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' \ Modified Approach Iris

Ciliary Body

Figure 8 Schematic of volume dilution method (A) and modified volume dilution method: microdialysis probe placement (B).

Figure 8 Schematic of volume dilution method (A) and modified volume dilution method: microdialysis probe placement (B).

Following anterior chamber placement of microdialysis probes into each eye of six rabbits and a >14-day recovery period, each rabbit received a tracer i.v. bolus of 14C-ascorbate (53). Basal ascorbate blood to aqueous transport, Ro, and ascorbate ocular clearance, CLAH were estimated. After a 1-hour washout, each rabbit received a series of three doses of 3H-propranolol (750-3000 mg, 16.5 mCi/mg) every 60 minutes into the lower cul-de-sac of each eye. Microdialysis probe effluent was analyzed for ascorbate with a spectrophotometric assay (53,54), and the time course of aqueous humor ascorbate was determined (Fig. 9) (54). Nonlinear least-squares regression analysis of the ascorbate time-course in aqueous humor was performed in order to estimate the altered CLAH following propranolol perturbation of aqueous humor production. The relative difference in the basal CLAH and the CLAH determined subsequent to propranolol administration was calculated. The major assumption for this approach was that this difference in CLAH could be attributed solely to inhibition of aqueous humor production. A ~ 47% reduction in flow was observed

Figure 9 Ascorbate aqueous humor concentrations following three doses of topically administered propranolol: (A) AH ascorbate as % baseline (bars indicate ± SE; n = 3) versus time profile for the 1500 mg dose (♦); (B) AH ascorbate as % baseline (bars indicate ± SE; n = 3 versus time profiles for the 750 mg (♦) and for the 3000 mg (□) doses. Arrows represent time for administration of 14C-ascorbate i.v. dose and the hourly propranolol topical doses. (From Ref. 57.)

Figure 9 Ascorbate aqueous humor concentrations following three doses of topically administered propranolol: (A) AH ascorbate as % baseline (bars indicate ± SE; n = 3) versus time profile for the 1500 mg dose (♦); (B) AH ascorbate as % baseline (bars indicate ± SE; n = 3 versus time profiles for the 750 mg (♦) and for the 3000 mg (□) doses. Arrows represent time for administration of 14C-ascorbate i.v. dose and the hourly propranolol topical doses. (From Ref. 57.)

following topical administration of propranolol (1500 mg). No modulation in the ascorbate aqueous humor time-course was observed for the 750 and 3000 mg groups. The model fit to three individual ascorbate aqueous concentration-time data is presented in Figure 10. Examination of iris-ciliary body tissue concentrations of ascorbate and propranolol provided evidence that a spectrum of intraocular responses to propranolol was observed in this experiment. It appeared that the 750 mg dose provided aqueous humor

Figure 10 Nonlinear least-squares regression fit of aqueous humor ascorbate concentration versus time profiles following three 1500 mg doses of topically administered propranolol in three individual rabbits. (From Ref. 54.)

propranolol concentrations below those that could inhibit aqueous humor production. Reduction in aqueous humor production was observed for the 1500 mg group, while the simultaneous inhibition of ascorbate ciliary tissue accumulation and aqueous humor formation resulting in no change in the ascorbate aqueous humor time course was observed for the 3000 mg group (54). Anterior segment microdialysis provided an excellent approach to examine complex ocular physiology and pharmacology.

VI. FUTURE CHALLENGES, MILESTONES, AND OPPORTUNITIES

The anterior segment microdialysis technique has provided the framework for novel approaches to the examination of the mechanisms involved in ophthalmic drug ocular pharmacokinetics and the pharmacodynamics of aqueous humor formation and modulation. Anesthetized and conscious animal models, in some cases involving the long-term placement of micro-dialysis probes into the anterior segment (i.e., anterior and posterior chambers), were established, which have provided a substantial advance in experimental approaches to ocular pharmacokinetic/pharmacodynamic experimentation. Reduction in the number of animals required for these studies (n = 48 reduced to n = 3 — 6) provides a clear market advantage via reduction in costs and in animal-sparing research approaches. Animal experimentation with probe placement in the anterior segment, pioneered by Sato et al. (49), Fukada et al. (48), and Ohtori et al. (50), up to papers describing conscious animal experimental with microdialysis probes in the anterior chamber for 5 to > 30 days (17,53,54), demonstrate the rapid development and increased utility of the microdialysis approach in the study of ocular drug delivery and disposition. Unique applications of the microdialysis approach in the examination of drug ocular disposition such as a dual probe implantation technique involving simultaneous examination of aqueous and vitreous drug disposition have been performed (51,52). The importance and broad-based applicability of the microdialysis sampling approach for the examination of ocular pharmacokinetics and dynamics of ophthalmics is of large impact. Reuse of animals is possible because of the long-term tolerability of the animals to probe implantation. A series of substrates could be examined using the same subject to assess phenomenon such as tolerance development. Possible reduction in inter-subject responses to ophthalmic drugs is possible via this approach. This approach also provided the framework for a detailed examination of in vivo basal blood to aqueous transport of ascorbate, a labile substrate of utmost importance to intraocular health and homeostasis. Moreover, the pharmacodynamics of beta-adrenergic antagonist-associated modulation of both aqueous humor formation and ascorbate ciliary accumulation was examined in detail using anterior segment microdialysis. Pharmacokinetic modeling was facilitated with this technique; by using alterations in the ascorbate aqueous humor time-course as a means of estimating aqueous humor flow, the modulatory effects of a model substrate, propranolol, on aqueous humor turnover, was examined.

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