Binding of gefitinib, an inhibitor of epidermal growth factor receptor-tyrosine kinase, to plasma proteins and blood cells: in vitro and in cancer patients

Summary Gefitinib exhibits wide inter-subject pharmacokinetic variability which may contribute to differences in treatment outcome. Unbound drug concentrations are believed to be more relevant to pharmacological and toxicological responses than total drug. Thus it is desirable to determine gefitinib binding in plasma and factors affecting this process. An equilibrium dialysis method using 96-well microdialysis plates was optimized and validated for determining the fraction unbound (fu) gefitinib in human plasma. Gefitinib binding in plasma from four different species and isolated protein solutions as well as drug partitioning in human blood cells were investigated. Unbound gefitinib plasma concentrations were measured in 21 cancer patients receiving daily oral gefitinib 250 mg or 500 mg. It was found that gefitinib was extensively bound in human rat mouse and dog plasma with mean fu values of 3.4%, 3.8%, 5.1% and 6.0% respectively. In isolated protein solutions approximately 90% and 78% of gefitinib was bound to human serum albumin (HSA) (40 mg/dL) and alpha1-acid glycoprotein (AAG) (1.4 mg/dL) with binding constants of 1.85 × 10⁴ M⁻¹ and 1.13 × 10⁵ M⁻¹ respectively. In whole blood 2.8% of gefitinib existed as the free drug while 79.4% and 17.8% was bound to plasma proteins and blood cells respectively. In plasma from cancer patients fu at pre-treatment varied 2.4-fold (mean 3.4 ± 0.6%; range 2.2–5.4%) and fu was constant over the 28-days of treatment (P > 0.05). Pre-treatment AAG concentration was negatively correlated with pre-treatment fu (R² = 0.28, P = 0.01). In conclusion gefitinib is highly protein bound (∼ 97%) in human plasma. Variable AAG concentrations observed in cancer patients may affect gefitinib fu with implications for inter-subject variation in drug toxicity and response.