INTRAOCULAR PHARMACOKINETICS USING MICRODIALYSIS

VI. INTRAOCULAR PHARMACOKINETICS USING MICRODIALYSIS

Gunnarson et al. (22) first utilized in vivo dialysis technique to sample the vitreous chamber. Studies were carried out to measure endogenous amino acids in the preretinal vitreous space. The effects of high potassium and nipecotic acid, a potent gamma-aminobutyric acid (GABA) inhibitor, on amino acid concentrations were measured. A dialysis probe was implanted in the vitreous of the eye of albino rabbits (Fig. 1). The integrity of the blood-retinal barrier was demonstrated by measuring the concentrations of

3 HOH and [ 14 C] j mannitol in the vitreous effluent following intracarotid injections. 3 HOH was detected in the vitreous within a few minutes, whereas

[ 14 C]mannitol was mostly excluded. Among the amino acids, glutamine had

a concentration similar to that in the plasma and cerebrospinal fluid (CSF). Vitreous concentration of all amino acids was lower than in plasma, the majority being below 50% of the plasma concentrations. The taurine level was approximately 70% that of plasma. A comparison with CSF shows that all amino acids except for glutamine and phosphoethanolamine (PEA) are present at higher concentrations in vitreous. Taurine was significantly ele- vated (fourfold) in the vitreous, as are valine and alanine. Perfusion with 125

262 Macha and Mitra

Figure 1 (Top) The positioner device for the dialysis probe on the rabbit eye. The Perspex cylinder (a) is sewn to the sclera. The probe (b) is placed in the guide channel (c). The prismatic lens (d) used for biomicroscopy is in contact with the cornea. (Bottom) The biomicroscopic view of the fundus and the inserted dialysis loop (e). The iris (f) and the retina (g) can also be observed through the lens.

Posterior Segment Microdialysis 263 mM KCl-containing media for 30 minutes, 4 hours after probe implantation

raised the taurine content by sixfold and PEA content by twofold. Other amino acids remained unchanged (Fig. 2). Perfusion with 60 mM nipecotic acid increased GABA concentration by 60 times and taurine levels by almost 10 times, while other amino acids remained fairly constant (Fig. 3).

Ben-Nun et al. (10) evaluated the intraocular pharmacokinetics of gentamicin after intravitreal administration. The experiments were carried out for a short duration in domestic cats weighing 2.5–5 kg. The animals were anesthetized and the pupils were dilated with tropicamide 0.5% and phenylephrine 10%. Lateral canthotomy was performed in both eyes and the area of the upper part of the sclera was exposed. The superior rectus muscle was divided and cotton wool was inserted into the gap between the posterior sclera and the superior margin of the orbit to stop bleeding. The cotton wool was fixed with a drop of cyanoacrylate glue. A rubber disk (5 mm in diameter and 1 mm thick) was glued to the sclera over the pars plana region in the superotemporal quadrant of each eye. A 1 mm diameter hole was made through the rubber disks and a 20 gauge needle was then passed through each hole into the eye. A sampling catheter for ocular dialysis was

Figure 2 Change of amino acid concentration with time. (*) Amino acid level on perfusion with Krebs-Ringer buffer. (*) Level on perfusion with high potassium.

264 Macha and Mitra

Figure 3 Change of concentration of taurine, phosphoethanolamine, and GABA in response to 60 mM nipecotic acid. The change from Krebs-Ringer buffer to nipecotic acid perfusion media is indicated by an arrow on the time axis.

passed into the sclerotomy site in each eye and glued by a drop of cyanoa- crylate glue, followed by a drop of rapid setting epoxy adhesive. The cathe- ters were connected in parallel to a double barrel Harvard pump and perfused at a rate of 3.5 mL=min (Fig. 4). Gentamicin was administered at

a site away from the sampling site. The experiments were carried in two groups of cats: normal and bacterial endophthalmitis (Staphylococcus

Figure 4 Diagram representing the bilateral simultaneous sampling of vitreous humor by ocular dialysis.

Posterior Segment Microdialysis 265

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 vitr- eous and its elimination from the vitreous (Fig. 5). The elimination rate constants were greater in infected eyes (0.107 hr ) than in controls (0.055 hr ), 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.

