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Characterization of a Novel Intraocular Drug-Delivery System Using Crystalline Lipid Antiviral Prodrugs of Ganciclovir and Cyclic Cidofovir

¹From the Shiley Eye Center, University of California San Diego, La Jolla, California; the ²Department of Medicine, San Diego VA Healthcare System and University of California San Diego, La Jolla, California; and ³Cirrus Pharmaceuticals, Durham, North Carolina.

	Abstract

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PURPOSE. In an earlier study, a novel intraocular drug-delivery system was reported in which hexadecyloxypropyl-phospho-ganciclovir (HDP-P-GCV) was used as a prototype. The hypothesis was that many biologically effective compounds could be modified to crystalline lipid prodrugs and could be delivered directly into the vitreous in a long-lasting, slow-release form. This study was undertaken to characterize this new drug-delivery system further, by using small particles of HDP-P-GCV and hexadecyloxypropyl-cyclic cidofovir (HDP-cCDV).

METHODS. HDP-P-GCV was microfluidized into 4.4-µm (median) particles, injected into rabbit vitreous. The vitreous drug level was then measured at different time points. Crystalline HDP-cCDV was synthesized, suspended in 5% dextrose, and injected into the rabbit’s vitreous at 10, 55, 100, 550, or 1000 µg in 50 µL vehicle per eye, to determine the highest nontoxic dose. The dose, 100 µg, was injected into 24 rabbit eyes, to evaluate pharmacokinetics; into 14 rabbit eyes with established HSV retinitis, to evaluate its efficacy; and into 58 rabbit eyes before herpes simplex virus (HSV) infection to evaluate its intraocular antiviral duration.

RESULTS. Microfluidized particles of HDP-P-GCV showed an increased drug release rate compared with the large-particle drug formulation, with area under concentration-time curve (AUC) of 219.8 ± 114.1 (n = 3) versus 108.3 ± 47.2 (n = 3) for unmodified HDP-P-GCV during the 12-week period after a 2.8-micromole intravitreal injection. There was a 103% increase of the drug released from the microfluidized formulation of HDP-P-GCV versus the unmodified formulation. Intravitreal injections of HDP-cCDV at doses of 100 µg/eye or lower were not toxic. After the 100 µg/eye injections, HPLC analysis showed a vitreous HDP-cCDV level of 0.05 µM at week 5, which declined to 0.002 µM at week 8. The concentration at week 8 (0.002 µM) remained above the IC₅₀ for cytomegalovirus (0.0003 µM). The pretreatment study demonstrated an antiviral effect that lasted 100 days after a single intravitreal injection.

CONCLUSIONS. This crystalline lipid prodrug intravitreal delivery system is an effective approach to achieving sustained, therapeutic drug levels in the eye. Small microfluidized particles of HDP-P-GCV provide more rapid dissolution and higher vitreous drug levels.

	Materials and Methods

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Synthesis of Compounds
HDP-P-GCV.
HDP-P-GCV was synthesized as previously reported.² To prepare a small-particle formulation and eliminate the population of large particles, HDP-P-GCV was suspended in distilled water, and the slurry was subjected to five passes through a microfluidizer (Microfluidics, Newton, MA). The slurry was then flash frozen in a 1-L round-bottomed flask and lyophilized overnight to remove the water. Unmodified HDP-P-GCV and microfluidized HDP-P-GCV were subjected to laser light-scattering particle size analysis at Cirrus Pharmaceuticals, Inc. (Durham, NC). Measurements were performed with a laser diffraction instrument (Helos; Sympatec, Lawrenceville, NJ), equipped with an R3 lens (0.5–175 µm; Sympatec). For each measurement, approximately 100 mg of dry sample was dispersed at a feed rate of 75% by a controlled feeder (Vibri; Sympatec), with dry dispersal (Rodos; Sympatec) attachments set at a main pressure of 4.0 bar and a venturi pressure of 100 mbar. The triggering conditions were set to start measurement when channel 25 reached 1% total signal and to stop when channel 25 dropped below 0.5%. Data were analyzed according to the Fraunhoffer method by means of analytical software (Windox, ver. 3.2, release 4; Sympatec).

