Introduction

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Human cytomegalovirus (HCMV¹) retinitis is the most common opportunistic infection occurring in about 15 to 42% of the patients with acquired immunodeficiency syndrome (AIDS), which often leads to blindness if untreated (Holland et al., 1983; Freeman et al., 1984; Gross et al., 1990). Ganciclovir (GCV), a 2'-deoxyguanosine analog, was the first Food and Drug Administration approved drug available in the United States with significant activity against HCMV. It was shown to be 26 times more potent than acyclovir against HCMV in vitro (Morse et al., 1993). Previous studies reported excellent in vitro activity of GCV against human herpes virus type 6, human herpes simplex viruses types 1 and 2, varicella-zoster virus, and Epstein-Barr virus (Smee et al., 1983; Andrei et al., 1991; Shigeta et al., 1991; Snoeck et al., 1991; Konno et al., 1993). It was approved for the induction and maintenance therapy of HCMV retinitis in AIDS patients in 1989 and has been widely used. GCV also gained significant importance in the treatment of HCMV infection in solid organ recipients (Dunn et al., 1991; Markham and Faulds, 1994; Tsinontides and Bechtel, 1996; Murray, 1997)

Intravenous administration of GCV alone or in combination with other anti-HCMV agents like foscarnet, cidofovir, zidovudine etc., is currently the treatment of choice for HCMV retinitis (Markham and Faulds, 1994; Manion et al., 1996). As it is only virostatic, indefinite maintenance therapy is required in addition to the induction therapy to prevent any relapse (Drew, 1992). The most common adverse effect in patients receiving intravenous GCV is the hematological toxicity, mainly neutropenia (up to 50%) (Faulds and Heel, 1990; Goodrich et al., 1993; Winston et al., 1993). Other toxic effects include granulocytopenia, thrombocytopenia, azoospermia, and rise in the serum creatinine (Faulds and Heel, 1990). Relatively high systemic toxicity caused by intravenous therapy has lead to the intravitreal administration of GCV (Henry et al., 1987; Daikos et al., 1988; Ussery et al., 1988; Cantrill et al., 1989; Cochereau-Massin et al., 1991), which enabled considerable improvement and/or stabilization of HCMV retinitis in most (80-100%) of the patients. Intravitreal administration has the advantage of maintaining high concentrations of the drug for effective control of ocular infections with minimal systemic adverse effects. Clinical trials reported good response rates to intravitreal induction therapy with GCV (Cochereau-Massin et al., 1991). However, very few studies have been carried out to date delineating the intravitreal pharmacokinetics of GCV (Henry et al., 1987; Ashton et al., 1992; Morlet et al., 1996). The major constraint to the development and assessment of posterior segment pharmacokinetics of drugs in animal models is the inaccessibility of the ocular fluids for continuous serial sampling. Microdialysis has gained wide recognition as a standard technique for sampling various tissues and fluids such as brain, liver, kidney, skin, tumor, bile, and blood (Diaz et al., 1992; Robinson, 1995; Maggs et al., 1997), as well as anterior and vitreous chambers of the eye (Stemples et al., 1993; Hughes et al., 1996; Sato et al., 1996; Waga and Ehinger, 1997; Rittenhouse et al., 1999; Macha and Mitra, 2001a,b). We have developed an animal model in our laboratory for simultaneous and continuous sampling of the vitreous and aqueous humors in rabbits using microdialysis technique (Macha and Mitra, 2001a). The proposed animal model would delineate complete ocular pharmacokinetics of drugs after intravitreal administration.

The present study was carried out to determine the intravitreal pharmacokinetics of GCV. Another objective was to achieve sustained concentrations of GCV, using its acyl monoester prodrugs, for a prolonged period of time. In this study, we have also demonstrated that the requirement for repeated intravitreal injections of GCV could be avoided by the prodrug approach. The detailed investigation of the pharmacokinetics of GCV and its monoester prodrugs may provide valuable information for the future development of more effective GCV therapy.

	Experimental Procedures

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Materials. GCV was a generous gift from F. Hoffman La Roche (Nutley, NJ). The monoester prodrugs of GCV [monoacetate (GCVMA), monopropionate (GCVMP), monobutyrate (GCVMB), monovalerate (GCVMV)] were synthesized according to a method previously published from our laboratory (Gao and Mitra, 2000).

