Recently, drug disposition in the anterior segment has been explored using microdialysis. Fukuda et al. (48-50) were the first to examine the utility of microdialysis sampling of anterior chamber aqueous humor. In their studies, linear probes inserted into the temporal cornea through the anterior chamber and exteriorized out of the nasal cornea were used to examine intraocular disposition of fluoroquinolones following oral or topical administration of ofloxacin, norfloxacin, or lomefloxacin in the anesthetized rabbit (48,49). Fukada et al. (48) characterized the ocular pharmacokinetics (Cmax, Tmax, T1/2) of ofloxacin. Sato et al. [of the same laboratory as Fukada (49)] were able to conclude that lomefloxacin penetrated into aqueous sooner and was eliminated faster than norfloxacin. In later experiments, Ohtori et al. [of the same laboratory as above (50)] examined the ocular pharmacokinetics of timolol and carteolol in rabbits shortly after recovery from anesthesia. A 5 mm cellulose membrane (50 kDa) linear probe of fused silica (0.2 mm o.d., 23 g tubing) was used. In vitro recoveries of 16-20% for norfloxacin/lomefloxacin and ~ 17—22% for timolol and carteolol were reported. Pigmented rabbits (1.5-3.0 kg) were studied. The surgery involved stitching the nictitating membrane in order to immobilize the eye followed by the insertion of a 23 gauge needle attached to one end of the probe in the temporal cornea and passing the needle through anterior chamber and out of nasal side. The exteriorized tubing was glued at the puncture sites with epoxy resin. The polyethylene tubing was taped to the face of the rabbit.
Rittenhouse et al. (16) developed an animal model for the evaluation of microdialysis sampling of aqueous humor to assess the ocular absorption and disposition of beta-adrenergic antagonist drugs. For this study using anesthetized dogs (n = 3) and rabbits (n = 3), microdialysis probes (10 mm CMA/20) were implanted in the anterior chamber. Immediately following probe implantation 30 min), a single dose of [3H]DL-propranolol was administered topically or intracamerally in order to estimate intraocular bioavailability of [3H]DL-propranolol. [3H]DL-Propranolol collected from probe effluent was assayed by liquid scintillation spectroscopy. The results of this study indicated a 10-fold higher intraocular exposure to propranolol in the rabbit relative to the dog (FAH ~ 0.55 vs. ~ 0.056). Time to peak was longer in the dog relative to the rabbit 87 vs. ~ 54 min), and the terminal rate constant for the dog was ~ twofold higher than the rabbit 0.0189 vs.
0.00983). Propranol recoveries of ~ 32-45% were reported. The results obtained in this initial examination of propranolol disposition in aqueous humor using microdialysis were highly variable. In general, aqueous humor protein concentrations would have minimal influence on ocular exposure (3) due to the low concentrations present. However, since propranolol is a highly protein-bound substrate (45), Rittenhouse et al. (17) examined the possibility that time-dependent protein binding might have been a contributing factor to variability in parameter estimates, due to surgical insult from probe implantation and subsequent increased influx of proteins into aqueous humor. In addition, anesthesia is a known contributor to alterations in the pharmacokinetics and pharmacodynamics of drugs (60). Thus, development of relevant experimental techniques for use in conscious animals was imperative.
Following redesign of the microdialysis probes for anterior or posterior chamber placement (4 mm, CMA/20 with 90° bend) (Fig. 1) in the conscious rabbit, studies were conducted with propranolol (17) to estimate the intraocular exposure (AUCah), time to peak (Tmax), and aqueous humor peak concentrations (Cmax) following a >5-day recovery. This minimum recovery period was established by following the time course of ocular wound healing and anterior segment resorption of fibrin, a phenomenon that could result in reduced substrate recovery via microdia-lysis aqueous humor sampling. Briefly, the surgical probe implantation procedure for New Zealand white rabbits (2.3-50 kg) proceeded as follows: A limbal-based conjunctival flap was created superior nasally or temporally ~ 3 mm from the limbus. A 10-12 mm conjunctival pocket was prepared, and the probe inlet/outlets were exteriorized to the top of head. A 20 gauge needle was inserted ~ 2-3 mm from limbus into the anterior chamber and removed. The microdialysis probe was then placed into the opening and the anchor of probe sutured to the sclera and covered with conjunctiva. Propranolol ocular pharmacokinetic parameter estimates obtained from a previous study (16) were compared to those obtained in the present study (17). It was observed that reduced dose-normalized AUCAH and Cmax were obtained in the previous study relative to the present study 1.9-fold relative to anesthetized results with >5-day recovery period). It was hypothesized that time-dependent aqueous humor protein concentrations may have been present immediately postsurgery, but that protein concentrations returned to basal levels given a sufficient recovery period. Increased aqueous humor protein concentrations have been reported in vivo shortly after cannulation of the eye (46). In order to examine this possible explanation for the apparent decrease in dose-normalized AUCah observed in the previous study (16), the time course of aqueous humor protein concentrations after microdialysis probe implantation in the anterior chamber was examined in 16 rabbits (17). Immediately following probe implantation, aqueous humor protein concentrations were comparable to control. At 30 minutes postimplantation, aqueous humor protein concentrations were maximal 30 mg/mL) and were maintained for up to 90 minutes. Aqueous humor protein concentrations were halfmaximal at 150 minutes. A simulation approach was used to examine the hypothesis that altered protein concentrations were responsible for differences in propranolol exposure between the two experiments. Results of the simulations indicated that time-dependent binding of propranolol in aqueous humor was probably the major contributor to the reduced aqueous humor intraocular exposure to propranolol observed in rabbits with a minimal recovery period postimplantation (2.4-fold for simulation results vs. ~ 1.9-fold difference observed in vivo). Another salient observation in this study was the appreciable difference between the ocular pharmacokinetics of propranolol in the conscious versus the anesthetized rabbit. Dose-normalized AUCAh was ^eight-fold lower in conscious rabbits as compared to anesthetized rabbits. Propranolol dose-normalized Cmax values for the conscious rabbits were appreciably lower than those reported in the literature for conscious animal experimentation (20,21). A careful examination of this question resulted in the hypothesis that traditional sampling procedures (euthanasia of conscious rabbits following topical administration of drug, with paracentesis sampling of aqueous humor as the last step) may result in artifactually higher intraocular exposures to topically administered xenobiotics. This work provided a framework for examination of ocular pharmacokinetics in a more physiologically relevant model.