HDP-cCDV.
cCDV was prepared from CDV, as described previously,³ except that the compound was isolated as the dicyclohexyl-morpholinocarboxamidine salt. The scheme of the synthesis is illustrated in Figure 1 . The purity (greater than 98%) of the compounds used in this study was confirmed by analytical thin-layer chromatography, nuclear magnetic resonance spectroscopy, and mass spectroscopy, as reported previously.^{4 5}

View larger version (11K): [in this window] [in a new window]   FIGURE 1. Synthesis of hexadecyloxypropyl-cyclic cidofovir (HDP-cCDV).

Intravitreal Pharmacokinetics of Small Particle Formulation of HDP-P-GCV.
Three rabbits received 2.8 micromoles of the drug in 50 µL of 5% dextrose in their left eyes. The dose had been determined to be nontoxic in an earlier study.² Vitreous sampling was performed at postinjection weeks 1, 2, 3, 5, 8, and 12. Rabbit eyes were well dilated before anesthesia by topical application of a combination of tropicamide 1% and phenylephrine hydrochloride 2.5%. Anesthesia was performed as previously described.⁶ Under anesthesia, the rabbit eye was proptosed through a hole made on a piece of latex rubber. A cornea ring (depth, 2.5 mm), made from a 5-mL plastic syringe barrel, was placed on the cornea. Methylcellulose was applied to the cornea within the inner area of the ring, followed by a glass coverslip. Under the direct view of a surgical microscope, 50 to 100 µL of vitreous fluid was aspirated through the pars plana with a 23-gauge needle attached to a 0.5-mL syringe. Vitreous fluid was obtained from a location deliberately chosen to be away from the drug depot.² Subsequent vitreous aspirations were performed with a 28.5-gauge needle. Vitreous samples were placed in a preweighed vial for HPLC analysis of HDP-P-GCV levels, as described before.²

Intravitreal Toxicity and Pharmacokinetics of HDP-cCDV.
For toxicity studies, five doses (10 µg, or 0.018 micromole; 55 µg, or 0.1 micromole; 100 µg, or 0.18 micromole; 550 µg, or 1.01 micromoles; 1000 µg, or 1.84 micromoles in 50 µL of 5% dextrose) were tested in eight rabbits (16 eyes) for 8 weeks. One eye of each animal was injected with drug in 50 µL of 5% dextrose, and the fellow eye was injected with 50 µL of 5% dextrose alone as the control. Before drug injection, baseline IOP and fundus examination were documented. Drug or 5% dextrose in a volume of 50 µL was injected into the vitreous cavity, as previously described.² After injection, eyes were monitored with a handheld tonometer (Tonopen; Medtronic, Jacksonville, FL), slit lamp, and indirect ophthalmoscope at day 3 and weeks 1, 2, 3, 5, and 8. Any change from the baseline was documented. All animals were killed at 8 weeks after the drug injection. Before death, full-field scotopic ERGs were obtained in all animals, as previously described.² After enucleation, globes were processed for paraffin-embedded sections and light microscopic examination, as described previously.⁶

For the pharmacokinetic studies, the highest nontoxic dose from the results of the studies, 100 µg/eye, was intravitreally injected into 24 eyes of 16 rabbits, and the remaining 8 eyes were injected with the same volume (50 µL) of 5% dextrose as the control. Two animals, three eyes with drug and one eye with 5% dextrose, were used at each time point: postinjection days 1 and 3 and weeks 1, 2, 3, 5, 8, and 10. After the animals were killed, the globes were enucleated and kept on ice before they were frozen and dissected. The globes were then submerged in –40°C 2-methylbutane in a beaker sitting in a dry ice-ethanol bath for 30 seconds. The frozen globe was cut into halves through the optic nerve, and the lens and anterior chamber blocks were removed immediately. The globe halves were then placed under the surgical microscope at room temperature for 40 to 70 seconds to allow the interface between retina and vitreous to thaw before the vitreous block was removed with forceps. The retina was gently scraped off the bed of retinal pigment epithelium (RPE)/choroid layer with a fine spatula. The remaining RPE/choroid layer was forcefully scraped off underneath the sclera. The dissection was completed under direct view of a surgical microscope within 2 to 3 minutes to avoid cross-contamination. These different tissues from the same eyes were stored separately in the preweighed and prelabeled glass vials. The vials were kept at –70°C until HPLC analysis.

