CONVENTIONAL TECHNIQUES TO STUDY OCULAR PHARMACOKINETICS

IV. CONVENTIONAL TECHNIQUES TO STUDY OCULAR PHARMACOKINETICS

Delineation of ocular pharmacokinetics is complicated due to the complex nature of the retina and the presence of blood-ocular barriers. In addition,

Posterior Segment Microdialysis 255 the small volume of the intraocular fluids aggravates the problem. Limited

volumes of the aqueous and vitreous humors does not allow multiple and continuous sampling. Most of the studies were carried out with an objective to decide a dosage regimen that can provide therapeutic concentrations of drugs in the eye. These studies were conducted in human subjects by collect- ing vitreous and/or aqueous humor samples prior to surgery (such as catar- act, paracentesis, vitrectomy, etc.) after administration of the drug by the route of interest (48). This method allows for collection of only one sample per subject at a specific time period. The data obtained can be interpreted only to determine whether the therapeutic concentrations of the drug have been achieved with the dose administered.

Ocular pharmacokinetic studies have been carried mainly in rabbits, the ocular physiology of which is most similar to the human eyes. Initially, the studies were carried out by collecting single samples from each animal at

a specified time point (13,14). The pooling of the data from individual rabbits requires at least 100 animals for each profile (12,13). Moreover, it introduces a significant amount of intersubject variability.

Serial sampling technique has been developed to obtain more reliable data with a significantly reduced number of animals, compared to the single sample approach, needed for the estimation of pharmacokinetic parameters (15–17). Miller et al. (15) have developed and validated on animal model by measuring the protein concentrations of the intraocular fluids for serial sampling of the aqueous and/or vitreous humors in New Zealand albino rabbits. Animals were anesthetized and vitreous samples of about 20 mL were collected using a 28 gauge needle inserted into the vitreous chamber,

4 mm below the limbus. For serial sampling of the aqueous humor a 30 gauge needle fused to a calibrated 25 mL capillary tube was gently inserted into the anterior chamber near the limbus, and approximately 7 mL of aqueous humor was withdrawn.

V. MICRODIALYSIS The in vivo microdialysis technique has gained significant importance in the

field of the neurochemistry and neurophysiology. This technique has been successfully adapted to sample various tissues and fluids such as skin, liver, kidney, blood, etc. Recently, microdialysis has found important applications in the fields of pharmacokinetics, (especially in the area of drug distribution and metabolism) and pharmacodynamics. It has also been used to study the in vitro protein and melanin binding of drugs (54).

Microdialysis is based on the principle of diffusion. It involves perfu- sion of a probe, containing a semipermeable membrane, implanted in a

256 Macha and Mitra tissue under nonequilibrium conditions. The driving force for the diffusion

of drugs across the semipermeable membrane is the concentration gradient. Endogenous compounds (harmones, neurotransmitters, etc.) and exogenous compounds (drugs and metabolites) diffuse into the probe, whereas com- pounds added to the perfusate diffuse out into the tissue. Therefore, the technique can be used not only to monitor the extracellular concentrations of the analyte, but also to deliver drugs to a specific tissue region (55,56).

Microdialysis offers a number of advantages: (a) it permits continuous monitoring of the tissue concentration of a drug with limited interference with the normal physiology; (b) no fluid is introduced nor is any withdrawn from the tissue, which is particularly important in sampling tissues/organs with limited volume; (c) concentration versus time profiles can be obtained from individual animals; (d) the method provides protein free samples, thus eliminating clean up procedures and ex vivo enzymatic degradation; (e) the samples can be analyzed by any analytical technique, which contributes to the selectivity and sensitivity of the method. Its disadvantages include a need to determine the recovery of the probe (which is still controversial), the diluting effect of dialysis, which requires sensitive analytical methods to measure small concentrations, and the invasive nature of the probe implan- tation.

A. Probe Design and Selection

A variety of probes have been employed to study posterior segment phar- macokinetics. Probes are selected mainly on the basis of the drug under investigation, surgical accessibility, type, and length. A vertical probe with

a concentric design, either as such or with modification, is most commonly used, both in fixed and repeated models, by reinsertion via a guide cannula. The molecular mass cut-off range of the commercially available probes is 5–

50 kDa, and selection of a particular probe is based on the molecular weight of the drug and/or metabolites being studied. The probe membrane type is one more important factor to be considered in microdialysis. Tao and Hjorth (57) demonstrated the difference in the recoveries of three different probe types: GF (regenerated cellulose cuprophan), CMA (polycarbonate ether), and HOSPAL (polyacrilonitril/sodium methallylsulfonate copoly- mer). GF and CMA probes exhibited maximal recovery immediately after the introduction of 5-HT in the artificial CSF, whereas it took about 2 hours for the HOSPAL probe. Landolt et al. (58) have shown that the CMA probe recovery of cysteine and glutathione varied with concentration.

