(T0930-02-12) Nanoparticle Delivery of Novel PDE4B Inhibitor for the Treatment of Alcoholic Liver Disease

Purpose: This study aims to develop a mPEG-b-P(CB-co-LA) nanoparticles (NPs) based delivery system and determine the efficacy of KVA-D88 loaded NPs formulation for alcoholic liver disease (ALD) treatment. ALD is a worldwide health issue currently only being treated by abstinence and nutrition support. Therefore, it is necessary to develop new therapeutics for treating ALD. Studies have revealed that inhibition of phosphodiesterase 4 (PDE4) is beneficial to ALD treatment in animal studies. However, the application of PDE4 inhibitors is limited due to the gastrointestinal adverse effects caused by PDE4D inhibition in the central nervous system. Therefore, we utilized a novel selective PDE4B inhibitor, KVA-D88, to treat ALD. To improve the poor aqueous solubility of KVA-D88, we employed mPEG-b-P(CB-co-LA) formed NPs as a drug carrier for the treatment of ALD in the mouse model.

Methods: Nanoparticles Formulation Preparation: NPs were prepared using mPEG-b-P(CB-co-LA) by solvent evaporation. KVA-D88 and mPEG-b-P(CB-co-LA) were dissolved in the mixture of dichloromethane and acetone, and 1% polyvinyl alcohol (PVA) was added. The solution was emulsified by a probe sonicator, the organic solvent was removed by evaporation, and the solution was centrifuged at 5000 rpm to remove the unencapsulated drug. The particle size distribution was measured by Zetasizer. For in vivo studies, the supernatant was further centrifuged at 20,000 rpm and the NPs were resuspended in PBS. For the drug release study, the formulation was dialyzed against 50 mL PBS containing 1% Tween 80 for sink condition at 37 ◦C, 120 rpm. KVA-D88 concentration was determined by HPLC. In Vivo Efficacy of KVA-D88 Loaded NPs Formulation: C57BL/6 mice were used to build the ALD model by feeding them with 5% (v/v) ethanol diet for 6 weeks. Mice were divided into four groups: control, EtOH feeding, KVA-D88, and KVA-D88 NPs. The control group was fed with the control diet and other groups were fed with ethanol diet. The treatment groups were intravenously injected with KVA-D88 free drug or NPs (5 mg/kg, twice a week) starting in the third week. At the end of week 6, treatment and control groups were given an oral gavage of 5 g/kg EtOH and 45% isocaloric maltose dextrin, respectively. All groups were sacrificed 9 h after the oral gavage. Mouse blood and major organs were harvested for analysis. Plasma aspartate transaminase (AST), alanine aminotransferase (ALT), and hepatic triglyceride (TG) levels were determined by commercially available kits. Relative gene expression levels in mouse liver tissues were determined by real time RT-PCT. Hematoxylin and eosin (H&E), Sirius red, oil red O, and immunohistochemical (IHC) staining were also carried out.

Results: The mean particle size of NPs was 135 ± 10 nm with the PDI of 0.146, and the drug loading was 4.2% w/w. NPs provided a sustained drug release profile with a burst release of 15% in the first 3 h and a sustained release of 60% after 48 h, followed by a slower and continuous release till 120 h. Chronic alcohol intake leads to various liver injuries, including inflammation, steatosis, oxidative stress, and fibrosis. KVA-D88 loaded NPs markedly decreased ALT and AST plasma levels compared to EtOH feeding. H&E staining showed that KVA-D88 loaded NPs ameliorated liver injury caused by EtOH as indicated by improved hepatic architecture and fewer lipid droplets accumulation. Systemic administration of KVA-D88 loaded NPs showed a pronounced anti-inflammatory effect as demonstrated by a significant decrease in TNF-α and IL-1β mRNA expression. KVA-D88 loaded NPs dramatically reduced hepatic lipid accumulation to the level of the control group as confirmed by Oil red O staining and hepatic TG level. Chronic alcohol intake leads to steatosis by suppressing fatty acid β-oxidation related genes, PPAR-α, PGC-1α, CPT1A, and ACOX1. The downregulation of PPAR-α, PGC-1α, CPT-1A, and ACOX mediated by alcohol was remarkably reversed by KVA-D88 loaded NPs. KVA-D88 loaded NPs ameliorated oxidative stress as indicated by increased anti-oxidative enzyme SOD1 and SOD2 levels and decreased lipid peroxidation product 4-HNE. Sirius red staining showed that KVA-D88 loaded NPs improved fibrosis induced by chronic alcohol consumption.

Conclusion: We have utilized a novel PDE4B inhibitor KVA-D88 loaded NPs as a therapeutic agent for ALD. The nanoparticle delivery system showed sustained drug release profiles. Delivery of KVA-D88 loaded NPs significantly improved alcohol-induced hepatic injury, inflammation, steatosis, oxidative stress, and fibrosis in mice. In conclusion, our results support that KVA-D88 loaded NPs are promising for ALD treatment and offer a new strategy for ALD therapy.

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Acknowledgements: University of Nebraska Medical Center Faculty Startup fund to Ram Mahato is duly acknowledged for providing financial support for this work. This work was also financially supported by the National Institute of Health (NIH) Grant (1K01AA029763-01) to Virender Kumar.

Figure1. Preparation, characterization, and in vivo efficacy of KVA-D88 loaded nanoparticles. (A) Synthesis of mPEGb-P(CB-co-LA). (B) Particle size distribution of KVA-D88 loaded nanoparticles. (C) Release profile of KVA-D88 loaded nanoparticles in PBS. (D) Representative microscopic pictures of H&E staining of mice liver tissue. Scale bars (20×), 200 µm. (E, F) Mice plasma AST and ALT levels. (G, H) Mice hepatic mRNA expression level of TNF-α and IL-1β. Results are presented as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.005.

Figure 2. KVA-D88 loaded nanoparticles alleviate alcohol-induced steatosis and promote lipolysis-related gene expression in the liver after systemic administration into mice. (A) Representative microscopic pictures of Oil red O staining of mice liver tissue. (B) Mice hepatic triglyceride (TG), also known as triacylglycerol (TAG) level. Mice hepatic mRNA expression levels of PPAR-α (C), CPT-1A (D), PGC-1α (E), and ACOX1 (F). Scale bars (20×), 200 μm. Results are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.005.

Figure 3. KVA-D88 loaded nanoparticles alleviate alcohol-induced oxidative stress and fibrosis after systemic administration into mice. (A, B) Mice hepatic mRNA expression levels of SOD1 and SOD2. (C) Representative microscopic pictures of IHC staining for 4-HNE. (D) Representative microscopic pictures of Sirius red staining of mice liver tissue. Scale bars (20×), 200 μm. Results are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.005.