Implications of plasma protein binding for pharmacokinetics and pharmacodynamics of the γ-secretase inhibitor RO4929097

Abstract

Purpose: Understanding of plasma protein binding will provide mechanistic insights into drug interactions or unusual pharmacokinetic properties. This study investigated RO4929097 binding in plasma and its implications for the pharmacokinetics and pharmacodynamics of this compound.

Experimental design: RO4929097 binding to plasma proteins was determined using a validated equilibrium dialysis method. Pharmacokinetics of total and unbound RO4929097 was evaluated in eight patients with breast cancer receiving RO4929097 alone and in combination with the Hedgehog inhibitor GDC-0449. The impact of protein binding on RO4929097 pharmacodynamics was assessed using an in vitro Notch cellular assay.

Results: RO4929097 was extensively bound in human plasma, with the total binding constant of 1.0 × 10(6) and 1.8 × 10(4) L/mol for α1-acid glycoprotein (AAG) and albumin, respectively. GDC-0449 competitively inhibited RO4929097 binding to AAG. In patients, RO4929097 fraction unbound (Fu) exhibited large intra- and interindividual variability; GDC-0449 increased RO4929097 Fu by an average of 3.7-fold. Concomitant GDC-0449 significantly decreased total (but not unbound) RO4929097 exposure. RO4929097 Fu was strongly correlated with the total drug exposure. Binding to AAG abrogated RO4929097 in vitro Notch-inhibitory activity.

Conclusions: RO4929097 is highly bound in human plasma with high affinity to AAG. Changes in plasma protein binding caused by concomitant drug (e.g., GDC-0449) or disease states (e.g., ↑AAG level in cancer) can alter total (but not unbound) RO4929097 exposure. Unbound RO4929097 is pharmacologically active. Monitoring of unbound RO4929097 plasma concentration is recommended to avoid misleading conclusions on the basis of the total drug levels.

Figure 1

RO4929097 binding to plasma proteins HSA and AAG. A and B, RO4929097 fraction unbound (Fu) as a function of HSA (5–50 mg/mL) and AAG (0.2–3.2 mg/mL) concentration. C and D, modified Scatchard plots of the observed unbound versus bound drug concentration as the total drug concentration ranging from 20 to 10,000 ng/mL (43–21,268 nmol/L) in isolated HSA (40 mg/mL or 615.4 μmol/L; C) and AAG (1.4 mg/mL or 31.8 μmol/L; D) solution. E and F, simulated RO4929097 Fu in plasma with varying clinically relevant AAG concentrations (i.e., 0.2, 1.4, 3.2 mg/mL) and a fixed HSA concentration (40 mg/mL) or with varying clinically relevant HSA concentrations (i.e., 20, 40, 50 mg/mL) and a fixed AAG concentration (1.4 mg/mL), using a 2-binding site model [Equation (D)].

Figure 2

Effect of GDC-0449 on RO4929097 binding to AAG in isolated AAG solution (1.4 mg/mL or 31.8 μmol/L). A, modified Scatchard plots of the observed unbound versus bound RO4929097 concentration and (B) RO4929097 fraction unbound (Fu), as the total drug concentration ranging from 43 to 21,304 nmol/L in AAG solution in the presence of 0 (■), 5 (◯), 25 (▲), and 125 (□) μmol/L of GDC-0449.

Figure 3

Observed RO4929097 total and unbound plasma concentration versus time profiles and the unbound fraction (Fu) as a function of time in individual patients receiving oral administration of RO4929097 20 mg alone (cycle 1) and in combination with oral administration of GDC-0449 150 mg (cycle 2). A and B, individual observed total RO4929097 plasma concentration–time profiles. C and D, individual observed unbound RO4929097 plasma concentration–time profiles. E and F, individual observed RO4929097 fraction unbound as a function of time. The different symbols in all graphs consistently represent individual patients.

Figure 4

Correlations between RO4929097 unbound fraction (Fu) and the pharmacokinetic parameters (C_max, AUC_0–24h, or CL/F) of total (A–C) and unbound (D–F) RO4929097 in patients receiving RO4929097 20 mg orally alone or in combination with GDC-0449 150 mg orally. Bivariate correlations were examined with the Spearman rank correlation test; ρ is the Spearman rank correlation coefficient; correlation is considered significant at P < 0.01 (2-tailed).

Figure 5

Impact of protein binding on in vitro Notch-inhibitory activity of RO4929097. A, the Notch-inhibitory activity of RO4929097 was expressed as the percentage of normalized luciferase activity in the treated cells relative to that in the untreated cells. Human U2OS cells were transiently cotransfected with a constitutively active NOTCH1 mutant construct and HES1-Luc reporter construct. The cells were treated with RO4929097 at 50, 500, and 5,000 nmol/L in the absence or presence of 0.5, 1.4, and 3.2 mg/mL AAG for 24 hours. The bars represent the mean ± SD from 2 independent experiments, with duplicate in each experiment. B, RO4929097 in vitro Notch-inhibitory activity as a function of the estimated unbound drug concentration was described by the inhibitory E_max model with an estimated IC₅₀ value of 109 nmol/L. The observed values are shown as ●, and the solid line represents the model-fitted curve. *, significantly different than RO4929097 treatment in the absence of AAG.