266 Macha and Mitra membrane length of 4 mm and the shaft bent at 60–908 was used. Adult

pigmented rabbits were anesthetized with Hypnorm vet 1 , and a small open- ing 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 125 I-labeled NGF to cross a previously implanted probe. The probes were perfused for different time periods with a solution containing NGF. With an inlet

M, the vitreous concentrations were found M when the solution was

M concentrations of NGF were perfused for 4 hours, the vitreous concen- M, respectively. The same model was used to delivery ganciclovir into the rabbit vitreous (60). Ganciclovir

used to administer 5-fluorouracil, benzyl penicillin, daunomycin, and dex- amethasone into the vitreal space of rabbits (25). The vitreal concentrations

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 micro- dialysis. 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%.

Posterior Segment Microdialysis 267

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 micro- dialysis 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 Ca 2+ 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

268 Macha and Mitra

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 repeat- edly 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

Posterior Segment Microdialysis 269

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 anesthe- sia 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 microdia- lysis probe was implanted across the cornea in the aqueous humor using a

25 gauge needle (Fig. 10). The probes were perfused with isotonic phos- phate 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

270 Macha and Mitra

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 elimina- tion half-lives of cephalexin, cefazolin, and cephalothin after intravitreal

mL) was found to generate higher concentrations in the aqueous humor parameters of cephalexin in the presence of gly-pro, i.e., AUC

m tively) (Fig. 11). In the case of cefazolin, the control parameters

7:31 min, respectively) were found to be similar, except the terminal elim- m

tively) (Fig. 12). Gly-sar was found to have no significant effect on the pharmacokinetics of both drugs. These studies indicated the involvement

Posterior Segment Microdialysis 271

Figure 11 Vitreous concentration-time profile of cephalexin (50 mg) in the presence of inhibitors after intravitreal administration. The line drawn represents the non- linear 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 tech- nique to delineate the ocular pharmacokinetics of ganciclovir (GCV) and its ester prodrugs (acetate, propionate, butyrate, and valerate). The pro- drugs generated sustained therapeutic concentrations of GCV over a pro- longed 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 intra- vitreal administration are shown in Figure 13. The vitreal terminal phase elimination half-life (t 1=2 b The proportion of GCV eliminating through the anterior chamber path- way was about 1%. The representative vitreous concentration-time profile

272 Macha and Mitra

Figure 12 Vitreous concentration-time profile of cefazolin (50 mg) in the presence of inhibitors after intravitreal administration. The line drawn represents the non- linear 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 ðt 1=2 k 10 ) of GCV, monoacetate, monopropio-

tionship was observed between the vitreal elimination rate constant ðk 10 Þ and the ester chain length. The C max for the regenerated GCV after the

rate 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 concen- trations of GCV in vivo and the MRT of GCV could be enhanced three- to-fourfold through prodrug modification.

Posterior Segment Microdialysis 273

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 con- centration-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.

274 Macha and Mitra

VII. CONCLUSIONS Microdialysis has been shown to be a very useful tool to study ocular

pharmacokinetics. The major strengths of the technique appear to be its simplicity and its ability to monitor drug and metabolite concentration and deliver the drugs. Despite its increasing popularity, microdialysis is still far from being a routine method in eye research. Until now, much work has gone into adapting and improving the technology involved. Future studies need to be focused on the methodological problems and limitations that could lead to erroneous data interpretation and conflicting results.

ACKNOWLEDGMENTS Supported by NIH grants R01 EY09171-08 and R01 EY10659-07.

REFERENCES 1. G. Raviola. (1977). The structural basis of the blood-ocular barriers. Exp. Eye

Res. 25:27–63. 2. S. P. Donahue, J. M. Khoury, and R. P. Kowalski. (1996). Common ocular infections. A prescriber’s guide. Drugs 52:526–540. 3. P. R. Pavan, E. E. Oteiza, B. A. Hughes, and A. Avni. (1994). Exogenous endophthalmitis initially treated without systemic antibiotics. Ophthalmology 101:1289–1297. 4. L. IH. (1984). Anti-infective agents. In: Pharmacology of the Eye. M. Sears,

ed. New York: Springer-Verlag, pp. 385–446. 5. B. D. Kuppermann, J. G. Petty, D. D. Richman, W. C. Mathews, S. C. Fullerton, L. S. Rickman, and W. R. Freeman. (1993). Correlation between CD4+ counts and prevalence of cytomegalovirus retinitis and human immu- nodeficiency virus-related noninfectious retinal vasculopathy in patients with acquired immunodeficiency syndrome. Am. J. Ophthalmol. 115:575–582. 6. J. G. Gross, S. A. Bozzette, W. C. Mathews, S. A. Spector, I. S. Abramson, J.