Ben-Nun et al. (10) used a sampling catheter for simultaneous sam- pling of the vitreous of both eyes for short-term experiments. Waga et al. (25,59,60) designed a new probe consisting of soft protecting tube (outer

Posterior Segment Microdialysis 257 diameter 0.6 mm) with a long opening toward one side and a dialysis mem-

brane mounted inside for long-term implantation in the vitreous chamber. The dialysis membrane consisted of a tube of polycarbonate-polyether copo- lymer, with an outer diameter of 520 mm and an inner diameter of 400 mm. In the later experiments the commercially available vertical probe (CMA 20), with a stiff plastic shaft, was used. The shaft length was set to 9 mm and was bent 60–908. Probes with a molecular weight cut-off of 20 and 100 kDA were selected for small and large molecules, respectively. Stempels et al. (23) used CMA probe with a shaft diameter of 0.6 mm, length 3 mm, semipermeable membrane diameter of 0.52 mm, and a cut-off value of 20 kDa.

Probe recovery is directly proportional to the dialysis membrane sur- face area (61–63). By increasing the area, low drug concentrations can be detected with reasonably high perfusion flow rates while maintaining an adequate time resolution. To obtain optimal recovery, Macha and Mitra (26,27) selected a commercially available CMA probe with a membrane length of 10 mm, shaft 14 mm, and a cut-off value of 20 kDa.

Recovery is shown to be independent of the extracellular analyte con- centration (61–63). A concentration gradient across the dialysis membrane changes in unison with the extracellular analyte concentration, thus main- taining a constant recovery.

B. Composition and Temperature of the Perfusate Solution

Intraocular fluid homeostasis is maintained by the highly perfused retina and iris-ciliary body. A perfusion fluid isosmotic with the plasma is pre- ferred, as it has direct access to the vitreous humor. Previous studies have revealed that perfusion with anisosmotic fluid changes the dialysate concen- tration of taurine in brain (64–66) and muscle (64) microdialysis. In addi- tion, problems arise when a compound already present in the perfusion medium is also measured using dialysis.

In vitro recovery is dependent on the temperature of the standard solution. Wages et al. (67) have shown that the in vitro recovery of 3,4- dihydroxyphenylacetic acid increased by approximately 30% when the solu- tion temperature was raised from 23 to 378C. Therefore, the in vitro probe calibration is always carried out at a constant temperature usually the phy- siological temperature.

C. Perfusion Flow Rate Recovery has been shown to improve with a decrease in the perfusion flow

rate. It is fairly high at the beginning but rapidly decelerates after 30–60

258 Macha and Mitra minutes of perfusion. The high dialysate concentrations immediately after

probe implantation may be due to the traumatic tissue response and also due to the steep concentration gradient across the dialysis membrane when the probe is first inserted into the medium. Although this may play an important role, it is usually neglected as the similar time-dependent decrease in recovery is observed in aqueous solutions (68,69).

D. Recovery Dialysate concentration of an analyte of interest is only a measure of its

concentration in the extracellular space. The ratio between the concentra- tion of a substance in the outflow solution following microdialysis of a tissue or a biological fluid and the undisturbed concentration of the same sub- stance in the solution outside the probe is defined as ‘‘recovery,’’ expressed either as a ratio or as a percentage (70).

Recovery factor of the probes is an important parameter in determin- ing the extracellular concentrations of the drug. In vitro recovery was found to be not only simple and time consuming, but also very appropriate for ocular microdialysis. In case of in vitro recovery technique, the recovery of a drug is usually determined by placing the probe in a standard solution. The probe is continuously perfused at a constant flow rate with saline containing no analyte. Samples are collected during fixed time intervals. The recovery of the substance of interest is calculated as follows:

C Recovery in vitro ¼ out ð1Þ

where C out is the concentration in the outflow solution and C i is the con- centration in the medium.

The dialysate substances concentrations are transformed into tissue concentrations as follows:

ð2Þ Recovery in vitro

out

where C i is the substance concentration in the tissue and C out is the con- centration of the dialysate.

Several other techniques have been used to assess probe recovery in vivo, especially for tissue microdialysis. Jacobsen et al. (71) calculated the extracellular concentrations by varying the perfusion flow during an in vivo experiment, measuring the change in the analyte concentration exiting the probe, and then extrapolating to zero flow rate. This method is called as flow-rate or stop-flow method. Lonnroth et al. (72) developed a method where in vivo recovery is estimated by perfusing the probes with varying

Posterior Segment Microdialysis 259 concentrations of the test analyte and then calculating the equilibrium con-

centration, i.e., the concentration at which the analyte in the perfusate does not change during the perfusion because it has the same concentration inside the probe as in the extracellular fluid. This is called as the concentration difference method or zero-net flux method. The two in vivo methods require that the drug concentration in the tissue remains constant during the experi- ment. Probe recovery has also been calculated using a reference substance in the perfusate (73). The method is based on the fact that recovery across the membrane is same in both directions. The percentage loss of the reference substance from the perfusate is used to calculate the concentration of the compound of interest in the tissues.

