Plasma protein binding (PPB) of drugs is a rapidly reversible and highly variable process that can change with species, disease states and/or age. According to the free drug hypothesis, only free, unbound drug – drug which has not bound to proteins and/or lipids in plasma and tissue – can permeate through membranes in the body. Additionally, if there is no active transport mechanism, the drug is assumed to be at steady state, meaning the free drug concentration is believed to be equal on both sides of the membrane and the unbound drug at the target site exerts the pharmacological effect. Because generally only unbound drug is permeable though membranes, PPB impacts the pharmacokinetics and distribution, as well as the resultant exposure and clearance processes.
There are two major plasma proteins that account for the majority of PPB; albumin and α1-acid glycoprotein (AAG). Albumin accounts for approximately 60% of plasma protein. It binds to acidic xenobiotics primarily, but can also bind to basic and neutral compounds. AAG comprises 1-3% of total plasma protein and predominantly binds to basic drugs. The binding potential that AAG exhibits can be disrupted by certain plasticisers such as Tris (2-butoxyethyl) phosphate (TBEP), which can be found in laboratory equipment and must be avoided during sample collection for PPB assays.
In this study, performed in conjunction with Epizyme, human plasma from healthy volunteers was used to establish the plasma protein binding potential of six compounds using equilibrium dialysis. The blood samples, from which the plasma was prepared, were collected into vessels made from one of two materials, either lithium heparin tubes made from polyethylene terephthalate which did not contain TBEP plasticisers or bags which contained polyvinylchloride with DEHP plasticisers but not latex. Compounds that were known albumin binders, such as testosterone, showed no difference in binding based on container material. However, compounds that bind to AAG showed clear differences in the fraction unbound (fu) depending on the type of plasticisers in sample collection materials.
This study highlights that the blood source and collection method is critical to the accurate determination of plasma protein binding in vitro.
This poster was presented at the 20th North American ISSX Meeting. Download a copy.
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