Background: Potential changes in free drug concentrations of antiretrovirals may alter the clinical effects of these drugs as it is the free drug fraction that is pharmacologically active. Therefore, the binding characteristics of nelfinavir and its active metabolite, M8, to human plasma proteins and the protein binding effects of concomitantly administered drugs that bind extensively to plasma proteins was investigated.

Methods: Free fractions of nelfinavir and M8 were separated by equilibrium dialysis and the unbound concentrations of nelfinavir and M8 were determined by a validated liquid chromatography/tandem mass spectroscopy method. Association constants were estimated using double reciprocal plots of the data. For interaction studies, various concentrations of drugs were added to α-1-acid glycoprotein (AAG) and serum albumin (HAS) solutions and plasma.

Results: The free fraction of nelfinavir and M8 in plasma averaged 0.42 ± 0.08 and 0.64 ± 0.07%, respectively. Association constants of nelfinavir and M8 were 7.25 X 10⁷ M¹ and 3.33 X 10⁷ M^-1 for AAG and 1.11 X 10⁶ M^-1 and 7.92 X 10⁵ M^-1 for HSA, respectively, indicating that these drugs bind extensively to both of these proteins. When ritonavir and saquinavir, drugs that bind extensively to AAG, were added to a solution of AAG, the free fraction of nelfinavir was significantly higher than that of control measuring 5.23 ± 1.27 and 1.18 ± 0.21% for ritonavir (10-μg/mL) and control respectively (p<0.01) and 2.55 ± 0.27 and 0.99 ± 0.16% for saquinavir (10-μg/mL) and control respectively (p<0.01). In contrast, the free fraction was not altered when ritonavir or saquinavir were added to whole plasma (p>0.05). Similarly, when salicylic acid or valproic acid, drugs that bind extensively to HSA, were added to a solution of HSA, there were significant differences compared with controls, whereas there was no difference in the free fraction when these drugs were added to plasma.

Conclusions: These in vitro results indicate that the protein binding of nelfinavir, a compound that binds extensively to both AAG and albumin, is not affected by concomitantly administered drugs exhibiting high protein binding. This is likely due to compensation of the alternate protein to bind up nelfinavir when the drug is displaced from AAG or albumin. These results suggest that free drug concentrations do not fluctuate due to drug interactions.