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Figure 5 Plots of vitreal gentamicin concentrations fitted with the pharmacokinetic model sampled over 8 hours from the time of injection. The top curve represents the gentamicin concentrations in control eye and the bottom curve the concentrations in the eye with endophthalmitis.

aureus)-induced eyes. Perfusate was collected over 30-minute periods for 3 hours and then hourly to 8 hours. Concentration-time data fitted into a one-compartment model that incorporated the diffusion of drug within the vitreous and its elimination from the vitreous (Fig. 5). The elimination rate constants were greater in infected eyes (0.107 hr_1) than in controls (0.055 hr_1), which might be due to increased permeability of the blood-retinal barrier. Aqueous humor gentamicin concentrations in control eyes were three to six times those in the infected eyes at the end of the experiment.

Waga et al. (59) developed the ocular microdialysis technique for long-term pharmacokinetic studies in rabbits (Fig. 6). A probe (CMA 20) with a

Figure 6 Diagrammatic representation of the microdialysis probe in the rabbit eye.

membrane length of 4 mm and the shaft bent at 60-90° was used. Adult pigmented rabbits were anesthetized with Hypnorm vet®, and a small opening was made in the sclera by conjunctival dissection, about one quarter of the circumference around the limbus. The beginning was at the nasal end of the superior rectus muscle, and the end was at the temporal side, before the lateral rectus. Sling sutures at the superior rectus and a U-shaped suture was made intrasclerally temporal to the rectus superior muscle. The tip of the U was pulled out and a loop was formed. At the loop the sclera was punctured with a 0.9 mm cannula, the probe was inserted, and the sutures were fixed. The tubes of the probe were led under the skin out between the ears. Ceftazidime was injected intramuscularly (1 mg/kg) (Fig. 7) or intravitreally (1 mg) (Fig. 8) in two groups: normal and the inflammation-induced eyes. The penetration of ceftazidime into the vitreous was higher (42%) in inflamed than in normal eyes (20%), suggesting an interference with the blood-retinal barrier. The vitreal half-life of ceftazidime after intravitreal administration was 8.1 hours and 11.7 hours in normal and inflamed eyes, respectively.

Microdialysis was also used to administer drugs into the vitreous chamber. Waga and Ehinger (78) investigated the ability of 125I-labeled NGF to cross a previously implanted probe. The probes were perfused for different time periods with a solution containing NGF. With an inlet NGF concentration of 8 x 10~n M, the vitreous concentrations were found to be 0.08 x 10-12, 0.87 x 10~12, and 0.86 x 10~12 M when the solution was perfused for 1, 4, and 6 hours, respectively. When 9 x 10~10 and 7.6 x 10 9 M concentrations of NGF were perfused for 4 hours, the vitreous concentrations were 012 10~n and 3.8 x 10 11 M, respectively. The same model was used to delivery ganciclovir into the rabbit vitreous (60). Ganciclovir concentration in the vitreous after microdialysis infusion of 120 ml 3.4 x 1 0~4 M solution was 10.5 x 10~7 ± 0.99 M. Microdialysis probe was also used to administer 5-fluorouracil, benzyl penicillin, daunomycin, and dex-amethasone into the vitreal space of rabbits (25). The vitreal concentrations achieved after perfusion were 5 x 10~5 M, 2 mM, 1.2 mM, and 1.2 x 10~7 M, respectively.

Stempels et al. (23) developed a removable ocular microdialysis system using scleral port for the first time for measuring the vitreous levels of biogenic amines. This model allowed long-term experiments using microdialysis. Dutch pigmented rabbits were equipped with a scleral entry port (internal diameter 0.6 mm) with a removal closing plug. The scleral port was sutured bilaterally about 2-3 mm from the limbus in the temporal superior quadrant and covered with conjunctiva. The light-adapted rabbits were intubated and maintained under halothane anesthesia with spontaneous breathing. The pupils were dilated with one drop of homatropine 1%.

Figure 7 Vitreous and blood concentration of ceftazidime after an intramuscular injection of 1 mg/kg in (a) healthy rabbit eye and (b) mildly inflamed eye.

The conjunctiva was reopened, the closing plug was removed and a microdialysis probe, with a shaft diameter of 0.6 mm and a cut-off value of 20 kDa, was inserted into the midvitreous. The position of the probe tip was confirmed by direct illumination through the pupil. The perfusion fluid used was Ringer's solution with a Ca2+ concentration of 0.75 mM. The perfusion fluid was pumped at a flow rate of 2 mL/min, and the samples were collected every 20 minutes. Using this model, the concentration dihydroxyphenyl acetic acid was found to be three times higher than in the bovine vitreous. No significant difference was observed between simultaneously taken left

Figure 8 Vitreous concentration of ceftazidime after an intravitreal injection of 1 mg ceftazidime in (a) healthy eye and (b) mildly inflamed eye.

and right eye samples nor between days 1, 7, 11, and 14 for dopamine, dihydroxyphenyl acetic acid (Fig. 9) and noradrenaline. This study proved that ocular microdialysis could be carried out over several hours and repeatedly in the same animal.

Macha and Mitra (27) have used the technique to study the ocular pharmacokinetics of cephalosporins after intravitreal administration and also investigated the presence of peptide transporters on the retina. New

Figure 9 Concentrations of dihydroxyphenyl acetic acid in the dialysates of rabbit vitreous on days 1, 7, 11, and 14.

