Drug-Target Residence Time Affects in Vivo Target Occupancy through Multiple Pathways

Abstract

The drug discovery and development process is greatly hampered by difficulties in translating in vitro potency to in vivo efficacy. Recent studies suggest that the long-neglected drug-target residence time parameter complements classical drug affinity parameters (K _I, K _d, IC₅₀, or EC₅₀) and is a better predictor of in vivo efficacy. Compounds with a long drug-target residence time are often more efficacious in vivo. The impact, however, of the drug-target residence time on in vivo efficacy remains controversial due to difficulties in experimentally determining the in vivo target occupancy during drug treatment. To tackle this problem, an in vivo displacement assay was developed using soluble epoxide hydrolase as a biological model. In this report, we experimentally demonstrated that drug-target residence time affects the duration of in vivo drug-target binding. In addition, the drug-target residence time plays an important role in modulating the rate of drug metabolism which also affects the efficacy of the drug.

Figure 1

Schematic diagram of the in vivo displacement assay. (a) Step 1, inhibitor A binds to the target enzyme after administration. (b) Step 2, the administrated inhibitor A in vivo is metabolized and/or excreted over time. (c) Step 3, high dose of displacement inhibitor B is administrated. The bound inhibitor A is competed and displaced by high concentration of inhibitor B. Inhibitor A is released to the blood and can be monitored by LC/MSMS. (d) Step 4, the expected PK profile of the in vivo displacement assay. The first peak in the PK profile corresponds to the blood concentration of inhibitor A after inhibitor A administration. The second peak of the PK diagram is hypothesized as the bound inhibitor A displaced by a high dose of inhibitor B. The area-under-the-curve (AUC) of the second peak reflects the amount of soluble epoxide hydrolase bound inhibitor A in vivo.

Figure 2

(a) The in vivo displacement assay between the WT mice (black line with solid circle, n = 6) and sEH KO mice (orange line with open circle, n = 6) of TPPU (0.3 mg/kg in PEG400, 100–110 μL based on the weight of the mouse, subcutaneous injection) indicates that the second displacement peak of TPPU in WT mice after administration of a high dose of the potent displacement sEH inhibitor (TCPU, 3 mg/kg in PEG400, 100–110 μL based on the weight of the mouse, subcutaneous injection) at 168 h, is the sEH-bound TPPU. For the structure of TPPU and TCPU, please refer to Table 1. (b) The result from the in vivo displacement assay in rat where the TCU (0.1 mg/kg in PEG400/olelate rich safflower oil/1:4, 1 mL, oral gavage) was displaced by a high dose of the weak inhibitor, DFPU (green line with open circle, n = 4, 3 mg/kg in PEG400/olelate rich safflower oil/1:4, 1 mL, oral gavage), or a high dose of potent inhibitor, TIPU (black line with solid circle, n = 4, 3 mg/kg, PEG400/olelate rich safflower oil/1:4, 1 mL, oral gavage), at 168 h indicates that the second displacement peak of TCU in rats is the sEH-bound TCU. For the structure of TCU, TIPU, and DFPU, please refer to Table S2. Panels (a, b) indicate that the in vivo displacement assay can be applied to other species. (c) TPPU level at postdosing day 7 correlated well with specific sEH activity in different tissues in WT mice. Please see Figure S15 for the relationship between the TPPU tissue level and sEH activity in different tissues in WT mice. R² was calculated based on the datum point close to the fitted line (black) except for thigh muscle. (d) The tissue level of TPPU at postdosing day 7 in different organs in WT mouse and sEH KO mouse (n = 6 per group). Unlike the WT mouse, there was no accumulation of TPPU in the sEH KO mouse. Please see Figure S16 for the tissue level of TPPU at postdosing day 7 in different organs in the Sprague–Dawley rat. The data are mean ± standard error of the mean (SEM).

Figure 3

t_R of the inhibitors impact the AUC of the second displacement peak (referenced to the displaced sEH-bound inhibitor) at day 7. (a) The AUC of the second displacement peak (the tested inhibitor displaced by a high dose of TCPU) changed with the t_R of the tested inhibitor in the in vivodisplacement assay. The AUC of second peak correlates well with inhibitors t_R except APAU which has a much shorter PK-T_1/2. The experimental protocol is detailed in Supporting Information. The data are mean ± SEM. (b) After normalization of the second peak AUC with PK-T_1/2 of the same inhibitor, the normalized AUC of the second peak correlates well (R² = 0.962) with the t_R of the inhibitors.

Figure 4

Drug-target residence time (t_R) modulates the pharmacokinetic profile of the inhibitors. The inhibitors with longer t_R bind to the sEH longer as compared to inhibitors with shorter t_R, therefore protecting the inhibitors from being metabolized and/or eliminated from the body. (a) TPPU, which has a long t_R (19.8 min), was administrated to both WT and sEH KO mice at the same dose (subcutaneous injection, 0.3 mg/kg). The PK profile of TPPU in WT mice (PK-T1/2 is 15 h) is very different from one in sEH KO mice (PK-T1/2 is 9 h) after 48 h. The TPPU is fully eliminated in sEH KO mouse within 72 h, while the elimination rate of TPPU is much slower in WT mice and the blood level of TPPU stayed above the K_i (2.5 nM) even at postdosing day 14. (b) TPAU, which has a short t_R (6.0 min), was administrated to both WT and sEH KO mice at the same dose (1 mg/kg). Unlike TPPU, the PK profiles of TPAU between WT (PK-T1/2 is 8.75 h) and sEH KO mice (PK-T1/2 is 8.35 h) is relatively similar to TPAU and is fully eliminated in WT mice within 168 h, while TPAU is fully eliminated in sEH KO mice within 144 h. These data suggest that t_R of the inhibitors affects its PK profiles, and inhibitors with a long t_R will have a slower PK elimination rate. Please refer to Figure S17 for a full PK profile of TPPU and TPAU in WT and sEH KO mice. The data are mean ± SEM.