Although a number of researchers have examined the blood-to-aqueous transport of ascorbate in ciliary body tissue and cell culture in vitro (61-68), the transport kinetics information derived from these studies, in most instances, does not correlate to in vivo determinations. In vivo investigations have been limited due to difficulties inherent in studies of ascorbate transport kinetics; Km, the blood concentration of ascorbate at half-maximal transport, is reputed to be at or below physiological blood concentrations (58,63). Rittenhouse et al. (53), following the development of an analytical procedure for assay of ascorbate in blood and aqueous humor, examined the transport kinetics of ascorbate using the recently developed conscious animal model with microdialysis sampling of aqueous humor.
Microdialysis probes were placed in the anterior chamber of one eye and the posterior chamber of the fellow eye (53). Basal blood-to-aqueous transport of 14C-ascorbate was established by the examination of aqueous humor ascorbate corrected for specific activity. Following a 30-day recovery period, the rabbits (n = 4) were placed in restraining devices, the marginal ear veins of respective ears were cannulated, and ascorbate was administered via an i.v. bolus loading dose followed by maintenance incremental infusions in order to characterize the linear-to-nonlinear kinetic profile in blood to aqueous humor transport. Blood and probe effluent were analyzed via UV spectrophotometry at 265 nm. A nonlinear least-squares regression analysis assessment of the transport kinetics of ascorbate was performed. Contrary to previous reports (58,63), ascorbate blood concentrations, which were increased in a stepwise fashion (an overall ^twofold increase), did not result in saturable ascorbate uptake into aqueous (blood concentrations from ~ 14 to ~ 21 to ~ 30 mg/L). Nonlinear least-squares regression analysis of a model that incorporated nonsaturable uptake into aqueous with first-order translocation from the posterior to the anterior chamber and first-order efflux from the anterior chamber, with an incorporated lag time of ~ 1 hour, appeared to describe the data best. The model fits to the serum, anterior, and posterior aqueous ascorbate concentration-time data are presented in Figure 3. Physiologically relevant parameter estimates were obtained with this approach. The analysis provided indications that reduced aqueous humor turnover occurred in this group of rabbits (translocation rate constants were ~ 0.005 min-1 as compared to 0.01 min-1 in intact animals). The parameter estimates were also in agreement with the model independent ascorbate ocular clearance determinations 39 mL/min or ~ 0.003 min-1, when divided by the estimated aqueous humor volume of 200 mL) (53). It is possible that the apparent transport of ascorbate was perturbed by surgery. Surgical trauma can result in increased peroxide generation as a result of the inflammatory cascade (69). There are no reports of studies evaluating basal ascorbate transport as a function of the degree of intraocular inflammation. It also is possible that time-dependent changes to ascorbate blood to aqueous transport were observed. In order to examine this possibility, the relationship between aqueous humor ascorbate concentrations and time post-probe implantation was examined (Fig. 4) (17,53,54). At 0 minutes, physiologically relevant ascorbate aqueous humor concentrations are observed 1.4 mM). An appreciable decrease 50%) was observed from day 1 to day 12. Hence, recovery periods were lengthened for subsequent experiments in order to examine ascorbate transport kinetics (>30 days). However, from
the data presented in Figure 5, it appeared that as early as ~ 21 days, ascorbate aqueous humor concentrations returned to physiologically relevant concentrations 1.3 mM). The relationship between basal blood to aqueous transport and time postsurgery is presented in Figure 5. Basal transport rates comparable to the expected value 27 mg/hr) were observed for rabbits following a minimum of 21 days of recovery. These data were examined retrospectively (53,54).
Macha and Mitra (51,52) explored an innovative approach in ocular microdialysis experimentation. A dual microdialysis probe experimental design provided the unique opportunity to examine intraocular drug disposition in both vitreous and aqueous humors simultaneously. Using this approach, the pharmacokinetics of systemically versus intravitreously administered fluorescein (51) was examined in the anesthetized rabbit. Concentric probes (CMA/20 with 0.5 x 10 mm, polycarbonate membrane and 14 mm shaft) were used for vitreous sampling. Linear probes (MD-2000, 0.32 x 10 mm, polyacrylonitrile membrane and 0.22 mm tubing)
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