Sudharshan Hariharan

Development and validation of a fast and sensitive bioanalytical method for the quantitative determination of glucocorticoids--quantitative measurement of dexamethasone in rabbit ocular matrices by liquid chromatography tandem mass spectrometry.

A sensitive, selective, accurate and robust LC-MS/MS method was developed and validated for the quantitative determination of glucocorticoids in rabbit ocular tissues. Samples were processed by a simple liquid-liquid extraction procedure. Chromatographic separation was performed on Phenomenex reversed phase C18 gemini column (50mmx4.6mm i.d.,) with an isocratic mobile phase composed of 30% of acetonitrile in water containing 0.1% of formic acid, at a flow rate 0.2mL/min. Dexamethasone (DEX), prednisolone (PD) and hydrocortisone (HD) were detected with proton adducts at m/z 393.20-->355.30, 361.30-->147.20 and 363.20-->121.0 in multiple reaction monitoring (MRM) positive mode respectively. Finally, 50microL of 0.1% novel DEX mixed micellar formulation was topically administered to a rabbit eye and concentrations were measured. The method was validated over a linear concentration range of 2.7-617.6ng/mL. Lower limit of quantitation (LLOQ) of DEX and PD was measured in the concentration range of 2.7 and 11.0ng/mL respectively. The resulting method demonstrated intra and inter-day precision within 13.3% and 11.1% and accuracy within 19.3% and 12.5% for DEX and PD, respectively. Both analytes were found to be stable throughout freeze-thaw cycles and during bench top and postoperative stability studies (r(2)>0.999). DEX concentrations in various ocular tissue samples i.e., aqueous humor, cornea, iris ciliary body, sclera and retina choroid were found to be 344.0, 1050.07, 529.6, 103.9 and 48.5ng/mg protein respectively. Absorption of DEX after topical administration from a novel aqueous mixed micellar formulation achieved therapeutic concentration levels in posterior segment of the rabbit eye.

Enhanced Corneal Absorption of Erythromycin by Modulating P-Glycoprotein and MRP Mediated Efflux with Corticosteroids

PurposeThe objectives were (i) to test in vivo functional activity of MRP2 on rabbit corneal epithelium and (ii) to evaluate modulation of P-gp and MRP2 mediated efflux of erythromycin when co-administered with corticosteroids.MethodsCultured rabbit primary corneal epithelial cells (rPCECs) was employed as an in vitro model for rabbit cornea. Cellular accumulation and bi-directional transport studies were conducted across Madin-Darby Canine Kidney (MDCK) cells overexpressing MDR1 and MRP2 proteins to delineate transporter specific interaction of steroids. Ocular pharmacokinetic studies were conducted in rabbits following a single-dose infusion of erythromycin in the presence of specific inhibitors and steroids.ResultsBi-directional transport of erythromycin across MDCK-MDR1 and MDCK-MRP2 cells showed significant difference between BL-AP and AP-BL permeability, suggesting that erythromycin is a substrate for P-gp and MRP2. Cellular accumulation of erythromycin in rPCEC was inhibited by steroids in a dose dependent manner. MK571, a specific MRP inhibitor, modulated the aqueous humor concentration of erythromycin in vivo. Even, steroids inhibited P-gp and MRP2 mediated efflux with maximum increase in ka, AUC0 − ∞, Cmax and Clast values of erythromycin, observed with 6α-methyl prednisolone.ConclusionMRP2 is functionally active along with P-gp in effluxing drug molecules out of corneal epithelium. Steroids were able to significantly inhibit both P-gp and MRP2 mediated efflux of erythromycin.

Ocular pharmacokinetics of acyclovir amino acid ester prodrugs in the anterior chamber: evaluation of their utility in treating ocular HSV infections.

