To further characterize the transscleral penetration process of TPT and to achieve high and long-term (24- to 48-hour) TPT concentrations in the vitreous, animal groups HL and HLv (
n = 6 and
n = 8 rabbits, respectively) receiving high-load implants (DDS containing 2.3 mg TPT;
Table 1) were included in the experiments. To evaluate the transscleral release kinetics of the drug after implanting the DDS in these two groups, two methods were used. First, to characterize the early vitreous penetration profile of the drug released from the DDS, a microdialysis-based multiple-sampling schedule was performed. Second, a terminal vitreous sample was aspirated 24 or 48 hours later. During the microdialysis sampling period, TPT concentrations were assessed by means of the principle by which drugs in the targeted tissue cross the capillary-like semipermeable microdialysis probe continuously perfused with a drug-free physiological fluid, until equilibrium was reached.
21 For these experiments, 2 hours after the surgical insertion of the DDS, in a location 3 mm away from the limbus and 120° away from the implant site, the surface of the sclera was exposed by making an incision in the conjunctiva, and the microdialysis probe (
Fig. 3) was inserted into the vitreous space through an incision made with a 25-gauge needle. A second probe was inserted into the contralateral eye in the same location. Both probes were fixed to the sclera with two vicryl 7-0 sutures (
Fig. 3). Microdialysis membranes were then perfused with PBS (pH 7.4) at a flow rate of 0.5 μL/min using an infusion pump (KDS230; KD Scientific, Holliston, MA) and were left for 30 to 40 minutes to equilibrate with the vitreous before sample collection started. Dialysates were collected every 40 minutes over a period of 160 minutes, covering the 160- to 320-minute time range after the DDS implantation, and total TPT was analyzed. At the end of the sample collection, a concentrated TPT solution (500 ng/mL) was perfused through the probe to calculate the probe recovery by the retrodialysis method.
22 The recovery value obtained for the probes was 30% ± 9% and 28% ± 6% (mean ± SD for the HL and HLv groups, respectively), which allowed calibration of the probes to calculate the actual intravitreal TPT concentrations. At the end of the microdialysis study, probes were withdrawn, a 5-0 suture was used to close the sclera, and animals were allowed to recover. Either 24 or 48 hours later, a vitreous sample (100 μL) was aspirated with a 25-gauge needle and was analyzed for total TPT and LTPT. During the experiment, plasma samples were obtained through a jugular catheter at 0.25, 0.5, 1, 2, 4, 6, and 24 (or 48) hours after the insertion of the DDS. Plasma exposure to the drug was defined as the area under the concentration-time curve (AUC), calculated as previously published.
16 At the end of the 24- or 48-hour experiment, DDS was removed. Thirty minutes later animals were euthanatized, eyes were enucleated, and tissues were processed as detailed for total TPT and LTPT content.