p70 S6K

A typical regimen calibration curve for the ATV assay with antiserum at an initial dilution of 1/500,000 is shown in Fig

A typical regimen calibration curve for the ATV assay with antiserum at an initial dilution of 1/500,000 is shown in Fig. 0.04 to 19). A significant increase Sav1 in the accumulation of ATV was BM-131246 noticed in the presence of P-glycoprotein and MRP1 inhibitors (dipyridamole, inter alia). Interestingly, efavirenz significantly increased the baseline accumulation of ATV, whereas nevirapine induced a marked reduction. This new enzyme immunoassay for measuring plasma and intracellular ATV levels was fully validated and provides an inexpensive and useful tool for routine therapeutic drug monitoring. Moreover, in vitro results suggested the implication of BM-131246 drug transporters and interactions with other antiviral drugs that should be further explored in human immunodeficiency virus-infected patients. The introduction of protease inhibitors (PIs) as treatment against human immunodeficiency computer virus (HIV) infection has led to a marked increase in the potency of antiretroviral therapy and therefore has reduced the rates of morbidity and mortality (22). Atazanavir (ATV; Reyataz) is the first azapeptide inhibitor of HIV type 1 (HIV-1) protease approved for treatment and has a half-life that allows once-daily dosing. ATV is currently indicated for use in combination therapy as part of a highly active antiretroviral therapy (HAART) regimen. It has been noticed, however, that PIs are associated with a range of drug-related side effects, such as lipodystrophy and metabolic disturbances (17). Moreover, patients receiving PI treatment exhibit wide variabilities in their virological responses, and many of them fail to achieve maximal viral suppression (14). In order both to increase the efficacy of the treatment and to reduce side effects, therapeutic drug monitoring is gaining increasing prominence in the management of HIV-positive subjects. Many studies have documented the associations between plasma PI concentrations and antiviral effects or drug toxicities, but few of them have resolved the intracellular concentrations of drugs. Indeed, only the fraction reaching the intracellular compartment is expected to have antiviral activity; therefore, antiviral drugs need to penetrate the cell at a concentration high enough to inhibit viral replication in order to be effective. Failure to do so may result in the establishment of a sanctuary for the computer virus. The accumulation of the drug within a target cell is controlled by influx and efflux processes (9). Most PIs are lipophilic and are assumed to enter cells by passive diffusion; moreover, a number of drug transporter proteins have been identified to expel drugs out of cells, including P-glycoprotein (P-gp) (15), multidrug resistance-associated proteins (MRPs) (9, 12), breast cancer resistance protein (9, 12), and organic anion transporters (OATs) (24). Thus, the intracellular concentration of the protease inhibitor ATV should be influenced by these processes, BM-131246 and an assay that enables determination of the concentration of the drug in cells may help provide an understanding of the mechanisms of intracellular accumulation. Moreover, the intracellular pharmacokinetics of the drug would be important for the better optimization of dosing regimens. Several high-performance liquid chromatographic (HPLC) assays combined with UV detection (6, 7, 18, 25) or liquid chromatography with tandem mass spectrometry (LC-MS-MS) (5, 8, 11, 21) have been described for the quantitative determination of ATV in plasma. Only a few of these assays have been validated for use for the measurement of intracellular concentrations (5, 11), and all of them involve the use of LC-MS-MS; but LC-MS-MS systems are not available in all routine laboratories that perform therapeutic drug monitoring and require expensive equipment. However, no immunoassay with a sensitivity, rapidity, and cost-effectiveness superior to those of LC-MS-MS has been published to date. In this report we describe the development and application of a competitive enzyme immunoassay (EIA) for the quantification of ATV in plasma and cells. This new assay is based on the use of specific anti-ATV polyclonal antibodies raised in rabbits and an enzyme tracer, prepared from a synthetic derivative of ATV. We took advantage of the high sensitivity of the assay to measure and compare intracellular ATV accumulation and the effects of drug transporter proteins and other antiretroviral drugs on.