A. McCutchan, T. Mendez, D. Munguia, and W. R. Freeman. (1990). Longitudinal study of cytomegalovirus retinitis in acquired immune deficiency syndrome. Ophthalmology 97:681–686.

7. D. A. Jabs, C. Enger, and J. G. Bartlett. (1989). Cytomegalovirus retinitis and acquired immunodeficiency syndrome. Arch. Ophthalmol. 107:75–80.

8. D. A. Jabs. (1992). Treatment of cytomegalovirus retinitis—1992. Arch. Ophthalmol . 110:185–187.

Posterior Segment Microdialysis 275 9. M. J. Daily, G. A. Peyman, G. Fishman. (1973). Intravitreal injection of

methicillin for treatment of endophthalmitis. Am. J. Ophthalmol. 76:343–350. 10. J. Ben-Nun, D. A. Joyce, R. L. Cooper, S. J. Cringle, and I. J. Constable. (1989). Pharmacokinetics of intravitreal injection. Assessment of a gentamicin model by ocular dialysis. Invest. Ophthalmol. Vis. Sci. 30:1055–1061.

11. A. G. Palestine, M. A. Polis, M. D. DeSmet, B. F. Baird, J. Falloon, J. A. Kovacs, R. T. Davey, J. J. Zurlo, K. M. Zunich, M. Davis, et al. (1991). A randomized controlled trial of foscarnet in the treatment of cytomegalovirus retinitis in patients with AIDS. Ann. Intern. Med. 115:665–673. 12. L. B. Sheiner, B. Rosenberg, and V. V. Marathe. (1977). Estimation of popu- lation characteristics of pharmacokinetic parameters from routine clinical data. J. Pharmacokinet. Biopharm. 5:445–479.

13. G. L. Drusano, W. Liu, R. Perkins, A. Madu, C. Madu, M. Mayers, and M. H. Miller. (1995). Determination of robust ocular pharmacokinetic para-

meters in serum and vitreous humor of albino rabbits following systemic administration of ciprofloxacin from sparse data sets by using IT2S, a popula- tion pharmacokinetic modeling program. Antimicrob. Agents Chemother. 39:1683–1687. 14. M. Mayers, D. Rush, A. Madu, M. Motyl, and M. H. Miller. (1991). Pharmacokinetics of amikacin and chloramphenicol in the aqueous humor of rabbits. Antimicrob. Agents Chemother. 35:1791–1798. 15. M. H. Miller, A. Madu, G. Samathanam, D. Rush, C. N. Madu, K. Mathisson, and M. Mayers. (1992). Fleroxacin pharmacokinetics in aqueous and vitreous humors determined by using complete concentration-time data from individual rabbits. Antimicrob. Agents Chemother. 36:32–38. 16. R. J. Perkins, W. Liu, G. Drusano, A. Madu, M. Mayers, C. Madu, and M.

H. Miller. (1995). Pharmacokinetics of ofloxacin in serum and vitreous humor of albino and pigmented rabbits. Antimicrob. Agents Chemother. 39:1493– 1498. 17. W. Liu, Q. F. Liu, R. Perkins, G. Drusano, A. Louie, A. Madu, U. Mian, M. Mayers, and M. H. Miller. (1998). Pharmacokinetics of sparfloxacin in the serum and vitreous humor of rabbits: physicochemical properties that regulate penetration of quinolone antimicrobials. Antimicrob. Agents Chemother. 42:1417–1423. 18. L. Bito, H. Davson, E. Levin, M. Murray, and N. Snider. (1966). The con- centrations of free amino acids and other electrolytes in cerebrospinal fluid in vivo dialysate of brain, and blood plasma of the dog. J. Neurochem. 13:1057– 1067. 19. M. Telting-Diaz, D. O. Scott, and C. E. Lunte. (1992). Intravenous micro- dialysis sampling in awake, freely-moving rats. Anal. Chem. 64:806–810. 20. J. E. Robinson. (1995). Microdialysis: a novel tool for research in the repro- ductive system. Biol. Reprod. 52:237–245.