PURPOSE To evaluate in vivo corneal absorption of the amino acid prodrugs of acyclovir (ACV) using a topical well model and microdialysis in rabbits. METHODS Stability of L-alanine-ACV (AACV), L-serine-ACV (SACV), L-isoleucine-ACV (IACV), gamma-glutamate-ACV (EACV) and L-valine-ACV (VACV) prodrugs was evaluated in various ocular tissues. Dose-dependent toxicity of these prodrugs was also examined in rabbit primary corneal epithelial cell culture (rPCEC) using 96-well based cell proliferation assay. In vivo ocular bioavailability of these compounds was also evaluated with a combination of topical well infusion and aqueous humor microdialysis techniques. RESULTS Among the amino acid ester prodrugs, SACV was most stable in aqueous humor. Enzymatic degradation of EACV was the least compared to all other prodrugs. Cellular toxicity of all the prodrugs was significantly less compared to trifluorothymidine (TFT) at 5mM. Absorption rate constants of all the compounds were found to be lower than the elimination rate constants. All the prodrugs showed similar terminal elimination rate constants (lambda(z)). SACV and VACV exhibited approximately two-fold increase in area under the curve (AUC) relative to ACV (p<0.05). Clast (concentration at the last time point) of SACV was observed to be 8+/-2.6microM in aqueous humor which is two and three times higher than VACV and ACV, respectively. CONCLUSIONS Amino acid ester prodrugs of ACV were absorbed through the cornea at varying rates (ka) thereby leading to varying extents (AUC). The amino acid ester prodrug, SACV owing to its enhanced stability, comparable AUC and high concentration at last time point (Clast) seems to be a promising candidate for the treatment of ocular HSV infections.

Interaction of Ocular Hypotensive Agents (PGF2α Analogs—Bimatoprost, Latanoprost, and Travoprost) With MDR Efflux Pumps on the Rabbit Cornea

PURPOSE The objectives of this work were (i) to screen ocular hypotensive prostaglandin (PGF2 alpha) analogs--bimatoprost, latanoprost, and travoprost as well as their free acid forms--for interaction with efflux pumps on the cornea and (ii) to assess the modulation of efflux upon co-administration of these prostaglandin analogs. METHODS Cultured rabbit primary corneal epithelial cells (rPCEC) were employed as an in vitro model for rabbit cornea. Transporter-specific interaction studies were carried out using Madin-Darby canine kidney (MDCK) cells overexpressing MDR1, MRP1, MRP2, MRP5, and BCRP. Freshly excised rabbit cornea was used as an ex vivo model to determine transcorneal permeability. RESULTS Cellular accumulation studies clearly showed that all prostaglandin analogs and their free acid forms are substrates of MRP1, MRP2, and MRP5. Bimatoprost was the only prostaglandin analog in this study to interact with P-gp. In addition, none of these molecules showed any affinity for BCRP. K (i) values of these prostaglandin analogs obtained from dose-dependent inhibition of erythromycin efflux in rPCEC showed bimatoprost (82.54 microM) and travoprost (94.77 microM) to have similar but higher affinity to efflux pumps than latanoprost (163.20 microM). Ex vivo studies showed that the permeation of these molecules across cornea was significantly elevated in the presence of specific efflux modulators. Finally, both in vitro and ex vivo experiments demonstrated that the efflux of these prostaglandin analogs could be modulated by co-administering them together. CONCLUSION Bimatoprost, latanoprost, travoprost, and their free acid forms are substrates of multiple drug efflux pumps on the cornea. Co-administration of these molecules together is a viable strategy to overcome efflux, which could simultaneously elicit a synergistic pharmacological effect, since these molecules have been shown to activate different receptor population for the reduction of intraocular pressure (IOP).

Biotin uptake by rabbit corneal epithelial cells: role of sodium-dependent multivitamin transporter (SMVT).

