Suresh Katragadda

Role of efflux pumps and metabolising enzymes in drug delivery

The impact of efflux pumps and metabolic enzymes on the therapeutic activity of various drugs has been well established. The presence of efflux pumps on various tissues and tumours has been shown to regulate the intracellular concentration needed to achieve therapeutic activity. The notable members of efflux proteins include P-glycoprotein, multi-drug resistance protein and breast cancer resistance protein. These efflux pumps play a pivotal role not only in extruding xenobiotics but also in maintaining the body’s homeostasis by their ubiquitous presence and ability to coordinate among themselves. In this review, the role of efflux pumps in drug delivery and the importance of their tissue distribution is discussed in detail. To improve pharmacokinetic parameters of substrates, various strategies that modulate the activity of efflux proteins are also described. Drug metabolising enzymes mainly include the cytochrome P450 family of enzymes. Extensive drug metabolism due to the this family of enzymes is the leading cause of therapeutic inactivity. Therefore, the role of metabolising enzymes in drug delivery and disposition is extensively discussed in this review. The synergistic relationship between metabolising enzymes and efflux proteins is also described in detail. In summary, this review emphasises the urgent need to make changes in drug discovery and drug delivery as efflux pumps and metabolising enzymes play an important role in drug delivery and disposition.

Amino acid prodrugs of acyclovir as possible antiviral agents against ocular HSV-1 infections: Interactions with the neutral and cationic amino acid transporter on the corneal epithelium

Purpose. The aim of this study was to explore the feasibility of improvement of ocular bioavailability of the antiviral agent acyclovir by designing amino acid prodrugs targeted to the amino acid transporters on the rabbit cornea. Materials and methods. Transcorneal flux of two water-soluble amino acid ester prodrugs of acyclovir (ACV), γ-glutamate-ACV (EACV) and L-tyrosine-ACV (YACV), was studied across freshly excised rabbit cornea. Chemical and enzymatic hydrolysis studies of the two prodrugs were also conducted. Results. EACV inhibited the uptake of [3H]L-Arg in rabbit primary corneal epithelial cells (rPCECs). The compound also exhibited longer half-life (t1/2) in cornea in comparison to YACV. Transcorneal flux of EACV was observed to be concentration-, energy-, and sodium-dependent and independent of pH within the range studied. EACV transport was inhibited by neutral and cationic amino acids, L-ornithine (specific for cationic amino acids), and BCH (2-aminobicyclo-[2,2,1]-heptane-2-carboxylic-acid) (specific inhibitor for L-type system and B0,+ system). On the other hand, YACV was not recognized by this amino acid transporter as it failed to inhibit the uptake of [3H]Arg, and also its transport across cornea was not inhibited by arginine. YACV and EACV exhibited excellent antiviral activity against HSV-1 and 2 and Varicella-Zoster Virus (VZV) in comparison to ACV. Conclusions. Analyses of the inhibition pattern of EACV transport suggests the involvement of a single transport system; namely, B0,+. Design of amino acid prodrugs seems to be an attractive strategy to enhance the solubility of the otherwise poorly aqueous soluble compounds and also to afford a targeted and possibly enhanced delivery of the active drug.

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.

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.

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.