HSV-1 Rabbit Retinitis Treatment Study of HDP-cCDV.
For the retinitis intervention study, 72 rabbits were used, including 14 for the treatment study and 58 for the pretreatment study. Only the right eye of each rabbit was used. Ophthalmoscopic retinitis grading was performed according to a previously reported method involving a standardized grading scheme.⁷ A very experienced observer graded retinitis throughout the experiments in an unmasked manner. 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 a 10^–7.6 mean tissue culture infective dose (TCID 50)/mL HSV-1. When retinitis developed and reached grade 1 (earliest detected retinitis grades are 1 or 2), five infected eyes received 100 µg HDP-cCDV in 50 µL 5% dextrose, four received an equivalent dose of free CDV in 50 µL 5% dextrose, and the other five received 50 µL of 5% dextrose. In the pretreatment study, 100 µg/eye was tested. Fifty-eight rabbits were divided into four groups: the 21-, 47-, 68-, and 100-day groups. Fifteen rabbits were used at each time point of the pretreatment study (except eyes of 13 rabbits were used at the 100-day time point). At each time point, five rabbits received 100 µg (in 50 µL 5% dextrose) of HDP-cCDV, five received an equivalent dose of CDV in 50 µL 5% dextrose, and five received 50 µL 5% dextrose (for the 100-day pretreatment only three rabbits received HDP-cCDV). HSV-1 virus was inoculated as scheduled at 21, 47, 68, or 100 days after the intraocular drug injections. The HSV-1 virus dose, injection method, and clinical retinitis grading were performed as previously described.^{2 7} Rabbits were killed 2 weeks after development of retinitis. Retinitis was graded on days 3, 6, 9, 11, and 14. Rabbits without retinitis 3 or 4 weeks after HSV-1 inoculation were killed.

Histologic Evaluation of Retinal Damage and Choroidal Inflammation.
After death, globes were enucleated and processed for light microscopy. After they were stained with hematoxylin and eosin (H&E), sections obtained next to the vertical meridian were selected from each eye. The section that was used to measure retinal thickness for each eye included the entire anterior and posterior retina in cross section. The thickness of the retina was measured at five locations in the inferior retina and three locations in the superior retina, as illustrated by the pointers in Figure 2 . Thickness was measured with a reticule installed in the eyepiece of the microscope. All measurements and grading were performed under 100x magnification. Each scale unit of the reticule is equivalent to 27.8 µm. The thickness of each retina was expressed as the mean of the measurements at all eight locations.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Schematic showing the eight locations in which thickness was measured.

	Results

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Effect of Microfluidization of HDP-P-GCV on Particle Size
We subjected an aqueous slurry of HDP-P-GCV to five consecutive cycles of microfluidization. After lyophilization and recovery of the powder, both the unmodified and the microfluidized HDP-P-GCV formulations were subjected to laser light-scattering particle size analysis (Fig. 3) . Unmodified HDP-P-GCV (Fig. 3A) showed a bimodal distribution, with a volume median diameter (x50) centered around 8.0 µm. After microfluidization (Fig. 3B) , a more monodispersed population of smaller particles was noted, with an x50 of 4.4-µm, a 99th percentile diameter (x99) of 20 µm, and a 90th percentile diameter (x90) of 10 µm. No large particles remained after microfluidization. Finally, we also analyzed an untreated formulation of HDP-cCDV powder (Fig. 3C) . This compound showed a population of particles with an x50 of 8.9 µm.

