HIV protease inhibitors are an important component of highly active antiretroviral therapy used to treat pregnant women infected with HIV. They have a low placental transfer and are highly plasma protein bound. This study was carried out to determine the unbound fraction of lopinavir in cord blood, and to characterize the binding of lopinavir to alpha(1)-acid glycoprotein (AAG) and human serum albumin (HSA), and displacement by ritonavir. Serum was obtained from cord blood from placentas obtained after cesarean section of healthy, non-HIV-infected women (n = 4). The unbound fraction of lopinavir in serum obtained from this cord blood was 0.022 +/- 0.011%. The unbound fraction of lopinavir in separately obtained maternal serum samples (n = 4) was 0.89 +/- 0.12%, which was not significantly different from that observed with cord serum samples. Varying concentrations of lopinavir, AAG, and HSA in buffer solutions were then used to characterize the lopinavir binding. The data were fit to obtain the number of binding sites (N) and equilibrium dissociation constant (K(D)). Binding of lopinavir to AAG (7-23 microM) was saturable with K(D) of 5.0 +/- 1.1 microM and N of 1.2 +/- 0.2. At low HSA concentrations (15-152 microM), lopinavir binding K(D) was 24.3 +/- 8.7 microM and N was 1.1 +/- 0.4; however, at 758 microM, lopinavir binding was essentially unsaturable. Lopinavir binding to AAG and HSA was not sensitive to ritonavir, and, thus, efforts to enhance fetal exposure to lopinavir should be focused on other issues such as efflux transporters.