Intracellular accumulation of Praziquantel in T lymphoblastoid cell lines, CEM (parental) and CEMvbl(P-gp-overexpressing)

Abstract

BackgroundPraziquantel (PZQ) is an antihelminthic drug whose P-glycoprotein (P-gp) substrate specificity has not been conclusively characterized. We investigated its specificity by comparing its in vitro intracellular accumulation in CEM (parental), and CEMvbl cells which over express P-gp, a drug efflux transporter. Saquinavir (SQV), a known substrate of efflux transporters was used as control.MethodsA reversed phase liquid chromatography method was developed to simultaneously quantify PZQ and SQV in cell culture media involving involved a liquid - liquid extraction followed by ultra-high performance liquid chromatography using a Hypurity C18 column and ultraviolet detection set at a wavelength of 215 nm. The mobile phase consisted of ammonium formate, acetonitrile and methanol (57:38:5 v/v). Separation was facilitated via isocratic elution at a flow rate of 1.5 ml/min, with clozapine (CLZ) as internal standard. This was validated over the concentration range of 1.6 to 25.6 μM for all analytes. Intracellular accumulation of SQV in CEMvbl was significantly lower compared to that in CEM cells (0.1 ± 0.031 versus 0.52 ± 0.046, p = 0.03 [p <0.05]).ResultsAccumulation of PZQ in both cell lines cells were similar (0.05 ± 0.005 versus 0.04 ± 0.009, p = 0.4) suggesting that it is not a substrate of P-gp in CEM cells. In presence tariquidar, a known inhibitor of P-gp, the intracellular accumulation of SQV in CEMvbl cells increased (0.52 ± 0.068 versus 0.61 ± 0.102, p = 0.34 in CEM cells and 0.09 ± 0.015 versus 0.56 ± 0.089, p = 0.029 [p < 0.05] in CEMvbl cells). PZQ did not significantly affect the accumulation of SQV in either CEM (0.52 ± 0.068 versus 0.54 ± 0.061, p = 0.77), or in CEMvbl cells (0.09 ± 0.015 versus 0.1 ± 0.031, p = 0.89) cells compared to tariquidar, implying that PZQ is not an inhibitor of P-gp in CEMvbl cells.ConclusionsPZQ is neither a substrate nor an inhibitor of the efflux drug transporter P-gp in T-lymphoblastoid cells, CEM and CEMvbl. We also report a simple, accurate and precise method for simultaneous quantification of PZQ and SQV.

Enhancement of saquinavir absorption and accumulation through the formation of solid drug nanoparticles

“…3 and Tables 4 . The intracellular accumulation of SQV in CEMVBL cells (which overexpress P-gp) was lower than CEM in all the formulations, suggesting that the formulations retained the P-gp substrate specificity as in our previous findings [ 47 ]. However, there was a significantly higher accumulation in CEM cells for three SDNs (SQV 05, 06 & 09) compared to that of standard SQV; 1.52 ± 0.19 versus 1.94 ± 0.16, p = 0.003 (SQV 05), vs 1.9 ± 0.29, p = 0.04 (SQV 06), and 1.87 ± 0.25, p = 0.04 (SQV 09) [Fig.…”

“…Increased dissolution/solubility would potentially improve the bioavailability of the drug, controlling for the efflux [ 5 , 24 , 65 – 67 ]. From the cellular accumulation results, intracellular accumulation was lower in CEMVBL than in CEM parental cells for all SDNs including the standard drug as in our previous results [ 47 ]. Nanodispersion did not therefore appear to significantly affect accumulation in CEMVBL cells in SDNs, suggesting that it did not significantly affect the P-gp mediated efflux transport.…”

“…CEM parental and CEMVBL cells were cultured in 175 cm 2 flasks containing RPMI (+ 10% FBS) in a humidified incubator (37 °C, 10% CO 2 ) as previously described [ 47 ]. The cells were counted and media containing 10 million cells centrifuged (2000 × g for 5 min at 4 °C) and the pellet reconstituted to a cell count of one million cells per ml in 10 ml of fresh RPMI.…”

“…The resulting pellets were reconstituted in 100 μl of distilled water and used to determine intracellular (INT) concentrations. The formulations were then assayed by HPLC and data expressed as cellular accumulation ratio (CAR) as described earlier [ 47 ].…”

“…The transport along the CCM of each SDN was compared to that of standard SQV using a method validated earlier in our laboratory [ 46 ]. In order to ascertain the effects of nanodispersion on the cellular accumulation of SQV, a P-glycoprotein [P-gp] substrate, the accumulation of the SDNs in CEM parental and CEMVBL cells (which overexpress P-gp) was compared to that of standard SQV using a method also developed earlier in our laboratory [ 47 ].…”