Nanda K. Mandava

Effect of short term and chronic administration of Sutherlandia frutescens on pharmacokinetics of nevirapine in rats.

Sutherlandia frutescens (sutherlandia), an African herbal supplement was recommended by the South African Ministry of Health for the treatment of AIDS patients. However, no reports yet exist delineating the effect of sutherlandia on pharmacokinetics of antiretroviral agents. Therefore, this investigation aimed at screening the effects of short term and chronic exposure of sutherlandia on oral bioavailability and pharmacokinetics of nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, in Sprague Dawley rats. NVP (6 mg/kg) was administered orally alone (control) and with co-administration of sutherlandia; short term (12 mg/kg single dose) and long term (12 mg/kg, once a day for 5 days). No significant difference in the pharmacokinetic parameters of NVP was found upon short-term co-administration of Sutherlandia. However, there was a 50% decrease (p<0.05) in the AUC and C(max) values of NVP after 5 days of chronic exposure with Sutherlandia. In addition, quantitative RT-PCR studies demonstrated a 2-3-fold increase in the hepatic and intestinal mRNA expression of CYP3A2, relative to vehicle control. To further confirm, if this could translate into a clinically relevant pharmacokinetic interaction in patients, we tested this hypothesis employing LS-180 cells as an in vitro induction model for human CYP3A4. Ninety-six hours post treatment, similar to positive control rifampicin (25 μM), sutherlandia extract (300 μg/mL) resulted in elevated m-RNA expression levels and functional activity of CYP3A4 (human homologue of rodent CYP3A2) in LS-180 cells. Taken together, these results suggest that a potential drug-herb interaction is possible when NVP is co-administered with S. frutescens, although this hypothesis still remains to be investigated in a clinical setting.

Interaction of dipeptide prodrugs of saquinavir with multidrug resistance protein-2 (MRP-2): Evasion of MRP-2 mediated efflux

Saquinavir (SQV), the first protease inhibitor approved by FDA to treat HIV-1 infection. This drug is a well-known substrate for multidrug resistance protein-2 (MRP-2). The objective of this study was to investigate whether derivatization of SQV to dipeptide prodrugs, valine-valine-saquinavir (Val-Val-SQV) and glycine-valine-saquinavir (Gly-Val-SQV), targeting peptide transporter can circumvent MRP-2 mediated efflux. Uptake and transport studies were carried out across MDCKII-MRP2 cell monolayers to investigate the interaction of SQV and its prodrugs with MRP-2. In situ single pass intestinal perfusion experiments in rat jejunum were performed to calculate intestinal absorption rate constants and permeabilities of SQV, Val-Val-SQV and Gly-Val-SQV. Uptake studies demonstrated that the prodrugs have significantly lower interaction with MRP-2 relative to SQV. Transepithelial transport of Val-Val-SQV and Gly-Val-SQV across MDCKII-MRP2 cells exhibited an enhanced absorptive flux and reduced secretory flux as compared to SQV. Intestinal perfusion studies revealed that synthesized prodrugs have higher intestinal permeabilities relative to SQV. Enhanced absorption of Val-Val-SQV and Gly-Val-SQV relative to SQV can be attributed to their translocation by the peptide transporter in the jejunum. In the presence of MK-571, a MRP family inhibitor, there was a significant increase in the permeabilities of SQV and Gly-Val-SQV indicating that these compounds are probably substrates for MRP-2. However, there was no change in the permeability of Val-Val-SQV with MK-571 indicating lack of any interaction of Val-Val-SQV with MRP-2. In conclusion, peptide transporter targeted prodrug modification of MRP-2 substrates may lead to shielding of these drug molecules from MRP-2 efflux pumps.

Amino Acid Prodrugs: An Approach to Improve the Absorption of HIV-1 Protease Inhibitor, Lopinavir

Poor systemic concentrations of lopinavir (LPV) following oral administration occur due to high cellular efflux by P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) and extensive metabolism by CYP3A4 enzymes. In this study, amino acid prodrugs of LPV were designed and investigated for their potential to circumvent efflux processes and first pass effects. Three amino acid prodrugs were synthesized by conjugating isoleucine, tryptophan and methionine to LPV. Prodrug formation was confirmed by the LCMS/MS and NMR technique. Interaction of LPV prodrugs with efflux proteins were carried out in P-gp (MDCK-MDR1) and MRP2 (MDCK-MRP2) transfected cells. Aqueous solubility studies demonstrated that prodrugs generate higher solubility relative to LPV. Prodrugs displayed higher stability under acidic conditions and degraded significantly with rise in pH. Uptake and transport data suggested that prodrugs carry significantly lower affinity towards P-gp and MRP2 relative to LPV. Moreover, prodrugs exhibited higher liver microsomal stability relative to LPV. Hence, amino acid prodrug modification might be a viable approach for enhancing LPV absorption across intestinal epithelial and brain endothelial cells which expresses high levels of P-gp and MRP2.

