Development of a Liver‐Targeted Drug Delivery Platform for Soluble Epoxide Hydrolase Inhibitors
Authors: Warner Jeffrey , Hardesty Josiah , Song Ying , Bauer Philip , Soni Chirag , Maldonado Claudio , McClain Craig , Warner Dennis , Kirpich Irina 
Published: May 2022

Background

Many drugs administered systemically suffer from drawbacks including off‐target effects. One approach to address this limitation is tissue‐ or cell‐targeted drug delivery, which can also improve therapeutic efficacy by increasing the “effective” dose. The goal of the current study was to develop a system to target a soluble epoxide hydrolase (sEH) inhibitor to the liver, since previous studies from our group and others demonstrated that elevated hepatic sEH contributes to the pathogenesis of liver diseases, including alcohol‐associated liver disease (ALD). We tested the hypothesis that encapsulation of an sEH inhibitor in fusogenic lipid vesicles (FLVs) would enable liver targeting and ameliorate liver injury in a mouse model of ALD.

Methods

FLVs were constructed with unsaturated lipids 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphate at a 3:2 molar ratio. FLVs were loaded with the sEH inhibitor t‐TUCB at doses ranging from 0‐9 mg/kg, then tagged with the fluorescent tracer DiD. Physical properties including size and zeta potential, as well as encapsulation efficiency were measured using a nanoparticle tracker analyzer and spectroscopy, respectively. To test the efficacy of t‐TUCB‐FLVs in vivo, we used a chronic‐binge mouse model of ALD. Specifically, male C57BL/6 mice were fed a liquid diet containing ethanol for ten days followed by a single ethanol binge nine hours prior to sacrifice. t‐TUCB‐FLVs were administered by intraperitoneal injection two hours prior to the ethanol binge. We measured endpoints related to liver injury, steatosis, inflammation, cell death, and endoplasmic reticulum (ER) stress. One‐way ANOVA was used to determine significance between groups (α = 0.05).

Results

Analysis of size, zeta potential, and encapsulation efficiency showed favorable physical characteristics for passively targeting t‐TUCB‐FLVs to the liver (129nm diameter, ‐55mV zeta potential, and 92% encapsulation efficiency). In a pilot dose response study, the presence of t‐TUCB‐FLVs in the liver was confirmed by fluorescent imaging and magnetic cell sorting followed by flow cytometry. In an experimental animal model, mice which received ethanol and 3.0 mg/kg t‐TUCB‐FLVs (observed to be the most effective dose) had significantly lower liver injury by plasma ALT levels compared to mice receiving ethanol and empty liposomes alone (47.75 U/L vs. 72.93 U/L, respectively). t‐TUCB‐FLV‐treated mice also had decreased liver parenchymal cell death (by TUNEL staining) and markers of ER stress (Atf4, Atf6, and spliced Xbp1), with no significant effects on steatosis.

Conclusions

We successfully developed a passive liver targeting system to deliver an sEH inhibitor to the liver in mice. In an ALD model, this drug formulation attenuated liver injury, cell death, and ER stress. This novel approach may help avoid clinical pitfalls which cause experimental drugs to fail in clinical trials, such as low efficacy or off‐target side effects. Future research will further evaluate the mechanisms of t‐TUCB‐FLVs in ALD, evaluate potential side effects, and consider an active targeting approach.