Methods.

Animal model New Zealand albino rabbits weighing 2 to 2.5 kg were used for the experiments. The animals were kept under anesthesia throughout the experiment using ketamine HCl (35 mg/kg) and xylazine (3.5 mg/kg) given intramuscularly every hour. Prior to the implantation of the microdialysis probes, pupils were dilated with two drops of 1% tropicamide. For the implantation of the concentric probe in the vitreous chamber, a 22-guage needle was inserted carefully into the eye about 3 mm below the corneal scleral limbus through the pars plana. The needle was then removed, and the probe was placed immediately and adjusted such that the membrane resides in the midvitreous as ascertained by microscopic examination. The linear probe was implanted in the anterior chamber using a 25-guage needle. The needle was inserted across the cornea just above the corneal scleral limbus so that it traverses through the center of the anterior chamber to the opposite end of the cornea as evidenced by microscopic examination. The sample-collecting end of the linear probe was inserted carefully into the bevel edge end of the needle. The needle was slowly retracted leaving the probe with the dialyzing membrane in the middle of the anterior chamber. The outlets of both the probes were fixed to prevent any disturbances during sample collection. A diagrammatic representation of the implanted microdialysis probes within the eye is presented in Fig. 1. The probes were perfused with isotonic phosphate buffer saline (pH 7.4) at a flow rate of 2 µl/min using a CMA/100 microinjection pump.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Diagrammatic representation of the microdialysis probes implanted in the anterior chamber and vitreous chambers of the eye.

In vitro probe calibration. Microdialysis probe recovery was determined in an aqueous solution containing a known concentration of the compound maintained at biological temperature. The probe was continuously perfused at a constant flow rate of 2 µl/min, and samples were collected every 20 min. The recovery of a compound of interest is calculated according to eq. 1.

(1)

C_out is the concentration in the outflow solution and C_i the concentration in the medium. The dialysate concentrations were transformed into the actual anterior and vitreous concentrations using eq. 2.

(2)

_i is the substance concentration in the vitreous and aqueous humors, and _out is the concentration of the compound in dialysate.

HPLC analysis. HPLC system (Waters 600 pump; Waters Corp, Milford, MA), equipped with a fluorescence detector (HP 1100) and a reversed phase C₁₂ column (4 µ, 250 × 4.6 mm, Synergy-max; Phenomenex, Torrance, CA), was used for the quantification of the samples. The GCV samples were analyzed using an isocratic method with an eluent containing 15 mM potassium phosphate buffer (pH 2.5) and 0.2% acetonitrile at a flow rate of 1 ml/min. The separation of GCV and its monoesters was achieved using a gradient method with 15 mM potassium phosphate buffer (pH 2.5): acetonitrile in proportions of 99.8:0.2 as phase A and 1:1 as phase B at a flow rate of 1 ml/min. All the samples were analyzed at an excitation wavelength of 265 nm and emission wavelength of 380 nm. The limit of quantification was 20 ng/ml for GCV and 50 to100 ng/ml for the prodrugs.

Data analysis. The experiments were carried out at least in triplicates, and the results are given as mean ± S.D. The statistical significance between the parameters was determined using analysis of variance at p < 0.05 unless otherwise specified.

	Results

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The representative anterior and vitreous chamber concentration-time profiles of GCV following intravitreal administration is shown in Fig. 2. Vitreous concentration of GCV appeared to decline biexponentially. The vitreous concentration-time profiles of GCV were modeled using a two-compartment open system (eq. 3) with the major component of elimination from the central compartment.

(3)

where,  +  = k₁₀ + k₁₂ + k₂₁,  = k₁₀k₂₁, X₀ is the dose administered and V₁ is the volume of the central compartment. The constant k₁₀ is the apparent first-order elimination rate constant from the central compartment, and k₁₂ and k₂₁ are the intercompartmental transfer rate constants.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Concentration-time profile of GCV (50.0 µg) following intravitreal administration. () represents vitreal and () anterior chamber concentrations. The line drawn represents the nonlinear least-squares regression fit of the model to the concentration-time data.