Zealand albino male rabbits, weighing 2-2.5 kg, were kept under anesthesia throughout the experiment. A concentric microdialysis probe was implanted into the midvitreous chamber using a 22 gauge needle about 3 mm below the limbus through the pars plana. Another linear microdialysis probe was implanted across the cornea in the aqueous humor using a 25 gauge needle (Fig. 10). The probes were perfused with isotonic phosphate buffer saline (pH 7.4) at a flow rate of 2 mL/min and the samples were collected every 20 minutes over a period of 10 hours. Animals were allowed to stabilize for 2 hours prior to initiation of a study. Ocular pharmacokinetics of cephalosporins were investigated following intravi-treal administration of 500 mg of cephalexin, cephazolin, and cephalothin. Inhibition experiments were carried in vivo using two dipeptides, gly-pro and gly-sar. The dipeptides were administered by a bolus injection intravi-treally 30 minutes prior to the administration of cephalosporins, followed by continuous perfusion through the vitreous probe to maintain the

Figure 10 Diagrammatic representation of the microdialysis probes implanted in the anterior chamber and vitreous of the eye.

steady-state dipeptide concentration during an experiment. Vitreal elimination half-lives of cephalexin, cefazolin, and cephalothin after intravitreal administration were found to be 185.38 ±27.25 min, 111.40 ± 17.17 min, and 146.68 ± 47.52 min, respectively. Cephalexin (224.39 ± 84.56 mg.min/ mL) was found to generate higher concentrations in the aqueous humor compared to cefazolin (85.37 ±45.11 mg.min/mL). The pharmacokinetic parameters of cephalexin in the presence of gly-pro, i.e., AUC (44452.06 ±3326.55 mg.min/mL), clearance (0.0013 ±0.0004 mL/min), and terminal elimination half-life (825.12 ±499.95 min), were found to be significantly different from that of the control (14612.83 ± 4036.47 mg.min/mL, 0.0036 ±0.0011 mL/min, and 187.96 ±65.12 min, respectively) (Fig. 11). In the case of cefazolin, the control parameters (30199.06 ±8819.07 mg.min/mL, 0.0018 ±0.0006 mL/min, and 229.53 ±3 7.31 min, respectively) were found to be similar, except the terminal elimination half-life, to those in the presence of gly-pro (26648.31 ±4156.56 mg.min/mL, 0.0019 ±0.0003 mL/min, and 881.03 ±469.67 min, respectively) (Fig. 12). Gly-sar was found to have no significant effect on the pharmacokinetics of both drugs. These studies indicated the involvement

Figure 11 Vitreous concentration-time profile of cephalexin (50 mg) in the presence of inhibitors after intravitreal administration. The line drawn represents the nonlinear least-squares regression fit of the model to the concentration-time data.

of a peptide carrier in the transport of cephalosporins across the retina. Although gly-pro inhibited the elimination of cephalexin from the vitreous, the effect of the a-amino group on the specificity of cephalosporins towards peptide carriers was not clearly established.

Furthermore, Macha and Mitra have utilized the microdialysis technique to delineate the ocular pharmacokinetics of ganciclovir (GCV) and its ester prodrugs (acetate, propionate, butyrate, and valerate). The prodrugs generated sustained therapeutic concentrations of GCV over a prolonged period of time after intravitreal administration. Drugs were administered (0.2 mmol) intravitreally and the samples were collected every 20 minutes over a period of 10 hours. The representative anterior and vitreous chamber concentration-time profiles of GCV following intravitreal administration are shown in Figure 13. The vitreal terminal phase elimination half-life (t1/2 P) of GCV was found to be 426 ± 109 minutes. The proportion of GCV eliminating through the anterior chamber pathway was about 1%. The representative vitreous concentration-time profile

Figure 12 Vitreous concentration-time profile of cefazolin (50 mg) in the presence of inhibitors after intravitreal administration. The line drawn represents the nonlinear least-squares regression fit of the model to the concentration-time data.

of the GCV butyrate is depicted in Figure 14. The hydrolysis rate and clearance of the prodrugs increased with the ascending ester chain length. Vitreal elimination half-lives (ti/2k10) of GCV, monoacetate, monopropio-nate, monobutyrate, and valerate esters of GCV were 170 ± 37, 117 ± 50, 122 ±13, 55 ±26, and 107 ±14 minutes, respectively. A parabolic relationship was observed between the vitreal elimination rate constant (k10) and the ester chain length. The Cmax for the regenerated GCV after the prodrug administration was found to be 2.75 ±0.431, 6.66 ±0.570, 8.03 ± 1.19, and 8.26 ± 1.76 mg for acetate, propionate, butyrate, and valerate esters, respectively. The mean residence time of the regenerated GCV after prodrug administration was found to be three to four times the value obtained after GCV injection. The low proportions of aqueous levels of GCV indicate the retinal pathway as the major route of elimination. These studies have shown that the ester prodrugs generated therapeutic concentrations of GCV in vivo and the MRT of GCV could be enhanced three-to-fourfold through prodrug modification.

Figure 13 Concentration-time profile of GCV (50.0 mg) following intravitreal administration: (*) vitreal and (~) anterior chamber concentrations. The line drawn represents the nonlinear least-squares regression fit of the model to the concentration-time data.
Figure 14 Concentration-time profile of GCV monobutyrate (63.3 mg) following intravitreal administration: (*) 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.
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