Although the recovery of a drug as determined in saline solution was used to calculate the drug concentrations in the extracellular space in ocular microdialysis, several studies have been carried out to determine the effect of extracellular milieu on probe recovery. In the case of brain microdialysis, in order to account for factors that may affect mass transport from brain ECF to membrane, in vivo recovery techniques have gained more popularity. In addition, complex solutions like agar gel or red blood cell media have been used to simulate the brain ECF conditions (74). However, these systems have limitations, since it is unlikely that a relatively simple solution like agar gel accurately reflects the complexity of the in vivo physiology. Vitreal microdialysis appears to be less complicated in terms of assessment of the actual vitreal drug concentration compared to other tissues/organs. Vitreous humor consists of almost 99% water. As a result, diffusion of drugs in the vitreous humor has been shown to be similar to that in water. As the microdialysis probe is surrounded by the vitreous humor without any direct contact with the tissue, in vitro recovery appears to be a good approxima- tion of in vivo recovery.

The readers are referred to a review article by de Lange et al. (56) for a detailed description of microdialysis recovery methods.

E. Surgical Trauma and Blood-Retinal Barrier Integrity Probe-induced inflammation at the site of implantation and subsequent

healing is of major concern in in vivo microdialysis. Such physiological changes may affect the intraocular pharmacokinetics significantly. The inflammatory response of the eye mainly depends on the precision of the surgical procedure; therefore, proper precautions should be taken during probe implantation. The time interval between the surgery and the onset of an experiment must be carefully determined, allowing the animal to completely recover.

260 Macha and Mitra Stempels et al. (23) reported that the scleral ports (internal diameter of

0.6 mm), implanted 2–3 mm from the limbus, were well tolerated during the observation period. A transient flare or minimal cell count was observed during the first few days following implantation at or near the entry port, but it was not considered to be due to intolerance. Endophthalmitis, the most common inflammatory response of the eye, and uveitis were not

observed for up to 6 months following probe implantation. Endophthalmitis was detected in 4 of the 23 insertions (17%) in which probes were reused without sterilization; uveitis was not observed when dialysis was conducted with new probes or probes treated with 25% ethanol.

According to Waga et al. (59) the probes were well tolerated for up to

30 days. Topical antibiotics effectively controlled the purulent discharge observed in few cases. Clinical observations and histopathological analysis demonstrated that the probes were well tolerated in majority of the cases. The inserted probe did not elicit any vitreous reactions and the retina in the posterior fundus remained normal. In a few cases when the probe touched the lens due to improper implantation, cataract formation was noticed.

Macha and Mitra (26) selected intraocular pressure (IOP) to determine the effect of microdialysis probe implantation in the anterior and vitreous

mmHg. A sharp fall in the IOP was observed immediately after the implan- level within 2 hours after the implantation and remained constant through-

out the duration of an experiment. The steady IOP after 2 hours following probe implantation suggests that there was no long-term effect of probe implantation on the aqueous humor dynamics.

Blood-aqueous and blood-retinal barriers restrict the passage of serum proteins into the aqueous and vitreous humors. Elevated protein levels and high enzymic activities in the ocular fluids indicate either a breakdown of the respective barrier or a leakage from the injured ocular tissue. Paracentesis (75) and vitrectomy (76) cause breakdown of the blood-ocular barriers, thereby producing elevated protein levels in the intraocular fluids. Integrity of the blood ocular barriers must be maintained following probe implantation, and this issue has been addressed in detail by Macha and Mitra (26). A change in the total protein concentration in the aqueous and vitreous humors was measured. Vitreal protein concentrations mea-

mL) after the probe implantation was not significantly different from the mg/mL). Although the aqueous humor total protein concentration was

Posterior Segment Microdialysis 261

of aqueous humor protein level was assumed to be mainly due to the trauma caused during probe implantation.

The blood-ocular barriers maintain the homeostasis of the intraocu- lar environment by restricting the movement of compounds from the sys- temic circulation to the retinal tissue and vitreous cavity. Several reports discussed the measurement of blood-ocular barrier integrity with the aid of posterior vitreous fluorophotometry (PVF) using fluorescein. Penetration of the dye depends on its concentration in the blood as well as its perme- ability across the blood-ocular barriers. Macha and Mitra (26) evaluated the blood-ocular barrier integrity by studying the fluorescein kinetics after probe implantation. The rate constant for fluorescein penetration into the anterior chamber was found to be significantly higher than into the vitr- eous, indicating that tighter barrier surrounds the vitreous compartment compared to the anterior chamber. Integrity of the blood-retinal and blood-aqueous barriers was ascertained by determining the permeability index (PI). PI of the anterior (9.48%) and the vitreous chamber (1.99%) determined using ocular microdialysis was found to be similar to the values reported using PVF (77).