Defense Date

2009

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmaceutics

First Advisor

Phillip Gerk

Abstract

HIV protease inhibitors are an important component of Highly Active Antiretroviral Therapy used to treat HIV infected pregnant women. They have a low placental transfer and are highly plasma protein bound. The purpose of this thesis was to characterize the factors limiting placental passage and fetal exposure to lopinavir. These factors include lopinavir plasma protein binding and uptake, cellular binding, and efflux of lopinavir in the placental trophoblast cells. First, we determined the unbound fraction of lopinavir in cord blood and characterized the binding of lopinavir to α1-acid glycoprotein (AAG) and human serum albumin (HSA), and displacement by ritonavir. Serum was obtained from cord blood from placentae 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 0.02 ± 0.01. The unbound fraction of lopinavir in separately obtained maternal serum samples (n=4) was 0.009 ± 0.001, 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 (KD). Binding of lopinavir to AAG (7-23 µM) was saturable with KD of 5.0 ± 1.1 µM and N of 1.2 ± 0.2. At low HSA concentrations (15-152 µM), lopinavir binding KD was 24.3 ± 8.7 µM and N was 1.1 ± 0.4; however at 758 µM, lopinavir binding was essentially unsaturable. Additionally, lopinavir binding to AAG and HSA was not sensitive to ritonavir within the range of therapeutic concentrations. Next, we examined lopinavir uptake, binding and efflux using the BeWo human trophoblast cell culture model. BeWo cells were treated with 3H-lopinavir in the absence or presence of inhibitors of ATP- Binding Cassette transporters. The radioactivity was then measured in the buffer and the cells after incubating for different time intervals and at two temperatures. Verapamil (100µM) stimulated apparent efflux of 3H lopinavir by two fold, possibly due to ABCC2. In addition, this efflux process was 75% inhibited by reduced temperature (4°C). Ritonavir (10 µM) also stimulated 3H-lopinavir efflux, whereas GF120918 (1 µM) had no effect. Reduced temperature (4°C), verapamil (100 µM) or ritonavir (10 µM) individually did not significantly affect the binding of 3H-lopinavir to cell homogenates. However, slight but significant binding displacement by verapamil at 4°C was observed. 3H lopinavir uptake was not sensitive to verapamil, bromosulfophthalein, taurocholate or to reduced temperature suggesting uptake involves diffusion rather than Organic Anion Transporting Polypeptide transporters. The results suggested that interplay between cellular binding and ABC efflux transporters, in addition to simple diffusion, determines the extent of 3H-lopinavir distribution into BeWo cells.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

July 2009

Share

COinS