Therapeutic targets, novel drugs, and delivery systems for diabetes associated NAFLD and liver fibrosis

Abstract

Type 2 diabetes mellitus (T2DM) associated non-alcoholic fatty liver disease (NAFLD) is the fourth-leading cause of death. Hyperglycemia induces various complications, including nephropathy, cirrhosis and eventually hepatocellular carcinoma (HCC). There are several etiological factors leading to liver disease development, which involve insulin resistance and oxidative stress. Free fatty acid (FFA) accumulation in the liver exerts oxidative and endoplasmic reticulum (ER) stresses. Hepatocyte injury induces release of inflammatory cytokines from Kupffer cells (KCs), which are responsible for activating hepatic stellate cells (HSCs). In this review, we will discuss various molecular targets for treating chronic liver diseases, including homeostasis of FFA, lipid metabolism, and decrease in hepatocyte apoptosis, role of growth factors, and regulation of epithelial-to-mesenchymal transition (EMT) and HSC activation. This review will also critically assess different strategies to enhance drug delivery to different cell types. Targeting nanocarriers to specific liver cell types have the potential to increase efficacy and suppress off-target effects.

Keywords: Cirrhosis; Diabetes; Hepatocellular carcinoma; Inflammation; Liver fibrosis; NAFLD.

Figure 1.. Peroxisome proliferator-activated receptors as targets to treat non-alcoholic fatty liver disease (NAFLD).

Various synthetic PPAR agonists are being evaluated for the treatment of NAFLD. PPAR-α activation leads to fatty acid β-oxidation in the mitochondria. The products can be later converted into ketone bodies (β-hydroxybutyrate or acetoacetate) or can be incorporated into TCA cycle as Acetyl CoA for further oxidation. PPAR- β/δ activation induces FOXO-1 transcription, which reduces hepatic gluconeogenesis and glucose uptake processes. Further, PPAR-β/δ activation suppresses inflammation by reducing IL-1β, IL-6, and NF-κB . Activation of PPAR-γ is linked to decreased hepatocyte lipogenesis and keeps HSCs in quiescent state. Dual or pan agonists targeting two or more of PPARs such as Lanifibranor has shown promising results in pre-clinical and clinical studies.

Figure 2.. Farnesoid X Receptor (FXR) agonists in non-alcoholic hepatitis.

A) The role of FXRs in bile homeostasis and liver disease. B) Various FXR binding agents. CYP7A1 activity affects the overall rate of bile acid synthesis. Hepatic FXR activation reduces CYP7A1 mRNA expression by activating small heterodimer partner (SHP). FXR signaling plays an essential role in the control of hepatic de novo lipogenesis via SHP- mediated inhibition of SREBP-1c. SHP is also expressed in HSCs, and FXR ligands inhibit their activation and collagen synthesis. In enterocytes, FXR through Ileal bile acid-binding protein (IBABP), induces intestinal hormone fibroblast growth factor 19 (FGF19), which activates FGF receptor 4 (FGFR4) signaling in hepatocytes to inhibit CYP7A1 via triggering the extracellular stress-activated receptor kinase 1/2 (ERK1/2) pathway. Obeticholic acid (OCA), a semi-synthetic bile acid analogue of 6α-ethyl-chenodeoxycholic acid (6-ECDCA), is used as a medication for treatment of primary biliary cholangitis. In REGENERATE trial, OCA improved fibrosis in NASH patients.

Figure 3.. Glucose-lowering effects of fibroblast growth factors (FGFs).

FGF21 is secreted by hepatocytes and stimulates glucose uptake in adipocytes while increases insulin secretions by pancreas. FGF21 requires the cofactor, b-Klotho (KLB) for binding its receptor FGFR and activation of the receptor auto-phosphorylation and signaling. In hepatocytes, via SIRT1 and AMPK dependent mechanism, FGF21 induces transcription coactivator, PGC-1α, which increases the mitochondrial activity and enhances oxidative capacity. In white adipose tissue, FGF21 induces the expression of glucose transporter, GLUT1, but prolonged exposure to FGF21 enhances lipolysis as well as increases thermogenesis. FGF21 induces insulin gene expression in islet cells, and this treatment alleviates β-cell dysfunction. A novel FGF21 version, LY2405319 with reduced tumorigenic potential and improved biophysical properties was investigated in Phase 1 clinical trial.

Figure 4.. The role of stearoyl-CoA desaturase 1 (SCD1) in the development of fatty liver diseases.

SCD1 converts saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs). SCD1 maintains a balance between SFAs and MUFAs in the cells. SFAs could be used for β-oxidation and energy production by mitochondria. MUFAs products of SCD1 may be further elongated or incorporated into a variety of complex lipids including triglycerides (TGs), phospholipids, and other lipid species or exported through VLDL mechanism. Excess TG accumulation in the cells leads to steatosis and onset of NAFLD. SCD1 inhibition produces a significant enrichment of SFAs and promotes fatty acid oxidation and increases insulin sensitivity. SCD1 inhibitor Aramchol was tested in Phase 3 clinical trial.