View larger version (61K): [in this window] [in a new window]   FIGURE 3. Laser light-scattering particle size analysis of HDP-P-GCV and HDP-cCDV formulations. (A) Unmodified HDP-P-GCV; (B) microfluidized HDP-P-GCV; (C) HDP-cCDV.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Micromolar concentration of HDP-P-GCV in vitreous aspirates at different time points after intravitreal injection of 2.8 micromoles of small-particle formulation (S2.8 micromoles) and unmodified particle size formulation (2.8 micromoles). Data are presented as the mean ± SD (n = 3).

View larger version (132K): [in this window] [in a new window]   FIGURE 5. Micrograph of a section of an eye that received a 550-µg HDP-cCDV intravitreal injection, showing localized retinal toxicity (between the two arrows), a disorganized outer nuclear layer, and marked RPE proliferation.

View larger version (71K): [in this window] [in a new window]   FIGURE 6. A fundus photograph taken at week 5 after an intravitreal injection of 100 µg crystalline HDP-cCDV into rabbit vitreous, showing a drug depot floating in the vitreous with an estimated size of 1 x 1.5 disc diameters.

View larger version (18K): [in this window] [in a new window]   FIGURE 7. A trend plot of HDP-cCDV elimination, determined in a pharmacokinetic study of rabbit vitreous over time, after 100-µg intravitreal injections. The estimated vitreous half-life was approximately 6.3 days. Data is presented as the mean ± SD (n = 3).

Pretreatment Study.
During the 3-week pretreatment study, all five rabbits that received intravitreal injection of 5% dextrose showed development of typical retinitis. In contrast, none of the rabbits that received the intravitreal injection of HDP-cCDV or CDV had retinitis. At each time point, retinitis scores were analyzed across three groups by the Kruskal-Wallis test, followed by a nonparametric Tukey-type test to locate the difference(s) if the null hypothesis was rejected by the Kruskal-Wallis test (Table 1 . For the 47-day pretreatment, at day 6 after virus inoculation all control eyes showed retinitis with a median grade of 4. The rabbits that received CDV pretreatment all had retinitis with a median grade of 3. In contrast, of the five rabbits that received HDP-cCDV pretreatment, only two had grade 2 retinitis. There is a significant difference in retinitis scores during the 14-day grading period between HDP-cCDV–pretreated eyes and CDV-pretreated eyes or control eyes (Table 1) . For the 68-day HDP-cCDV pretreatment study, at day 14 after virus inoculation, only two rabbits had grade 3 or grade 4 retinitis. The remaining three rabbits had complete protection from HSV-1 infection. The other two groups (10 eyes) with dextrose or CDV pretreatment all had grade 4 retinitis (Table 1) . For the 100-day pretreatment study, one of four rabbits in the HDP-cCDV–pretreated group died before virus inoculation. Among the other three rabbits, one did not have retinitis, and the other two had grade 3 retinitis at day 6. The HDP-cCDV pretreated group had lower retinitis scores than the retinitis scores of the dextrose-pretreatment group (Table 1) .

	Discussion

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Injection of lower drug levels could eliminate the local retinal toxicity resulting from contact of drug depot with retina due to gravitational effects and positioning.^{2 8} The current studies were conducted within nonvitrectomized eyes. The drug aggregation and release profile could be quite different if injected into a vitrectomized eye. Further studies are warranted in vitrectomized eyes. Based on our findings, we hypothesize that controlled release could be achieved by using mixtures of different sizes of crystalline drug in an intravitreal administration. These mixtures could be designed to have release profiles tailored to treat different kinds of vitreoretinal diseases.

