HUMAN CLINICAL TRIALS EVALUATING PROTEIN DISULFIDE ISOMERASE AS AN ANTITHROMBOTIC
TARGET: PHARMACODYNAMIC AND PHARMACOKINETIC STUDIES OF ORAL QUERCETIN AND
ISOQUERCETIN.
Authors: Jack Stopa, Bruce Furie, Robert Flaumenhaft, Jeffrey Zwicker
Background:
Protein disulfide isomerase (PDI) regulates both platelet accumulation and fibrin generation during thrombus formation following vascular injury in animal models. A high-throughput drug screen previously identified the flavonoid quercetin-3-rutinoside (and related analogs) as potent inhibitors of
PDI activity in vitro and in vivo.
Aims:
In anticipation of clinical trials to evaluate PDI as a novel antithrombotic target, we performed a pharmacokinetic study to assess whether oral quercetin or isoquercetin achieves therapeutic concentrations and pharmacodynamic studies to measure PDI inhibitory activity.
Methods:
A total of 10 healthy volunteers were enrolled and received either quercetin 500 mg or isoquercetin 500 mg as a single dose. Serial phlebotomies were performed at 0 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. Total quercetin (aglycone) in plasma was measured by HPLC following enzymatic hydrolysis. In order to assess the pharmacodynamic inhibition of PDI following oral administration of the flavonoids, we developed a plasma-based assay utilizing a fluorescence-based eosin probe coupled to glutathione, with relative fluorescence measured in the presence of PDI.
Results:
Isoquercetin demonstrated improved bioavailability (maximal concentration was 3.45

M 95% CI 0.52-
6.39

M, Tmax 2.6 hours, half-life 9.14 hours, AUC 17.54) compared with quercetin aglycone (Cmax 0.77

M 95% CI 0.4 -1.12

M, Tmax 3.8 hours, half-life 10.79 hours, AUC 5.14). The formulation of isoquercetin or quercetin with ascorbic acid (500 mg) to prevent oxidation did not alter pharmacokinetic profiles. Significant PDI inhibitory activity in plasma was detected following the ingestion of isoquercetin

but not quercetin. The PDI inhibitory activity in plasma increased significantly by 4 hours following the oral administration of isoquercetin (P=0.001) with a mean difference of 59% relative to baseline. By 6 hours, the mean PDI inhibitory activity neared baseline values (mean difference of 16% compared to baseline).
Conclusions:
These studies provide proof-of-principle for the use of quercetins to inhibit extracellular PDI in humans and represent an important first step in the development of a novel class of antithrombotic agents. We conclude that oral isoquercetin has improved bioavailability compared with quercetin in healthy adults and results in detectable PDI inhibition as measured by a novel plasma-based PDI assay. The antithrombotic efficacy of isoquercetin will be evaluated in cancer-associated thrombosis for high risk cancer populations and in individuals with antiphospholipid antibodies.