Srinivasan Senthilkumari

Evaluation of the Modulation of P-glycoprotein (P-gp) on the Intraocular Disposition of its Substrate in Rabbits

Purpose: To evaluate the functional role of P-gp and ocular tissue distribution of intravitreally injected Rhodamine-123 (Rho-123) in the presence of P-gp specific blocker (GF 120918) in normal as well as rifampicin-fed rabbits using microdialysis and direct sampling technique. Methods: Intravitreal pharmacokinetics of Rho-123 were conducted in male New Zealand albino rabbits. Direct sampling and microdialysis were employed to study the disposition of Rho-123 in normal as well as rifampicin-fed conditions. Control animals received Rho-123 at the concentration of 350 ng in PBS (0.05 ml) intravitreally, and the blocker-treated group received GF 120918 intravenously at the dose of 3.5 mg/kg 30 min prior to intravitreal injection of Rho-123. In case of direct sampling, four eyes were enucleated at different time points, and ocular tissues and humors were stored at –86°C until analysis by HPLC with fluorescence detection. Results: In direct sampling, the blocker group showed significant increase (2.6 fold) in the mean vitreous concentration of Rho-123. Other tissues like ret-choroid, iris, and cornea also showed significant increase in their mean concentration. Microdialysis did not significantly predict the changes observed with direct sampling. Rifampicin-fed rabbits showed a vitreous pharmacokinetic profile comparable with non-fed (control) animals, and the pharmacokinetic parameters were unaffected by the blocker pretreatment. Conclusion: Intravenously injected blocker significantly altered the ocular disposition of intravitreally injected P-gp substrate. Rifampicin pretreatment did not upregulate P-gp transporters of the retina to the extent to affect the intravitreal kinetics of Rho-123 significantly.

Evaluation of the Impact of P-Glycoprotein (P-gp) Drug Efflux Transporter Blockade on the Systemic and Ocular Disposition of P-gp Substrate

PURPOSE The impact of P-glycoprotein (P-gp) blockade on the intravenous (i.v.) pharmacokinetics of rhodamine-123 (Rho-123), and the subsequent effect on its disposition in ocular and nonocular tissues, was studied by using rabbits. METHODS Three (3) control rabbits received only an i.v. bolus dose of Rho-123 (1.52 mg/kg). Three (3) blocker-pretreated rabbits received an i.v. dose of GF120918 (3.5 mg/kg) 30 min before the i.v. bolus of Rho-123. The plasma concentration of Rho-123 at different time points was subjected to a pharmacokinetic compartmental analysis, using WinNonlin (Scientific Consultants, Lexington, KY). For tissue-distribution study, a drug treatment similar to the i.v. kinetic study was followed by having 5 rabbits in each group. The animals were sacrificed at 30 min with an excess of anesthesia. Plasma and tissues samples were analyzed by using a validated high-performance liquid chromatographic IV method with a fluorescent detector. RESULTS The method validated was sensitive enough to estimate Rho-123 up to 1.94 ng/mL in plasma. I.v. Rho-123 data fitted well into the three-compartment model, and P-gp blocker treatment changed it into a two-compartment model. The P-gp blockade significantly increased the mean tissue concentrations in the lungs and spleen, whereas the rise in mean tissue levels in the heart, liver, and kidney and in all ocular tissues were found to be statistically insignificant. CONCLUSIONS Increasing the ocular concentration of systemically given drugs may not be possible with the degree of P-gp blockade achieved when using GF120918 at the studied concentration after an i.v. administration.

Single and Multidose Ocular Kinetics and Stability Analysis of Extemporaneous Formulation of Topical Voriconazole in Humans

Purpose: The purpose of the present study was to evaluate the kinetics of single and multiple doses of topical, non-preserved voriconazole (VZ) in human eyes. Methods: For single dose kinetics, 119 patients undergoing cataract surgery were divided into group I and group II and each group received a single drop (30 µl) of either 1% or 0.1% VZ formulation. Aqueous humor was collected at designated time intervals. For multidose kinetics, a single drop of 1% VZ was instilled 5 times either hourly or every 2 hr. The aqueous humor was tested for VZ at the 5th hr and 9th hr, respectively, after initial instillation. The stability and efficacy of the reconstituted VZ formulations were also evaluated after 30 days. Results: Single dose ocular kinetics of 1% VZ resulted in a maximum mean aqueous concentration of 3.333 ± 1.61 µg/ml in 30 min whereas 0.1% showed a maximum mean aqueous concentration of 0.817 ±.36 µg/ml. In the multidose kinetic study, hourly and bi-hourly dosing resulted in mean aqueous concentrations of 7.47 ± 2.14 µg/ml and 4.69 ± 2.7 µg/ml, respectively. The reconstituted VZ formulations were stable at all studied temperatures, and their efficacy was maintained throughout the study period. Conclusion: The present study showed that the achieved mean concentration of VZ in both single dose and multi dose kinetic studies satisfactorily met the MIC90 for almost all causative fungal organisms. The frequency of instillation may be designed for an “every 2 hr regimen” to maintain a therapeutic concentration for successful therapy.