21. D. G. Maggs, W. P. Borg, and R. S. Sherwin. (1997). Microdialysis techniques in the study of brain and skeletal muscle. Diabetologia 40(Suppl. 2):S75–82. (1987).

276 Macha and Mitra 22. G. Gunnarson, A. K. Jakobsson, A. Hamberger, and J. Sjostrand. (1987).

Free amino acids in the pre-retinal vitreous space. Effect of high potassium and nipecotic acid. Exp. Eye Res. 44:235–244. 23. N. Stempels, M. J. Tassignon, and S. Sarre. (1993). A removable ocular microdialysis system for measuring vitreous biogenic amines. Graefes Arch. Clin. Exp. Ophthalmol . 231:651–655. 24. P. M. Hughes, R. Krishnamoorthy, and A. K. Mitra. (1996). Vitreous dis- position of two acycloguanosine antivirals in the albino and pigmented rabbit models: a novel ocular microdialysis technique. J. Ocul. Pharmacol. Ther. 12:209–224. 25. J. Waga and B. Ehinger. (1997). Intravitreal concentrations of some drugs administered with microdialysis. Acta Ophthalmol. Scand. 75:36–40. 26. S. Macha and A. K. Mitra. (2001). Ocular pharmacokinetics in rabbits using a novel dual probe microdialysis technique. Exp. Eye Res. 72:289–299. 27. S. Machal and A. K. Mtira. Ocular pharmacokinetics of cephalosporins using microdialysis. J. Ocul. Pharmacol. Ther. (in press). 28. K. D. Rittenhouse, R. L. Peiffer, Jr., and G. M. Pollack. (1999). Microdialysis evaluation of the ocular pharmacokinetics of propranolol in the conscious rabbit. Pharm. Res. 16:736–742.

29. V. H. Lee and J. R. Robinson. (1986). Topical ocular drug delivery: recent developments and future challenges. J. Ocul. Pharmacol. 2:67–108. 30. S. S. Chrai, M. C. Makoid, S. P. Eriksen, and J. R. Robinson. (1973). Drop size and initial dosing frequency problems of topically applied ophthalmic drugs. J. Pharm. Sci. 63:333–338. 31. S. S. Chrai, T. F. Patton, A. Mehta, and J. R. Robinson. (1973). Lacrimal and instilled fluid dynamics in rabbit eyes. J. Pharm. Sci. 62:1112–1121.

32. A. K. Mitra and T. J. Mikkelson. (1988). Mechanism of transcorneal permea- tion of pilocarpine. J. Pharm. Sci. 77:771–775. 33. R. Abel, Jr., G. L. Boyle, M. Furman, and I. H. Leopold. (1974). Intraocular penetration of cefazolin sodium in rabbits. Am. J. Ophthalmol. 78:779–787. 34. S. J. Faigenbaum, G. L. Boyle, A. S. Prywes, R. Abel, Jr., and I. H. Leopold. (1976). Intraocular penetrating of amoxicillin. Am. J. Ophthalmol . 82:598–603.

35. B. M. Faris and M. M. Uwaydah. (1974). Intraocular penetration of semi- synthetic penicillins: methicillin, cloxacillin, ampicillin, and carbenicillin stu- dies in experimental animals with a review of the literature. Arch. Ophthalmol. 92:501–505.

36. G. A. Peyman, D. R. May, P. I. Homer, and R. T. Kasbeer. (1977). Penetration of gentamicin into the aphakic eye. Ann. Ophthalmol. 9:871–880. 37. L. Salminen. (1978). Ampicillin penetration into the rabbit eye. Acta Ophthalmol. (Copenh.) 56:977–983. 38. M. Barza, A. Kane, and J. L. Baum. (1979). Intraocular levels of cefamandole compared with cefazolin after subconjunctival injection in rabbits. Invest. Ophthalmol. Vis. Sci. 18:250–255.

Posterior Segment Microdialysis 277 39. M. Barza, A. Kane, and J. Baum. (1980). Oxacillin for bacterial endophthal-

mitis: subconjunctival, intravenous, both or neither? Invest. Ophthalmol. Vis. Sci . 19:1348–1354. 40. W. M. Jay, R. K. Shockley, A. M. Aziz, M. Z. Aziz, and J. P. Rissing. (1984). Ocular pharmacokinetics of ceftriaxone following subconjunctival injection in rabbits. Arch. Ophthalmol. 102:430–432.

