Lamivudine permeability study: a comparison between PAMPA, ex vivo and in situ Single-Pass Intestinal Perfusion (SPIP) in rat jejunum.

In order to reach the bloodstream and thus the target receptor, an orally-administered drug must first cross the intestinal barrier, which can occur via a paracellular, passive transcellular, or carrier-mediated uptake and/or efflux process (active or concentration gradient-driven). Our work aimed to explore the transport mechanism of the antiretroviral lamivudine (deoxycytidine nucleoside analogue), using a three-part strategy: in vitro, an ex vivo and an in situ method, represented by PAMPA, rat jejunum patches and rat Single Pass Intestinal Perfusion (SPIP), respectively. The determined permeability coefficients were compared with those from a published Caco-2 and MDCK study. Computational prediction of human jejunal permeability was explored, using various non-human permeability coefficients as descriptors. The ex vivo technique was performed in Franz-type diffusion cells, mounted with male Wistar rat jejunum segment patches. PAMPA was performed with an acceptor solution simulating the binding of serum proteins, an artificial membrane impregnated with egg lecithin/cholesterol and a gradient of pH between donor and acceptor solutions. The SPIP was conducted by proximal jejunum cannulation and drug perfusion in a constant flow rate of 0.2 mL/min. The outcomes of our studies showed the following predicted pattern for lamivudine effective jejunal permeability: P(eff)(exvivoA>B)>P(eff)(SPIP)>P(eff)(exvivo B>A)>P(eff)(Caco-2)≈P(eff)(MDCK)≈P(eff)(PAMPA), strongly suggesting that this compound has carrier-mediated uptake as its dominant transport mechanism. Notwithstanding, Caco-2 cells may indicate an under-expression of uptake transporters and possibly an over-expression efflux transporters, compared to that found in the rat jejunum.

Simultaneous quantitation of intracellular zidovudine and lamivudine triphosphates in human immunodeficiency virus-infected individuals.

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with human immunodeficiency virus (HIV). The most common HAART regimen consists of the combination of at least one protease inhibitor (PI) with two nucleoside reverse transcriptase inhibitors (NRTIs). Contrary to PIs, NRTIs require intracellular activation from the parent compound of their triphosphate moiety to suppress HIV replication. Simultaneous intracellular determination of two NRTI triphosphates is difficult to accomplish due to their relatively small concentrations in peripheral blood mononuclear cells (PBMCs), requiring large amounts of blood from HIV-positive patients. Recently, we described a method to determine intracellular zidovudine triphosphate (ZDV-TP) concentrations in HIV-infected patients by using solid-phase extraction and tandem mass spectrometry. The limit of quantitation (LOQ) for ZDV-TP was 0.10 pmol, and the method was successfully used for the determination of ZDV-TP in HIV-positive patients. In this study, we enhanced the aforementioned method by the simultaneous quantitation of ZDV-TP and lamivudine triphosphate (3TC-TP) in PBMCs from HIV-infected patients. The LOQ for 3TC-TP was 4.0 pmol, with an interassay coefficient of variation and an accuracy of 7 and 12%, respectively. This method was successfully applied to the simultaneous in vivo determination of the ZDV-TP and 3TC-TP pharmacokinetic profiles from HIV-infected patients receiving HAART.