The anterior chamber concentration-time profiles of GCV after intravitreal administration were analyzed using a noncompartmental model, in which the initial anterior chamber concentration was considered to be zero.

The vitreous concentration-time profiles of the monoesters are depicted in Figs. 3 to 6. The anterior chamber concentrations of the monoesters after intravitreal administration were below the quantitation limits. The vitreous concentration of acetate and butyrate esters of GCV was observed to decline biexponentially, and their disposition was modeled according to a two-compartment system. The propionate and valerate esters showed a monoexponential decline and were modeled as one-compartment system. The distribution phase of the metabolite (GCV) was obscured by the kinetics of its formation in vivo; therefore, a single compartment was used to model the vitreous concentration-time profiles of the metabolite.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Concentration-time profile of GCV monoacetate (57.8 µg) following intravitreal administration. () represents vitreal GCV monoacetate and () vitreal GCV concentrations. The line drawn represents the nonlinear least-squares regression fit of the model to the concentration-time data.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Concentration-time profile of GCV monopropionate (60.6 µg) following intravitreal administration. () represents vitreal GCV monopropionate and () vitreal GCV concentrations. The line drawn represents the nonlinear least-squares regression fit of the model to the concentration-time data.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Concentration-time profile of GCV monobutyrate (63.3 µg) following intravitreal administration. () represents vitreal GCV monobutyrate and () vitreal GCV concentrations. The line drawn represents the nonlinear least-squares regression fit of the model to the concentration-time data.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Concentration-time profile of GCV monovalerate (66.1 µg) following intravitreal administration. () represents vitreal GCV monovalerate and () vitreal GCV concentrations. The line drawn represents the nonlinear least-squares regression fit of the model to the concentration-time data.

The three-compartment system used to describe the disposition of acetate and butyrate esters of GCV is depicted in Fig. 7. Two of the compartments are for the monoester, which was assumed to be eliminated from the central compartment (compartment 1). In this model, the monoesters distribute into the peripheral compartment (compartment 2) and are also metabolized in the central compartment to GCV, which was assumed to distribute instantaneously (compartment 3). The metabolite formed also undergoes elimination from the central compartment. The vitreous concentration-time profiles of acetate and butyrate esters were described based on the eq. 4 and the regenerated GCV using eq. 5.

(4)

(5)

where,  +  = k₁₀ + k₁₂ + k₂₁ + k₁₃,  = k₁₀k₂₁ + k₁₃k₂₁,  = k₃₀, and X₀ is the intravitreal dose. The constant k₁₀ is the apparent first-order elimination rate constant of the drug from the central compartment, k₁₂ and k₂₁ are the intercompartmental transfer rate constants, and k₁₃ and k₃₀ are the apparent first-order formation and elimination rate constants, respectively, of the metabolite.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Pharmacokinetic model for GCV monoesters and their metabolite.

The disposition of the propionate and valerate esters of GCV was described by a two-compartment system. The monoesters were assumed to distribute instantaneously (compartment 1) and simultaneously metabolize to GCV, which is represented by the compartment 3. The vitreous concentration-time profiles of propionate and valerate esters were described based on eq. 6 and the regenerated GCV using eq. 7.

(6)

where,  = k₁₀ + k₁₃.

(7)

where,  +  =k₁₀ + k₃₀ + k₁₃, and  = k₁₀k₃₀ + k₁₃k₃₀. The constants k₁₀, k₁₃, and k₃₀ are as defined previously.

The concentration-time data of all the drugs was fitted into their respective models and the final pharmacokinetic parameters were determined using nonlinear, least-squares program Kinetica 2000 (version 3.1). The pharmacokinetic model parameters for GCV and its monoesters are shown in Table 1.

The distribution (t_1/2) and the terminal elimination (t_1/2) half-lives of GCV were 43 ± 19 and 426 ± 109 min, respectively. The area under the anterior and vitreous chamber concentration-time profiles of GCV following intravitreal administration were found to be 225 ± 91 and 20822 ± 4187 µg · min/ml, which indicates that only 1% of the administered dose was eliminated through the aqueous humor pathway. The V_C and V_SS of the central compartment were found to be 0.59 ± 0.11 and 1.3 ± 0.23 ml, respectively.