Purpose: The objective of this research was to investigate the presence of sodium-dependent multivitamin transporter (SMVT) on rabbit corneal epithelial cells. Methods: Primary cultured rabbit corneal epithelial cells (rPCECs)and freshly excised rabbit corneas were used for characterization of biotin uptake and transport, respectively. Reverse transcription–polymerase chain reaction (RT-PCR) was performed to confirm the molecular identity of SMVT. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis was performed to examine the presence of biotin in rabbit tears. Results: Uptake of biotin by rPCECs was found to be time and concentration dependent with Km of 32.52 μ M and Vmax of 10.43 pmol min− 1 mg protein− 1. Biotin was significantly inhibited in the presence of pantothenic acid and lipoic acid. Biotin uptake was found to be energy and Na+ dependent but H+ and Cl− independent. The uptake was inhibited by valeric acid in a concentration-dependent manner but not much affected in the presence of biotin methyl ester and biocytin with no free carboxyl group. Modulators of both PKC- and PKA-mediated pathways had no effect on biotin uptake, but calcium-calmodulin inhibitor significantly inhibited its uptake. Sodium-dependent multivitamin transporter was identified by RT-PCR in rPCECs. Transport experiments across the rabbit corneas revealed the functional localization of SMVT on the apical side of the cornea, and thereby corroborating with in vitro results with cultured corneal cells. Finally, LC-MS/MS analysis showed the presence of biotin in rabbit tears. Conclusions: Results obtained from both in vitro and ex vivo studies suggest the possible role of SMVT expressed on corneal epithelial cells for the uptake of biotin, which co-transports pantothenic acid and lipoic acid. Further, the presence of biotin in tears suggests the physiological significance of this transporter in rabbit corneal epithelium.

Pharmacokinetics of amino acid ester prodrugs of acyclovir after oral administration: interaction with the transporters on Caco-2 cells.

In vivo systemic absorption of the amino acid prodrugs of acyclovir (ACV) after oral administration was evaluated in rats. Stability of the prodrugs, L-alanine-ACV (AACV), L-serine-ACV (SACV), L-isoleucine-ACV (IACV), gamma-glutamate-ACV (EACV) and L-valine-ACV (VACV) was evaluated in various tissues. Interaction of these prodrugs with the transporters on Caco-2 cells was studied. In vivo systemic bioavailability of these prodrugs upon oral administration was evaluated in jugular vein cannulated rats. The amino acid ester prodrugs showed affinity towards various amino acid transporters as well as the peptide transporter on the Caco-2 cells. In terms of stability, EACV was most enzymatically stable compared to other prodrugs especially in liver homogenate. In oral absorption studies, ACV and AACV showed high terminal elimination rate constants (lambda(z)). SACV and VACV exhibited approximately five-fold increase in area under the curve (AUC) values relative to ACV (p<0.05). C(max(T)) (maximum concentration) of SACV was observed to be 39+/-22 microM in plasma which is 2 times better than VACV and 15 times better than ACV. C(last(T)) (concentration at the last time point) of SACV was observed to be 0.18+/-0.06 microM in plasma which is two times better than VACV and three times better than ACV. Amino acid ester prodrugs of ACV were absorbed at varying amounts (C(max)) and eliminated at varying rates (lambda(z)) thereby leading to varying extents (AUC). The amino acid ester prodrug SACV owing to its enhanced stability, higher AUC and better concentration at last time point seems to be a promising candidate for the oral treatment of herpes infections.

Identification and functional expression of a carrier-mediated riboflavin transport system on rabbit corneal epithelium.

Purpose: To investigate the functional expression of a carrier-mediated transport mechanism for riboflavin (vitamin B2) across cultured rabbit primary corneal epithelial cells (rPCECs) and intact rabbit cornea. The secondary objective was to understand the physiological significance behind the presence of such a transport system for riboflavin on the apical side of the corneal epithelium. Methods: rPCECs and freshly excised rabbit corneas were selected as in vitro and ex vivo models, respectively. Transport and uptake characteristics of [3H]riboflavin were determined at various time points, concentrations, temperatures, and pH. Substrate specificity, energy, and ion dependence studies were carried out to characterize the translocation mechanism. Rabbit tear analysis was done with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to understand the physiological relevance of this transporter. Results: The uptake process in rPCECs was found to be concentration dependent and saturable at higher concentrations. The process was also independent of pH, Na+, and Cl− but dependent on energy and temperature. Unlabeled riboflavin and its structural analogues caused significant inhibition, whereas unrelated vitamins did not interfere with the process. Transport of [3H]riboflavin across rabbit cornea was also saturable at higher concentration and energy dependent but Na+ independent. Substrate specificity studies across intact rabbit cornea produced results similar to the uptake studies in cultured rPCECs. LC-MS/MS analysis of rabbit tears showed the presence of riboflavin. Conclusions: Results suggest the presence of a specialized, high-affinity transport mechanism for riboflavin that is expressed on the apical side of rabbit corneal epithelium and may in turn be responsible for influx of riboflavin from tears to cornea.

