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Direct intravitreal injection of drug is a common method for treating diseases of the retina or vitreous. The stagnant nature of the vitreous humor and surrounding tissue barriers creates concentration gradients within the vitreous that must be accounted for when developing drug therapy. The objective of this research was to study drug distribution in the vitreous humor of the rabbit eye after an intravitreal injection, using a finite element model. Fluorescein and fluorescein glucuronide were selected as model compounds due to available experimental data. All required model parameters were known except for the permeability of these compounds through the retina, which was determined by fitting model predictions to experimental data. The location of the intravitreal injection in the experimental studies was not precisely known; therefore, several injection locations were considered, and best-fit retinal permeability was determined for each case. Retinal permeability of fluorescein and fluorescein glucuronide estimated by the model ranged from 1.94 x 10(-5) to 3.5 x 10(-5) cm s(-1) and from 0 to 7.62 x 10(-7) cm s(-1), respectively, depending on the assumed site of the injection. These permeability values were compared with values previously calculated from other models, and the limitations of the models are discussed. Intravitreal injection position was found to be an important variable that must be controlled in both experimental and clinical settings.

The evaluation of the blood-ocular barrier for fluorescein requires the measurement of free and unconjugated fluorescein in plasma. This study introduces a new and simple method for the determination of free fluorescein in plasma on the basis of determined total free plasma fluorescence and the free fraction of fluorescence. An excellent good correlation between differential spectrofluorophotometry and this new method is demonstrated. After intravenous administration of sodium fluorescein, the contribution of fluorescein glucuronide to total free plasma fluorescence was evaluated on basis of the areas under the plasma concentration/time curves for fluorescein and fluorescein glucuronide, respectively. After 1 h 8.2% of total free fluorescence in plasma was found to originate from fluorescein glucuronide and after 24 h 18.3% originated from this metabolite. It was concluded that although plasma fluorescein glucuronide measurements are important in the exact evaluation of the blood-ocular barrier, the contribution of fluorescein glucuronide to vitreous fluorescence after intravenous fluorescein administration seems to be of minor magnitude.

To characterize quantitatively the active transport mechanism of the blood-retinal barrier (BRB), we estimated the inward (Pin) and outward permeability (Pout) of the BRB in monkey eyes using vitreous fluorophotometry. Pin values for fluorescein (F) and fluorescein glucuronide (FG) were simulated by computer in separate experiments following measurements of intraocular fluorescence at 1 h following the intravenous injection of F or FG. The estimated mean Pin values for F and FG were 4.7 +/- 1.6 and 5.9 +/- 1.7 x 10(-6) cm/min, respectively, in ten eyes of five monkeys. At 1 month thereafter, F was injected into the right vitreous cavity and the same amount of FG was injected into the left vitreous cavity. The intraocular kinetics of fluorescence were measured at 6-24 h postinjection. As estimated by the simulation model, the mean Pout values were 7.7 +/- 2.6 and 1.7 +/- 0.9 x 10(-4) cm/min for F and FG, respectively. The Pout/Pin ratio was 160 +/- 78 for F and 26 +/- 9 for FG. The intraperitoneal injection of probenecid resulted in a significant decrease in the Pout value for F (P less than 0.005) but had no significant effect on that for FG, suggesting that F is excreted from the retina via an active transport mechanism; as characterized by the Pout/Pin ratio, the actual magnitude of the latter is far greater than that previously reported.

The permeability of the corneal endothelium and its aqueous-cornea distribution ratio were reevaluated in the rabbit eye. Both parameters were determined in an individual eye by applying the dye first by iontophoresis and then by intravitreal injection, which allows the influence of fluorescein glucuronide on the fluorophotometric measurements to be excluded. The corneal endothelial permeability coefficient was 5.13 +/- 1.64 X 10(-4) cm min-1, and the aqueous-cornea distribution ratio was 0.25 +/- 0.06 (mean +/- S.D., n = 11) on the average, and the former was considerably greater than the previous results, while the latter was considerably smaller.

Comparative studies of fluorescein and fluorescein glucuronide were carried out. Binding to human serum protein was studied using an Amicon MPS-3 ultrafiltration unit; it averaged 63% for fluorescein glucuronide and 85% for fluorescein. Intracameral penetration of both compounds was studied in the human eye, and the concentration changes of both compounds in the plasma ultrafiltrate and in the anterior chamber were analyzed, based on Davson's equation. The coefficient of entry into the anterior chamber (ki) was 0.018 +/- 0.007 h-1 (mean +/- SD, n = 10) for fluorescein glucuronide and 0.054 +/- 0.033 h-1 for fluorescein, and the former was significantly lower than the latter (P less than 0.005). The rate of loss from the vitreous (kv) was studied by injecting each compound into the vitreous of the pigmented rabbit and following the fluorescein intensity changes in it. It was 0.042 +/- 0.008 h-1 (mean +/- SD, n = 8) for fluorescein glucuronide and 0.17 +/- 0.01 h-1 for fluorescein, and the former was significantly smaller than the latter (P less than 0.001). Intraperitoneal injection of probenecid significantly decreased the kv of fluorescein but had little effection that of fluorescein glucuronide. It was suggested that fluorescein glucuronide is lost from the vitreous mainly by a passive mechanism.

After intravenous injection of fluorescein the time-course of the plasma concentrations of fluorescein (F) and fluorescein glucuronide (FG) was studied in 18 insulin-dependent diabetics with various degrees of nephropathy, and in two non-diabetic subjects. Fifteen minutes after injection the molar concentrations of free (non-protein bound) F and FG were almost identical. After one hour the concentration curve integrals of the two substances were of the same magnitude. There was, however, considerable interindividual variation. In diabetic patients with renal insufficiency an increase was found in both integrals, the F integral being less increased (27%) than the FG integral (44%). It appears from the variation in absolute and relative concentrations of F and FG that a separate determination of the two fluorophores in plasma is desirable, when F is used by intravenous administration as an indicator of blood-ocular barrier function. Previous observations of an increase with time after injection of the free fraction of plasma fluorescence are explained by the finding of a higher free fraction of FG (34%) than of F (11%) and a change in relative concentrations of the two fluorophores.

Fluorescein (F) and fluorescein glucuronide (FG) were determined in the vitreous of four diabetic patients by a double-filter slit-lamp fluorophotometric technique. Determinations were performed 60-80 min after i.v. injection of fluorescein. F and FG were also determined in plasma ultrafiltrate 5, 15, 30, 60 and 120 min after injection by high-pressure liquid chromatography. The concentration of FG in the vitreous was 3 times that of F. After correction for plasma concentrations of FG higher than those of F, the penetration index of FG through the blood-retinal barrier was found to be twice the penetration index of F. This is not what would be expected if passive transport alone were involved. Accordingly, it is suggested that active transport mechanisms contribute to the movement of F and FG across the blood-retinal barrier.