Ravi S. Talluri

Synthesis, metabolism and cellular permeability of enzymatically stable dipeptide prodrugs of acyclovir

The objective of this study was to synthesize and evaluate novel enzymatically stable dipeptide prodrugs for improved absorption of acyclovir. l-Valine-l-valine-acyclovir (LLACV), l-valine-d-valine-acyclovir (LDACV), d-valine-l-valine-acyclovir (DLACV) and d-valine-d-valine-acyclovir (DDACV) were successfully synthesized. The uptake and transport studies were conducted on a Caco-2 cell line. Buffer stability and metabolism of the prodrugs in Caco-2, rat intestine and liver homogenates were studied. Structure and purity of the all compounds were confirmed with LC-MS/MS and NMR spectroscopy. Uptake and transport of [(3)H] glycylsarcosine was inhibited by all prodrugs except DDACV. DLACV and DDACV exhibited no measurable degradation in Caco-2 homogenate. Except DDACV other three prodrugs were hydrolyzed in rat intestine and liver homogenates. The order of permeability across Caco-2 was LDACV>LLACV>DDACV>DLACV. A linear correlation between the amount of prodrug transported and over all permeability of acyclovir was established. This study shows that the incorporation of one d-valine in a dipeptide did not abolish its affinity towards peptide transporters (PEPT). Moreover, it enhanced enzymatic stability of prodrug to a certain extent depending on the position in a dipeptide conjugate. This strategy improved both the cellular permeability and the amount of intact prodrug transported which would enable targeting the nutrient transporters at blood ocular barrier (BOB).

Identification and Characterization of a Na+-Dependent Neutral Amino Acid Transporter, ASCT1, in Rabbit Corneal Epithelial Cell Culture and Rabbit Cornea

Purpose: The aim of this study was to investigate the presence of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit primary corneal epithelial cell culture and rabbit cornea. Methods: Uptake studies were carried out on rabbit primary corneal epithelial culture (rPCEC) cells using 12-well plates. Transport studies were conducted with isolated rabbit corneas at 34°C. Uptake and transport of L-alanine was determined at various concentrations. Inhibition studies were conducted in presence of various L- and D-amino acids, metabolic inhibitors like ouabain and sodium azide, and in the absence of sodium to delineate the functional characteristics of L-alanine uptake and transport. Reverse transcription–polymerase chain reaction (RT-PCR) was performed on total RNA harvested from rabbit cornea and rPCEC cells for identification of ASCT1. Results: Uptake of L-Ala was found to be saturable with a Km of 0.71 mM and a Vmax value of 0.84 μ moles min−1 mg−1 protein. Uptake was independent of pH and energy but depends on sodium. It was inhibited by serine, threonine, cysteine, and glutamine but did not respond to BCH (2-aminobicyclo [2,2,1] heptane-2-carboxylic acid) and MeAIB (α -methylaminoisobutyric acid). Transport of L-Ala across rabbit cornea was also saturable (Km 6.52 mM and Vmax 1.09 × 10−2 μ moles min−1 cm−2), energy independent, and subject to similar competitive inhibition. Presence of ASCT1 on rPCEC and on rabbit cornea was identified by RT-PCR. Conclusions: L-Alanine, the chosen model substrate, was actively transported by Na+-dependent, neutral amino acid exchanger ASCT1, which was identified and functionally characterized on rPCEC cells and rabbit cornea.

Mechanism of L-Ascorbic Acid Uptake by Rabbit Corneal Epithelial Cells: Evidence for the Involvement of Sodium-Dependent Vitamin C Transporter 2

Purpose: To investigate the mechanism of L-ascorbic acid uptake by rabbit corneal epithelial cells and to functionally characterize the specific transporter involved in this translocation process. Methods: Uptake studies were carried out with SIRC (Statens Seruminstitut Rabbit Cornea) and rPCEC (rabbit Primary corneal epithelial cell culture) in 12-well plates using [14C] Ascorbic acid (AA). Uptake was done in the presence of L-ascorbic acid and D-isoascorbic acid to delineate stereospecificity. Inhibition studies were performed in the presence of D-glucose a substrate for GLUT and also para amino hippuric acid (PAHA) a substrate for organic anion transporter. Effects of pH and sodium on the uptake of AA were characterized. Concentration dependency studies were performed. Energy dependence of AA uptake was investigated with ouabain and sodium azide in rPCEC. Reverse Transcription-polymerase chain reaction (RT-PCR) was also performed. Results: Uptake of AA was inhibited by about 90% and 50% respectively in the presence of L-ascorbic acid and D-isoascorbic acid in both SIRC and rPCEC. Uptake was unaltered by D-glucose and PAHA. The process was sodium dependent and saturable at higher concentrations. Ouabain and sodium azide significantly diminished the uptake process. It also decreased with a reduction in pH. The RT-PCR results showed the presence of SVCT2 but not SVCT1. Conclusions: Uptake of AA across rabbit corneal epithelial cells appears to be a carrier mediated active process. A saturable, sodium dependent, and pH sensitive transporter with high specificity for L-ascorbic acid was functionally characterized and was identified as SVCT2.

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.

Disposition kinetics of a dipeptide ester prodrug of acyclovir and its metabolites following intravenous and oral administrations in rat.

The objective of this work was to study the disposition kinetics of valine-valine–acyclovir (VVACV), a dipeptide ester prodrug of acyclovir following intravenous and oral administrations in rat. A validated LC-MS/MS analytical method was developed for the analysis VVACV, Valine-Acyclovir (VACV), and Acyclovir (ACV) using a linear Ion Trap Quadrupole. ACV was administered orally for comparison purpose. In the VVACV group, both blood and urine samples and in the ACV group only blood samples were collected. All the samples were analyzed using LC-MS/MS. The LLOQ for ACV, VACV, and VVACV were 10, 10, and 50 ng/ml, respectively. Relevant pharmacokinetic parameters were obtained by non-compartmental analyses of data with WinNonlin. Following i.v. administration of VVACV, AUC0-inf (min*μM) values for VVACV, VACV, and ACV were 55.06, 106, and 466.96, respectively. The AUC obtained after oral administration of ACV was 178.8. However, following oral administration of VVACV, AUC0-inf values for VACV and ACV were 89.28 and 810.77, respectively. Thus the exposure of ACV obtained following oral administration of VVACV was almost 6-fold higher than ACV. This preclinical pharmacokinetic data revealed that VVACV has certainly improved the oral bioavailability of ACV and is an effective prodrug for oral delivery of ACV.