Development of the Analytical Methodology for Pyrimethamine and Its Application to Studies of Partitioning and Binding in the Subcompartments of Blood

Author

Anita C. RudyFollow

DOI

https://doi.org/10.25772/F7Y2-YZ34

Defense Date

1987

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmaceutical Sciences

First Advisor

Wesley J. Poynor

Abstract

An original HPLC assay was developed for pyrimethamine (PYR) in plasma, RBCs, and buffer for the purpose of studying its plasma protein binding and RBC partitioning.

Equilibrium dialysis (ED) was used to study protein binding. Isotonic phosphate buffer used in ED did not prevent small volume shifts. The pH of the plasma affected the protein binding of PYR although it was not significant for the comparison of binding at pH 7.4 vs. 8.0. PYR at 1000 ng/ml averaged 93.1% bound to plasma proteins. Binding to pure human albumin was 86.5% at lower levels of albumin and PYR (350 ng/ml). There was a significant difference (p<.03) in the plasma binding at two levels in the therapeutic range, with more free at higher levels. There was also concentration dependent binding at higher concentrations; the drug did not follow to law of mass action when binding increased at higher concentrations. This is a solubility phenomenon. Linear regression at the effect at albumin concentration on plasma binding yielded the equation percent free = -0.467(albumin g/L) + 23.5. The binding to pure albumin was only slightly above that predicted by this equation (83.1%). The first and second stoichiometric binding constants are K₁ = 2.83 x 10⁴ and K₂ = 1.74 x 10⁴ M^-1 from nonlinear regression at data. There was no binding to normal levels at α₁-acid glycoprotein.

PYR is preferentially bound to plasma proteins in comparison to RBCss. The mean RBC/plasma ratio was 0.42 (10.2% CV, n=5). when plasma was removed and pH 7.4 isotonic buffer substituted, mean RBC/buffer ratio was 5.2 (11.8% CV, n=2). Mean percent bound to hemolysate was 42.5% (19% CV, n=10). Binding to hemoglobin did not account for all the RBC uptake. Therefore, PYR binds to RBC membranes.

Comments

Scanned, with permission from the author, from the original print version, which resides in University Archives.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-1-2017