In the previous report, we first described this novel intraocular drug-delivery system using HDP-P-GCV as a prototype.² We reasoned that the hexadecyloxypropanol moiety could be coupled to many nucleoside phosphates or phosphonates to form solid hydrophobic crystals that slowly dissolve in water. The dissolved molecules enter the cells and are cleaved intracellularly by phospholipase C into hexadecyloxypropanol and the parent drug.⁹ In the present study, we used the same concept and technique to synthesize an ether lipid ester of cyclic cidofovir, HDP-cCDV. Intravitreal injection of 100 µg or lower doses of HDP-cCDV crystals provided an ideal drug depot that floated in the inferior vitreous cavity without disturbing the visual axis. The vitreous elsewhere was clear and no toxicity was found in the eyes with 100 µg or lower doses. Local toxicity with higher doses was caused by the contact of drug depot with retina or lens, which was similar to the local retinal toxicity caused by intravitreal high-dose HDP-P-GCV.² The higher dose forms a larger drug depot in the vitreous, which tends to contact intraocular tissues and cause toxicity. An IOP decrease associated with intravitreal injection of CDV was not observed in the eyes that received 100-µg or lower doses in the present study. The eyes with higher doses showed a mild decrease in IOP at the last time point (8.7 ± 2.1 vs. 11.3 ± 2.3 mm Hg, P = 0.01). However, no hypotony was found. Hypotony (IOP of 5 mm Hg or lower with associated retinal edema) is a well-known complication after local or systemic cidofovir administration.^{10 11} The absence of hypotony may be because cyclic CDV and HDP-cCDV are not picked up avidly by organic anion transporters in the ciliary body. Intravitreal pharmacokinetics showed that HDP-cCDV was still detectable at week 10 after a single intravitreal injection of 100 µg per eye. The estimated vitreous half-life for HDP-cCDV was 6.3 days, which favorably compares to 20 hours for CDV or 10 hours for cCDV.¹² The detected concentration at week 8 was 0.002 µM, which is above the IC₅₀ for CMV. In this study, we did not detect HDP-cCDV in the retina, which could be due to low sensitivity of HPLC and fast conversion of HDP-cCDV into cCDV then into CDV by cellular phosphohydrolases. Using HDP-P-GCV, we have shown that the prodrug may be metabolized by participation of vitreous cells. There was little parent drug detectable when prodrug was incubated with a heat-inactivated vitreous sample, but conversion was detected readily by native vitreous that contains cells.⁹ It has been known that CDV is phosphorylated to cidofovir diphosphate, the active form of cidofovir that has a long intracellular half-life.¹³ Indeed, the pretreatment studies indicated at least 100 days of pharmacologic effect against HSV-1 infection of the rabbit retina. In the current studies, retinitis was graded in an unmasked manner. It could lead to bias if retinitis severity were similar to that in the treatment study. Therefore, we performed further objective analysis of retinal thickness from the pathologic examination for the treatment groups to confirm the findings. In the pretreatment studies, the severity of retinitis among the three groups was so obvious that it is unlikely that unmasked grading influenced the results.

In summary, this novel intraocular drug-delivery system has promise for challenging refractory chronic vitreoretinal diseases that require prolonged drug treatment. Small crystals of HDP-P-GCV have been found to release more drug over time than large unmodified particles, and the delivery system has been extended to crystalline HDP-cCDV. The concept and technique described in this study can be applied to many compounds, including antiproliferative drugs such as phosphonomethoxyethylguanine (PMEG), arabinofuranosylguanine (Ara-G), and 5-fluoro-2'-deoxyuridine (5-FUdR), and other antiproliferative drugs that we are investigating.

	Footnotes

 
Supported by National Eye Institute Grants EY07366 (WRF) and EY11832.

Submitted for publication January 22, 2004; revised April 6 and June 7, 2004; accepted June 15, 2004.

Disclosure: L. Cheng, None; K.Y. Hostetler, None; J. Lee, None; H.J. Koh, None; J.R. Beadle, None; K. Bessho, None; M. Toyoguchi, None; K. Aldern, None; J.-M. Bovet, Cirrus Pharmaceuticals (E); W.R. Freeman, None

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: Lingyun Cheng, Department of Ophthalmology, Shiley Eye Center, University of California, San Diego, 9415 Campus Point Drive, La Jolla, CA 92093-0946; cheng{at}eyecenter.ucsd.edu.

	References

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