Recent Patents and Advances in Ophthalmic Drug Delivery

Ophthalmic drug delivery has long been a challenging task for pharmaceutical scientists seeking to alleviate various ocular diseases affecting the anterior and posterior segments. In order to deliver therapeutic agents to target tissues, the unique anatomical barriers of the eye must be circumvented effectively, without causing any patient discomfort or alteration in protective physiological mechanisms. This challenge is currently being met with the development of novel non-invasive delivery methods as well as improvements over existing techniques. Over the past decade many advanced technologies have been patented. Nevertheless a need for additional research and continuous innovation is still warranted. Patent literature is often essential for promoting new directions in research as well as for elucidating possibilities for future technologies. Hence, the aim of this review article is to discuss several recently filed patents on non-invasive modes of drug delivery to the ocular tissues. This review will also focus on the role of colloidal/particulate systems in ocular drug delivery and formulation. Recent patents filed on prodrugs as an efficient ophthalmic drug delivery mechanism also have been discussed. As a whole, this article is intended to provide a valuable insight into current trends in the field of ocular drug delivery and highlights advances made in patent literature.

Drug Delivery to Cornea and Conjunctiva: Esterase- and Protease-Directed Prodrug Design

Drug entry across cornea and conjunctiva following topical administration is a formidable challenge due to highly lipophilic epithelial membrane with tight junctions. Prodrug strategy targeted toward esterases and amidases for bioreversion has been found to be an effective and promising approach for improving drug permeation. Esterases and proteases are known to be present ubiquitously in most of the ocular tissues. The ability of therapeutically inactive prodrug to generate the active parent drug following corneal/conjunctival absorption determines its efficacy and utility. Lipophilic ester prodrug design – though successful in terms of achieving enhanced corneal/conjunctival permeability – is limited by solubility and formulation constraints. Recently, nutrient transporter-mediated prodrug delivery has gained attention in ophthalmic drug delivery. Prodrugs targeted toward nutrient transporters for absorption followed by hydrolyzing enzymes toward activation can be an effective strategy to improve ocular drug absorption.

Pharmacokinetics of Stereoisomeric Dipeptide Prodrugs of Acyclovir Following Intravenous and Oral Administrations in Rats: A Study Involving In vivo Corneal Uptake of Acyclovir Following Oral Dosing

Objective To delineate the plasma pharmacokinetics and determine the corneal uptake of valine based stereoisomeric dipeptide prodrugs of acyclovir (ACV) in rats. Methods Male Sprague-Dawley rats were used for the study. Pharmacokinetics of ACV, L-valine-acyclovir (LACV), L-valine-D-valine-acyclovir (LDACV) and D-valine-L-valine acyclovir (DLACV) prodrugs were delineated. These compounds were administered intravenously as a bolus via jugular vein cannula and orally by gavage. Samples were purified by protein precipitation method and analyzed by LC-MS/MS. Pertinent pharmacokinetic parameters were obtained by using WinNonlin. Corneal uptake studies of LDACV and LACV were studied following oral administration. Results Following i.v. administration, the area under the curve (AUC) in μM*min of generated ACV was in the order of LACV > LDACV > DLACV indicating their rate of metabolism. The AUC values of total drug obtained in the systemic circulation after oral administration LACV and LDACV were 1077.93 ± 236.09 and 1141.76 ± 73.67 μM*min, respectively. DLACV exhibited poor oral absorption. Cmax (μM) and AUC of the intact prodrug obtained in the systemic circulation following oral administration of LDACV were almost 4–5 times higher than LACV. Moreover, concentrations achieved in the cornea after oral administration of LDACV were almost two times of LACV. Conclusions LDACV increased both the oral bioavailability and subsequent in vivo corneal uptake of ACV Hence, LDACV can be considered as the most promising drug candidate for delivery of ACV, in treatment of both genital herpes and ocular herpes keratitis after oral administration.