because of the short vitreous half-life of GCV. Frequent injection may cause endophthalmitis21 or retinal detachment20 21 and also decrease quality of life. Currently the only nonfrequent intravitreal injectable antiviral drug for local therapy is cidofovir (HPMPC).23 24 Cidofovir could provide 5- to 6-week intervals between injections, but, unfortunately, it causes iritis and even irreversible visually significant hypotony.23 Decreasing the dose does decrease the incidence of iritis and hypotony, but the treatment effect decreases.24
HDP-P-GCV provided a 4- to 6-week period of antiviral effect in our retinitis model in this study. It should be noticed that retinitis in this model is much more severe and progressive than CMV retinitis or other viral retinitis seen in humans. So, it may provide even longer antiviral duration if used to treat human viral retinitis. Compared with liposomal formulations of a lipid prodrug of foscarnet (1-O-octadecyl-sn-glycerol-3-phosphonoformate, ODG-PFA)6 and lipid prodrug of acyclovir (acyclovir diphosphate dimyristoylglycerol),14 HDP-P-GCV also demonstrated longer antiviral duration after a single injection in the same retinitis rabbit model (Table 4) .
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Footnotes |
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Submitted for publication July 14, 1999; revised November 30, 1999; accepted December 20, 1999.
Commercial relationships policy: N.
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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