Figure 5.. Hepatocyte apoptosis in NAFLD.

Lipids accumulation in hepatocytes makes them more susceptible to multiple secondary hits such as ROS and LPS that can lead to hepatocyte apoptosis and NASH progression. Apoptosis can occur by two main mechanisms: extrinsic and intrinsic pathways. In the extrinsic pathway, death receptors including Fas, TNFR1, and TNF-related apoptosis-inducing ligand (TRAIL) are activated. These receptors initiate intracellular cascades that activate death-inducing proteolytic enzymes, especially caspases. APO-1/Fas (CD95) -mediated apoptosis is one of the mechanisms for hepatocyte apoptosis. Intrinsic apoptotic pathway is initiated by damage of the intracellular organelles such as mitochondria, lysosomal permeabilization, ER stress, and nuclear DNA damage. Blocking apoptosis pathways may prevent hepatic fibrosis by reducing inflammation. Emricasan a pan-caspase inhibitor show antiapoptotic and anti-inflammatory effects. It was evaluated in a randomized, double-blind Phase 2b clinical trial in patients with decompensated NASH.

Figure 6.. Various vascular adhesion protein (VAP-1) and semi carbazide-sensitive amine oxidase (SSAO) inhibitors for treating NAFLD and fibrosis.

Neutrophil’s activation is a characteristic feature of inflammatory liver disease. Neutrophils can perform a series of functions, including degranulation, reactive oxygen species (ROS) generation, phagocytosis, and the formation of neutrophil extracellular traps (NETs)Upon injury, endothelial cells express P and E selectins, which serve as an anchoring ligands for neutrophils to adhere the endothelium layer. Amine oxidase (AO) are the enzymes that catalyze the oxidation of endogenous amines. One of the isoform AOC-3 also known as VAP-1 and SSAO catalyzes oxidation of primary amines of lymphocytes and enables them to transmigrate through the endothelial cell lining into the underlying parenchyma. In contrast, neutrophils can also inhibit liver injury by phagocytosing necrotic cellular debris and secrete hepatocyte growth factor (HGF) to induce hepatocyte regeneration. To inhibit neutrophil migration via their adhesion process, several small molecules are being developed. PXS-4728 was evaluated under a phase 2a study in patients with NASH. Although the treatment was well tolerated and showed inhibition of AOC3 activity in the plasma, compared to placebo, it was not developed further due to drug-drug interactions.

Figure 7.. Chemokines in the progress of NAFLD.

Chemokines mediate the liver inflammation by controlling the migration of various hepatic and immune cells. The C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their respective ligands (CCL2 and CCL3–5) are involved in the development of NAFLD and NASH. Therefore, CCR2 and CCR5 have been established as promising therapeutic targets for NASH. TAK-779 was the first non peptide CCR5 receptor antagonist. To improve the oral bioavailability, cenicriviroc (CVC) was developed for dual CCR2 and CCR5 inhibition. CVC demonstrated anti-fibrotic effects in the Phase 2b clinical study in adults with NASH and liver fibrosis.

Figure 8.. Adenosine receptor (AR) agonists for treatment of NAFLD.

A) The role of ARs in inflammatory responses in NAFLD. ARs are G protein-coupled receptors (GPCRs) that regulate adenylyl cyclase (AC) activity. A3AR stimulation triggers increase of AC activity and cAMP production. cAMP activates protein kinase B (PKB), which is a negative regulator of inflammatory NF-kB pathway and glycogen synthase kinase 3 beta (GSK-3β) mediated cell apoptosis. B) There are various AR inhibitors. Namodenoson, a potent and selective A3AR agonist was evaluated in Phase 2 trials for the treatment of NAFLD and NASH. The drug successfully met the endpoints with 60 patients and the optimal dosage. The Phase IIb NASH trial of namodenoson is currently underway.

Figure 9.. Role of transforming growth factor beta 1 (TGF-β1) in progression of liver fibrosis.

Kupffer cells (KCs), monocytes, and injured hepatocytes release TGF-β, the main cytokine in fibrosis, which causes the activation of HSCs and more accumulation of ECM protein in the liver. In the hepatocytes, TGF-β promote accumulation of lipids and promote steatosis.

Figure 10.. Drug delivery systems used for treating liver fibrosis.

Cell-specific receptors could be used for targeted delivery of nanomedicine for fibrosis/cirrhosis. (B) Ligand decoration on nanocarries facilitate receptor binding and uptake of compounds resulting in target cell-specific actions.