Cell experiment:

Cell monolayers that exceeds a resistance of 300 Ω cm-2 are incubated with either 1 μM or 10 μM of TPPU solution in DMSO on the apical side. Medium samples on the apical and basolateral side are collected and frozen immediately after 1, 3 and 6 hours. The apparent permeability coefficient is calculated for t=1 h[2].

Animal experiment:

Rats: Water is provided ad libitum containing 0.2% PEG400 with and without TPPU at a concentration of 0.2 mg/L, 1 mg/L and 5 mg/L during the treatment period (8 days). Before (0 h) and after 2 h, 4 h, 8 h, 1 d, 2 d, 4 d and 8 d, 10 μL blood are sampled from the tail vain. The blood is directly mixed with 50 μL deionzed water28 and frozen until analysis. On day 8, the animals are sacrificed by cardiac puncture after anesthesia. Plasma and whole blood are sampled[2]. Monkeys: TPPU is prepared in 0.3, 1 and 3 mg/kg doses and is administered to four animals with 48 h dosing intervals based on the t1/2 of the compound obtained from the first cassette dosing. Blood is collected from animals at time points 0, 0.25, 0.5, 1, 2, 3, 4, 8, 24 and 48 h after each dosing for analysis[1].

IC50: human and mouse sEH (IC50 = 3.7 and 2.8 nM, respectively)

TPPU is a potent inhibitor of both human and mouse sEH.

The soluble epoxide hydrolase (sEH) can convert epoxides to the corresponding diols by the catalytic addition of a water molecule. sEH is implicated in various disease states for its ability to metabolize fatty acid epoxides such as epoxyeicosatrienoic acids and leukotoxin, important endogenous signaling lipids, to less active dihydroxyeicosatrienoic acids and toxic, proinflammatory leukotoxin diols, respectively. sEH inhibitors are of growing interest for therapeutic use since they have been shown to increase the in vivo concentration of EETs and other fatty acid epoxides.

In vitro: TPPU was identified as a potent inhibitor of both human and mouse sEH with IC50 of 3.7 and 2.8 nM, respectively [1].

In vivo: Animal study found that oral administration of 13 1-aryl-3-(1-acylpiperidin-4-yl)urea inhibitors including TPPU in mice showed substantial improvements in pharmacokinetic parameters over previously reported 1-adamantylurea based inhibitors. For example, 1-(1-(cyclopropanecarbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea, a close analog of TPPU, had a 7-fold increase in potency, a 65-fold increase in Cmax, and a 3300-fold increase in AUC over its adamantine analogue 1-(1-adamantyl)-3-(1-propionylpiperidin-4-yl)urea. This sEH inhibitor displayed a 1000-fold increase in potency when compared to morphine by reducing hyperalgesia using the in vivo carrageenan induced inflammatory pain model [1].

Clinical trial: So far, no clinical study has been conducted.

Reference:
[1] Rose, T. E.,Morisseau, C.,Liu, J.Y., et al. 1-aryl-3-(1-acylpiperidin-4-yl)urea inhibitors of human and murine soluble epoxide hydrolase: Structure-activity relationships, pharmacokinetics, and reduction of inflammatory pain. J Med Chem. 2010 Oct 14;53(19):7067-75.