The effect of protein binding on antiretroviral drug distribution and antiviral effect among diverse anatomic compartments.

摘要

This thesis demonstrates the impact of protein binding on antiretroviral drug distribution and efficacy. Antiretroviral drugs (ARVs) are highly effective in treating HIV infection through suppression of viral replication. For many ARVs, however, there is a lack of understanding of drug distribution beyond the blood plasma and into other extravascular compartments. This greatly limits our ability to understand drug toxicity and efficacy within anatomic compartments.;It has been widely demonstrated that ARVs do not adequately penetrate the male genital tract (MGT) raising concern it may be a pharmacological sanctuary in which ARVs have no effect on HIV. Protein binding was examined as an explanation for the large gradient reported in total EFV concentration between blood and seminal plasma. A highly sensitive ultra performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) method was developed for the detection of very low concentrations of protein-free EFV, and a novel ultrafiltration method for the separation of protein-free EFV from protein-bound EFV. Using these techniques, it has been demonstrated that the protein-free EFV concentration is in equilibrium in the blood plasma and seminal plasma. Since most potent HIV drugs are similarly highly protein bound, this negates prior concerns of the MGT as a pharmacological sanctuary from ARVs.;To confirm the functional impact of these findings, it was vital to prove that only free, not total, ARV concentrations are responsible for ARV effect. Since protein binding influences the distribution of many ARV drugs, it should also impact local ARV efficacy. To explore the relationship between ARVs, protein binding, and the impact on infectivity, a single-round infectivity assay using an HIV-GFP reporter virus was used to infect the cell of interest, CD4+ T cells. Increasing extracellular protein correlates with both decreased extracellular protein-free EFV concentration and decreasing in intracellular drug penetration, which results in increased HIV infection. This concept was generalizable to additional ARVs, of varying protein binding and mechanism of action.;To further generalize the usefulness of these protein binding results to another anatomic drug sanctuary, protein-free EFV concentrations were described in the cerebrospinal fluid (CSF), a significant target of HIV associated disease. Through an application of the law of mass action, established from the prior description of the distribution of EFV into the MGT, the protein binding of EFV was mathematically predicted and subsequently confirmed by direct observation within CSF. This method will ultimately provide a predictive tool for protein binding within additional sub-compartments. Combined with our infectivity models, we may also provide estimates of ARV effect in the distant compartment.;In conclusion, new methodologies have been established for the quantitation of protein-free drug within anatomic compartments. This thesis has applied these novel methods to demonstrate the impact of protein binding on local ARV distribution and efficacy, and provided a tool for the prediction of protein binding in an extravascular compartment. Combined, these methodologies provide a greater understanding on the impact of protein binding on ARV pharmacokinetics and pharmacodynamics in diverse and clinically relevant compartments.