Following the intravitreal administration of the monoester prodrugs of GCV, the distribution rate constant (k₁₂) of GCV monobutyrate was significantly higher compared with that of GCV. The high k₁₂ of monobutyrate and monoexponential decline of the vitreous concentrations of propionate and valerate esters might be due to the more rapid distribution of these prodrugs into the tissues compared with GCV. The rate of formation of the metabolite increased with the ester chain length of the prodrug. The vitreal elimination half-life of the propionate ester was found to be similar to that of GCV, where as the acetate, butyrate, and valerate esters exhibited shorter half-lives. The prodrugs showed higher clearance rates and lower AUC compared with GCV, which can be attributed to their simultaneous elimination and metabolism in the vitreous chamber. Comparatively, the propionate ester demonstrated lower clearance rate, similar elimination half-life (122 ± 13 min), and AUC (18050 ± 2231 µg · min/ml) as that of GCV (170 ± 37 min and 20822 ± 4187 µg · min/ml, respectively).

Following prodrug administration, the C_max of the regenerated GCV increased with the ester chain length, in accordance with the metabolism rate. The acetate and propionate esters were found to generate sustained concentrations of GCV with a C_max of 2.75 ± 0.431 and 6.66 ± 0.57 µg and C_last of 2.22 ± 0.685 and 5.07 ± 1.03 µg, respectively. In case of the other prodrugs, the C_last for GCV levels were almost half the C_max. The mean residence time (MRT) of the metabolite decreased in accordance with the increased in vivo hydrolysis and elimination rate constants of the prodrugs.

	Discussion

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Cytomegalovirus retinitis is the most common opportunistic infection in AIDS patients. Intravenous administration of antiviral agents has been the conventional mode of administration in AIDS patients. Kuppermann et al., (1993) reported a mean intravitreal GCV concentration of 0.93 ± 0.39 µg/ml from a daily dose of 6.1 ± 2.0 mg/kg i.v. for a period of 3 months, which falls within the borderline or subtherapeutic levels. Moreover, the toxic side effects of systemically administered antiviral agents limit their use and necessitate direct intravitreal injection to preserve visual function. However, the short intravitreal half-life of GCV makes weekly injections necessary to maintain the therapeutic concentrations over the treatment period. Various strategies have been explored to achieve prolonged therapeutic concentrations of GCV, such as chemical modification, intravitreal implants etc., to minimize the frequent administration of the antiviral agent. Intravitreal GCV implants require complicated surgery for the implantation and replacement, which might result in retinal detachment. Morlet et al. (1996) have shown that multiple high dose injections of GCV in albino rabbit eyes resulted in no electroretinogram or histopathological evidence of toxicity. Based on our previous studies, the chemical modification approach was considered to have some merit. Synthesis of short-chain ester prodrugs of GCV has been reported from our laboratory (Gao and Mitra, 2000). The ester prodrugs possess adequate lipophilicity to cross the blood-retinal barrier, which prevents the entry of relatively hydrophilic molecules like GCV. The solution stability and aqueous solubility enables their formulation in an appropriate dosage form for direct intravitreal injections. The ocular homogenate studies showed that the hydrolysis rate of the prodrugs increased with the ester chain length (Dias et al., 2002). The prodrugs generated GCV primarily due to the esterase activity, acetylcholine and butyrylcholine esterases, in all the ocular tissues (i.e., retina-choroid, iris-ciliary, lens, vitreous and aqueous humors) (Lee et al., 1985).

Previously, pharmacokinetic studies have been carried out in human patients by collecting a sample of the vitreous and/or aqueous humors prior to intraocular surgery. In case of animal studies, the subjects were sacrificed for collecting the vitreous and/or aqueous humors at each time point. These techniques yield inadequate data and, moreover, introduce a considerable amount of intersubject variability. A dual probe microdialysis technique has been developed in our laboratory to study the ocular pharmacokinetics in rabbits following intravitreal or systemic administration. The animal model was validated by measuring intraocular pressure, protein concentrations in the aqueous and vitreous humors, and fluorescein kinetics after systemic and intravitreal administration. These studies have proved the integrity of the blood ocular barriers during the term of an experiment.