41. F. P. Furgiuele, J. P. Smith, and J. G. Baron. (1978). Tobramycin levels in human eyes. Am. J. Ophthalmol. 85:121–123. 42. D. M. Maurice, and S. Mishima. (1984). Ocular pharmacokinetics. In: Pharmacology of the Eye II . M. L. Sears, ed. New York: Springer-Verlag, p. 19. 43. M. Barza, A. Kane, and J. Baum. (1983). Pharmacokinetics of intravitreal carbenicillin, cefazolin, and gentamicin in rhesus monkeys. Invest. Ophthalmol. Vis. Sci . 24:1602–1606.

44. V. N. Reddy, B. Chakrapani, and C. P. Lim. (1977). Blood-vitreous barrier to amino acids. Exp. Eye Res. 25:543–545. 45. A. G. Schenk and G. A. Peyman. (1974). Lincomycin by direct intravitreal injection in the treatment of experimental bacterial endophthalmitis. Albrecht Von Graefes Arch. Klin. Exp. Ophthalmol . 190: 281–291. 46. S. C. Pflugfelder, E. Hernandez, S. J. Fliesler, J. Alvarez, M. E. Pflugfelder, and R. K. Forster. (1987). Intravitreal vancomycin. Retinal toxicity, clear- ance, and interaction with gentamicin. Arch. Ophthalmol. 105:831–837.

47. F. M. Ussery, 3rd, S. R. Gibson, R. H. Conklin, D. F. Piot, E. W. Stool, and A. J. Conklin. (1988). Intravitreal ganciclovir in the treatment of AIDS-asso-

ciated cytomegalovirus retinitis. Ophthalmology, 95:640–648. 48. K. Henry, H. Cantrill, C. Fletcher, B. J. Chinnock, and H. H. Balfour, Jr. (1987). Use of intravitreal ganciclovir (dihydroxy propoxymethyl guanine) for cytomegalovirus retinitis in a patient with AIDS. Am. J. Ophthalmol. 103:17–23.

49. G. A. Peyman, D. R. May, E. S. Ericson, and D. Apple. (1974) Intraocular injection of gentamicin. Toxic effects of clearance. Arch. Ophthalmol. 92:42– 47. 50. R. K. Shockley, W. M. Jay, T. R. Friberg, A. M. Aziz, J. P. Rissing, and M. Z. Aziz. (1984). Intravitreal ceftriaxone in a rabbit model. Dose- and time-depen- dent toxic effects and pharmacokinetic analysis. Arch. Ophthalmol. 102:1236– 1238.

51. A. G. Schenk, G. A. Peyman, and J. T. Paque. (1974). The intravitreal use of carbenicillin (Geopen) for treatment of pseudomonas endophthalmitis. Acta Ophthalmol . 52:707–717. 52. S. K. Gardner. (1987). Ocular drug penetration and pharmacokinetic princi- ples. In: Clinical Ophthalmic Pharmacology. D. W. Lamberts and D. E. Potter, eds. 53. T. S. Lesar and R. G. Fiscella. (1985). Antimicrobial drug delivery to the eye. Drug Intell. Clin. Pharm . 19:642–654. 54. J. D. Wright, F. D. Boudinot, and M. R. Ujhelyi. (1996). Measurement and analysis of unbound drug concentrations. Clin. Pharmacokinet. 30:445–462.

278 Macha and Mitra 55. U. Ungerstedt. (1991). Microdialysis—principles and applications for studies

in animals and man. J. Intern. Med. 230:365–373. 56. E. C. de Lange, M. Danhof, A. G. de Boer, and D. D. Breimer. (1997). Methodological considerations of intracerebral microdialysis in pharmacoki- netic studies on drug transport across the blood-brain barrier. Brain Res. Brain Res. Rev . 25:27–49. 57. R. Tao and S. Hjorth. (1992). Differences in the in vitro and in vivo 5-hydro- xytryptamine extraction performance among three common microdialysis membranes. J. Neurochem. 59:1778–1785. 58. J. Landolt, H. Langemann, T. H. Lutz, and O. Gratzl. (1991). Non-linear recovery of cysteine and glutathione in microdialysis. In: H. Rollema, et al., eds. Meppel, Netherlands: Krips Repro, pp. 63–65. 59. J. Waga, I. Nilsson-Ehle, B. Ljungberg, A. Skarin, L. Stahle, and B. Ehinger. (1999). Microdialysis for pharmacokinetic studies of ceftazidime in rabbit vitreous. J. Ocul. Pharmacol. Ther. 15:455–463. 60. J. Waga. (2000). Ganciclovir delivery through an intravitreal microdialysis probe in rabbit. Acta Ophthalmol. Scand. 78:369–371.