Evidencing the Modulation of P-glycoprotein at Blood-Ocular Barriers using Gamma Scintigraphy

Purpose: To evaluate the effect of P-glycoprotein modulation at blood-ocular barriers using gamma scintigraphy. Methods: Ofloxacin, a fluoroquinolone, was selected as a substrate to study the drug efflux transporter (P-glycoprotein) modulation after labeling with Technetium (99mTc) in rabbits. New Zealand albino rabbits were randomized into two groups of 4 each. Group I received labeled ofloxacin intravitreally (100 μ Ci) and Group II animals were given verapamil intravitreally 15 min before the labeled ofloxacin. Static imaging was done at predetermined time, and dynamic images were also taken after 30 min of intravitreal injection. Results: The radio-chemical purity of labeled ofloxacin was found to be 90–95% with the labeling efficiency of 90%. The static anterior planar images of verapamil pre-treated group showed marginal increase in the uptake of labeled ofloxacin, and dynamic images showed less systemic pool as compared to its control. Conclusion: This study further confirms the findings of our laboratory regarding the involvement of P-glycoprotein in the intraocular disposition of susceptible drugs.

Identification of glaucomatous optic nerve head changes in Indian donor eyes without clinical history

Purpose: The purpose of this study is to develop methods to identify glaucoma by examining the optic nerve head (ONH) of donors eyes when information on the preexisting ocular disease is unavailable. Materials and Methods: The ONH of the donors eyes was evaluated under a stereomicroscope for the cup-disc ratio (CDR) and focal retinal rim thinning. The vertical diameter of the cup and disc was also measured using a precalibrated eyepiece micrometer. The suspect eyes were subjected to histological analysis to confirm the presence of specific glaucomatous changes. Results: A total of 202 eyes from 119 donors (68 males and 51 females, aged 42–96) were evaluated for glaucoma. Among them, 190 (94%) eyes showing vertical CDR in the of 0.0–0.6 range were considered nonglaucomatous and the remaining eyes with >0.6 as glaucoma suspect. The calculated mean CDR of the two groups (0.3 ± 0.16, 0.62 ± 0.27) was highly significant (P = 0.0003). Of 12 eyes suspected of glaucoma, 7 eyes from 5 donors showed specific glaucomatous changes by histology. The prevalence of glaucoma was 4.2% among the donors studied. Conclusions: A simple method of screening fresh donor eyes for selecting those with glaucoma features using CDR and histological analysis was reported. This method helps to obtain biologically active human ocular tissue for glaucoma research on gene expression, ultrastructural/proteome changes, and outflow mechanism.

SB772077B, A New Rho Kinase Inhibitor Enhances Aqueous Humour Outflow Facility in Human Eyes

We investigated the effect of a new Rho kinase inhibitor, SB772077B (SB77) on aqueous outflow facility (OF) in human eyes using human organ-cultured anterior segment (HOCAS). IOP was monitored for 24 h post-treatment with either SB77 (0.1/10/50 µM) or vehicle after a stable baseline pressure. The hydrodynamic pattern of aqueous outflow was analysed by labelling outflow pathway with red fluorescent microspheres. The effect of SB77 on cell morphology, actin stress fibers, focal adhesions, ECM, status of RhoA activation and myosin light chain phosphorylation (p-MLC) were evaluated and compared with Y27632, by immunostaining using primary human trabecular meshwork (HTM) cells. Following 24 h treatment, SB77 increased OF by 16% at 0.1 µM (N = 6), 29% at 10 µM (N = 8; p = 0.018) and 39% at 50 µM (N = 8; p = 0.004) in human eyes. There was an overall increase in tracer quantity and in area along inner wall of Schlemm’s canal. Treatment with SB77 showed no evidence of cytotoxicity and caused a significant reduction in the expression of fibrotic markers compared to Y27632. The present findings indicate that SB77 treatment was effective in enhancing OF and reducing fibrotic markers in an ex vivo model. Thus SB77 may be a potential clinical candidate for the management of glaucoma.

Epalrestat, an Aldose Reductase Inhibitor Prevents Glucose-Induced Toxicity in Human Retinal Pigment Epithelial Cells In Vitro

PURPOSE Aldose reductase (ALR), the first and rate-limiting enzyme involved in polyol pathway plays a central role in diabetes and its related complications, including diabetic retinopathy (DR). Inhibition of ALR may also be an ideal target for reducing the deleterious effects of DR. Therefore, the purpose of the present study was to investigate the protective effect of epalrestat (EPL), ALR inhibitor on glucose-induced toxicity in ARPE-19 cells. METHODS ARPE-19 cells were challenged with normal glucose (NG, 5 mM) and high glucose (HG1, 25 mM and HG2, 50 mM) in the presence or absence of EPL. ALR and VEGF165 expression in retinal pigment epithelial (RPE) cells under experimental conditions were quantified by real-time polymerase chain reaction using SYBR Green chemistry. Vascular endothelial growth factor (VEGF) secretion in the cell supernatant was measured by Sandwich ELISA. Cytotoxicity of EPL was assessed by MTT assay. ALR inhibitory activity, apoptosis, and sorbitol accumulation were also investigated. RESULTS EPL at studied concentration did not show any toxicity to RPE cells and showed as maximum as 65% ALR inhibition under high glucose condition (HG1). The presence of EPL significantly reduced ALR expression and VEGF levels as induced by high glucose in ARPE-19 cells. CONCLUSION Inhibition of ALR appeared to be beneficial in reducing diabetes-related complications in RPE cells under high glucose condition.