The terminal elimination (t_1/2) half-life of GCV after intravitreal administration was found to be 426 ± 109 min, which is consistent with a previous report (Lopez-Cortes et al., 2001). Lopez-Cortes et al. (2001) have reported terminal half-lives of 7.14 and 8.66 h after the intravitreal administration of 196 and 800 µg of GCV, respectively. The biexponential decline of GCV indicates its distribution/penetration into the intraocular tissues such as retina, lens, iris-ciliary body etc. The proportion of GCV eliminated through the anterior chamber pathway was found to be only 1%, thus indicating that the retinal pathway is the major route of elimination for this drug. The vitreal elimination half-life of GCV obtained in our studies support intravitreal administration of ganciclovir twice a week to maintain vitreous levels above minimum inhibitory concentration of 0.25 to1.22 µg/ml for the treatment of cytomegalovirus retinitis. The monoester prodrugs of GCV would provide therapeutic concentrations over prolonged periods of time. The in vivo rate of hydrolysis of the prodrugs increased with the ester chain length, which is in accordance with the in vitro data. The elimination rate constant (k₁₀) of the ester prodrugs showed a parabolic relationship with lipophilicity (Fig. 8). It increased from acetate to butyrate followed by a fall in case of the valerate ester. The AUC of the valerate ester was found to be lower than that of the butyrate ester, although the elimination rate constant (k₁₀) was higher, possibly due to its high in vivo metabolism rate (k₁₃) compared with the other prodrugs.

View larger version (8K): [in this window] [in a new window]   Fig. 8.   Relationship between the ester chain length and elimination rate constant (k10).

GCV was detected in the vitreous within 10 to 30 min after the administration of the prodrugs. The T_max for GCV regeneration following prodrug administration was observed to range from 150 to 210 min, and the therapeutic concentrations of GCV were achieved within 1 h. C_max values for the regenerated GCV increased with the ester chain length possibly due to an ascending metabolism rate from acetate to valerate esters. The acetate and propionate ester prodrugs degraded at an optimum rate with matching GCV generation and elimination rates thus providing sustained concentrations of GCV over the experimental time period. In case of butyrate and valerate esters, the C_max values were found to be twice the C_last values, probably due to the rapid metabolism in vivo and elimination of the prodrug as such. MRT of the regenerated GCV from acetate and propionate esters was found to be three to four times as compared with the direct administration of GCV.

The percentage of the acetate, propionate, butyrate, and valerate ester prodrugs metabolized to produce GCV in vivo were 26, 27, 35, and 31%, respectively. The percentage metabolized increased with the ester chain length and the hydrolysis rate of the prodrugs. These results indicate that the prodrugs are also eliminated as such from the vitreous, and their elimination rate accelerates with lipophilicity. GCV formed in vivo has lower rate of elimination compared with the prodrugs, thus improving the residence time of GCV in the vitreous chamber. In addition, it has been assumed that equilibrium is established between the vitreous humor and retina, and the drug levels become similar in both the tissues (Henry et al., 1987; Morlet et al., 1996). The in vitro tissue hydrolysis studies carried out in our laboratory showed that the prodrugs are hydrolyzed in the retina at a much faster rate compared with that in the vitreous humor. A major proportion of the prodrugs penetrating as such into the retina might hydrolyze to generate more GCV in the tissue, thus providing higher retinal concentrations of GCV than detected in the vitreous humor.

In conclusion, the microdialysis technique can be effectively used for determining the drug and metabolite concentrations simultaneously. Chemical modification of GCV was found to provide effective concentrations of GCV over a prolonged period of time. Based on the MRT values, the frequency of administration could be reduced at least by three to four times using acetate and propionate esters of GCV, with proper dosage adjustments. This approach appears to be useful in development of new formulations for direct intraocular administration, as the prodrugs studied have demonstrated optimal physicochemical properties. Ocular drug delivery through prodrugs appears to be a better approach compared with the sustained release implants, which require complicated surgery for the implantation and removal of the device from the vitreous chamber.