61. A. Hamberger, C. H. Berthold, B. Karlsson, A. Lehmann, and B. Nystrom. (1983). Extracellular GABA, glutamate and glutamine in vivo perfusion dia- lysis of the rabbit hyppocampus. In: Glutamine, Glutamate and GABA in the Central Nervous System . New York: Alan R. Liss Inc., pp. 473–492. 62. M. Sandberg and S. Lindstrom. (1993). Amino acids in the dorsal lateral geniculate nucleus of the cat—collection in vivo. J. Neurosci. Methods. 9:64–74. 63. U. Tossman and U. Ungerstedt. (1986). Microdialysis in the study of extra- cellular levels of amino acids in the rat brain. Acta Physiol. Scand. 128:9–14.

64. A. Lehmann. (1989). Effects of microdialysis-perfusion with anisoosmotic media on extracellular amino acids in the rat hippocampus and skeletal mus- cle. J. Neurochem. 53:525–535. 65. J. M. Solis, A. S. Herranz, O. Herreras, J. Lerma, and R. Martin del Rio. (1988). Does taurine act as an osmoregulatory substance in the rat brain? Neurosci. Lett . 91:53–58. 66. J. V. Wade, J. P. Olson, F. E. Samson, S. R. Nelson, T. L. Pazdernik. (1988).

A possible role for taurine in osmoregulation within the brain. J. Neurochem. 51:740–745. 67. S. A. Wages, W. H. Church, and J. B. Justice, Jr. (1986). Sampling considera- tions for on-line microbore liquid chromatography of brain dialysate. Anal. Chem . 58:1649–1656. 68. J. M. Delgado, J. Lerma, R. Martin del Rio, and J. M. Solis. (1984). Dialytrode technology and local profiles of amino acids in the awake cat brain. J. Neurochem. 42:1218–1228.

69. G. Amberg, and N. Lindefors. (1989). Intracerebral microdialysis: II. Mathematical studies of diffusion kinetics. J. Pharmacol. Methods 22:157–183. 70. T. Zetterstrom, L. Vernet, U. Ungerstedt, U. Tossman, B. Jonzon, and B. B. Fredholm. (1982). Purine levels in the intact rat brain. Studies with an implanted perfused hollow fibre. Neurosci. Lett. 29:111–115.

Posterior Segment Microdialysis 279 71. I. Jacobson, M. Sandberg, and A. Hamberger (1985). Mass transfer in brain

dialysis devices—a new method for the estimation of extracellular amino acids concentration. J. Neurosci. Methods 15:263–268. 72. P. Lonnroth, P. A. Jansson, and U. Smith. (1987). A microdialysis method allowing characterization of intercellular water space in humans. Am. J. Physiol . 253:E228–231.

73. D. Scheller and J. Kolb. (1991). The internal reference technique in micro- dialysis: a practical approach to monitoring dialysis efficiency and to calculat- ing tissue concentration from dialysate samples. J. Neurosci. Methods 40:31– 38.

74. H. Benveniste, A. J. Hansen, and N. S. Ottosen. (1989). Determination of brain interstitial concentrations by microdialysis. J. Neurochem. 52:1741– 1750.

75. G. Raviola. (1974). Effects of paracentesis on the blood-aqueous barrier: an electron microscope study on Macaca mulatta using horseradish peroxidase as a tracer. Invest. Ophthalmol. 13:828–858. 76. F. P. Killey, H. F. Edelhauser, and T. A. Aaberg. (1980). Intraocular fluid dynamics. Measurements following vitrectomy and intraocular sulfur hexa- fluoride administration. Arch. Ophthalmol 98:1448–1452. 77. L. L. Knudsen, T. Olsen, and F. Nielsen-Kudsk (1988). Anterior chamber fluorescein kinetics compared with vitreous kinetics in normal subjects. Acta Ophthalmol. Scand . 76:561–567. 78. J. Wagar and B. Ehinger. (2000). NGF administered by microdialysis into rabbit vitreous. Acta Ophthalmol. Scand. 78:154–155.