Amino acid prodrug of quinidine: An approach to circumvent P-glycoprotein mediated cellular efflux

In the present study, we investigated the effect of large neutral amino acid modification in overcoming P-gp mediated cellular efflux of quinidine. L-isoleucine ester prodrug of quinidine (Ile-quinidine) was synthesized in our laboratory. [14C]-erythromycin was selected as a model substrate to study interaction of quinidine and Ile-quinidine with P-gp. Transport studies were conducted to study translocation kinetics of quinidine and Ile-quinidine in MDCK-MDR1 cells. In cellular accumulation study, uptake rate of [14C]-erythromycin elevated drastically in presence of cyclosporine A and GF 120918. This result indicates that [14C]-erythromycin is an excellent substrate of P-gp. Similarly, uptake rate of [14C]-erythromycin was enhanced significantly in presence of quinidine (25 and 50 μM). However, [14C]-erythromycin uptake rate remained fairly constant in presence of Ile-quinidine (25 μM). Apparent A-B and B-A permeability of quinidine observed in MDCK-MDR1 cells were 1.6 ± 0.2 × 10(-6) and 7.0 ± 0.4 × 10(-6)cm/s, a 4.4-fold difference. Moreover, A-B permeability of quinidine increased dramatically in the presence of cyclosporine A and GF 120918. Apparent permeability values of Ile-quinidine observed from A-B and B-A direction were 4.3 ± 0.9 × 10(-6) and 5.5 ± 0.4 × 10(-6)cm/s, a 1.3-fold difference. Importantly, A-B transport of Ile-quinidine did not change dramatically in the presence of cyclosporine and GF 120918. Based on these results, it was apparent that quinidine displayed higher substrate affinity toward P-gp relative to Ile-quinidine. Chemical or enzymatic hydrolysis of Ile-quinidine resulted in regeneration of low quantities of quinidine during transport studies. Competitive inhibition studies demonstrated that Ile-quinidine was recognized by multiple amino acid transporters such as LAT1, LAT2 and cationic amino acid transporter. In conclusion, chemical modification of quinidine with neutral amino acids results in circumvention of P-gp mediated drug efflux. Hence, amino acid transporter targeted prodrug delivery seems to be a viable strategy for improving drug accumulation in P-gp overexpressing cells.

Transporter targeted drug delivery

The pharmacological behavior of various drugs is severely affected by biological barriers such as epithelial tight junctions, efflux proteins and metabolizing enzymes. Apart from the biological barriers, physicochemical properties of drug molecules such as molecular weight, lipophilicity, surface charge and solubility also play an important role in absorption characteristics of drug candidates. Pharmacological properties affected by efflux pumps such as P-gp and MRPs include bioavailability, hepatobiliary and urinary excretion of drugs as well as drug metabolites. This leads to sub-therapeutic concentrations of various potential drugs at the target site. One of the strategies to overcome these biological barriers is transporter targeted prodrug design. Prodrug derivatization targeting membrane transporters and receptors improves drug absorption. Various prodrugs which have been synthesized so far demonstrated enhanced bioavailability and tissue specificity. This review mainly focuses on the efflux pumps which play an important role in drug absorption and a few strategies to overcome these efflux pumps.

Recent patents and emerging therapeutics for HIV infections: a focus on protease inhibitors

The inclusion of protease inhibitors (PIs) in highly active antiretroviral therapy has significantly improved clinical outcomes in HIV-1-infected patients. To date, PIs are considered to be the most important therapeutic agents for the treatment of HIV infections. Despite high anti-HIV-1 potency, poor oral bioavailability of PIs has been a major concern. For achieving therapeutic concentrations, large doses of PIs are administered, which results in unacceptable systemic toxicities. Such severe and long-term toxicities necessitate the development of safer and potentially promising PIs. Recently, considerable attention has been paid to the development of newer compounds capable of inhibiting wild-type and resistant HIV-1 protease. Some of these PIs have displayed potent HIV-1 protease inhibitory activity. In this review, we have made an attempt to provide an overview on clinically approved and newly developing PIs, and related recent patents in the development of novel PIs.

Dipeptide prodrug approach to evade efflux pumps and CYP3A4 metabolism of lopinavir

Oral absorption of lopinavir (LPV) is limited due to P-glycoprotein (P-gp) and multidrug resistance-associated protein2 (MRP2) mediated efflux by intestinal epithelial cells. Moreover, LPV is extensively metabolized by CYP3A4 enzymes. In the present study, dipeptide prodrug approach was employed to circumvent efflux pumps (P-gp and MRP2) and CYP3A4 mediated metabolism of LPV. Valine-isoleucine-LPV (Val-Ile-LPV) was synthesized and identified by LCMS and NMR techniques. The extent of LPV and Val-Ile-LPV interactions with P-gp and MRP2 was studied by uptake and transport studies across MDCK-MDR1 and MDCK-MRP2 cells. To determine the metabolic stability, time and concentration dependent degradation study was performed in liver microsomes. Val-Ile-LPV exhibited significantly higher aqueous solubility relative to LPV. This prodrug generated higher stability under acidic pH. Val-Ile-LPV demonstrated significantly lower affinity toward P-gp and MRP2 relative to LPV. Transepithelial transport of Val-Ile-LPV was significantly higher in the absorptive direction (apical to basolateral) relative to LPV. Importantly, Val-Ile-LPV was recognized as an excellent substrate by peptide transporter. Moreover, Val-Ile-LPV displayed significantly higher metabolic stability relative to LPV. Results obtained from this study suggested that dipeptide prodrug approach is a viable option to elevate systemic levels of LPV following oral administration.

Microdialysis in Ocular Drug Development

Development of novel treatments for ocular diseases demands a complete understanding of ocular drug pharmacokinetics. Microdialysis is a continuous sampling technique that has been frequently applied for assessing ocular pharmacokinetic parameters. This sampling technique has gained considerable attention in ocular pharmacokinetics since it considerably reduces the requirement of large number of animals needed to obtain a complete pharmacokinetic profile. Remarkable progress has been made in ocular anterior and posterior segment microdialysis in recent years. These developments have simplified sampling of ocular fluids such as vitreous and aqueous humor. In this book chapter, the principle and application of microdialysis in measuring drug disposition in various segments of the eye will be discussed. This chapter will also highlight on recent progress on the development of various animal models and probe designs in ocular microdialysis.