NAFLD is related to Post-prandial Triglyceride-enrichment of HDL Particles in Association with Endothelial and HDL Dysfunction.

NAFLD is closely related with the metabolic syndrome (MetS) and increased risk of cardiovascular disease. Liver fat associates with post-prandial hypertriglyceridemia, potentially contributing to triglyceride-enrichment of high-density lipoproteins (HDL-TG), and subsequent HDL dysfunction. We assessed liver fat by MR spectroscopy, and its association with HDL physiochemical properties, and endothelial function, measured as flow-mediated dilation (FMD), before and following three consecutive meals, in 36 men with type 2 diabetes mellitus (T2DM), with the MetS, and controls. Plasma triglycerides increased significantly following the meals (P < .001). Fasting HDL-TG was highest in T2DM, relative to MetS and controls (P = .002), and increased post-prandially in all groups (P < .001). HDL function was negatively associated with HDL-TG following three meals (r = -.32, P<.05). Liver fat associated with HDL-TG after three meals (r = .65, P < .001). HDL-TG was independently associated with FMD following three consecutive meals (r = -.477, P = .003). We conclude liver fat is associated with post-prandial HDL-TG enrichment which was closely related with endothelial and HDL dysfunction.

Feasibility of SPECT-CT Imaging to Study the Pharmacokinetics of Antisense Oligonucleotides in a Mouse Model of Duchenne Muscular Dystrophy.

Antisense oligonucleotides (AONs) are promising candidates for treatment of Duchenne muscular dystrophy (DMD), a severe and progressive disease resulting in premature death. However, more knowledge on the pharmacokinetics of new AON drug candidates is desired for effective application in the clinic. We assessed the feasibility of using noninvasive single-photon emission computed tomography-computed tomography (SPECT-CT) imaging to determine AON pharmacokinetics in vivo. To this end, a 2'-O-methyl phosphorothioate AON was radiolabeled with 123I or 111In, and administered to mdx mice, a rodent model of DMD. SPECT-CT imaging was performed to determine AON tissue levels, and the results were compared to data obtained with invasive analysis methods (scintillation counting and a ligation-hybridization assay). We found that SPECT-CT data obtained with 123I-AON and 111In-AON were qualitatively comparable to data derived from invasive analytical methods, confirming the feasibility of using SPECT-CT analysis to study AON pharmacokinetics. Notably, also AON uptake in skeletal muscle, the target tissue in DMD, could be readily quantified using SPECT-CT imaging, which was considered a particular challenge in mice, due to their small size. In conclusion, our results demonstrate that SPECT-CT imaging allows for noninvasive characterization of biodistribution and pharmacokinetics of AONs, thereby enabling quantitative comparisons between different radiolabeled AON drug candidates and qualitative conclusions about the corresponding unmodified parent AONs. This technology may contribute to improved (pre)clinical drug development, leading to drug candidates with optimized characteristics in vivo.

Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON.

Preclinical studies on intestinal administration of antisense oligonucleotides as a model for oral delivery for treatment of duchenne muscular dystrophy.

Antisense oligonucleotides (AONs) used to reframe dystrophin mRNA transcripts for Duchenne muscular dystrophy (DMD) patients are tested in clinical trials. Here, AONs are administered subcutaneously and intravenously, while the less invasive oral route would be preferred. Oral delivery of encapsulated AONs supplemented with a permeation enhancer, sodium caprate, has been successfully used to target tumor necrosis factor (TNF)-α expression in liver. To test the feasibility of orally delivered AONs for DMD, we applied 2'-O-methyl phosphorothioate AONs (with or without sodium caprate supplementation) directly to the intestine of mdx mice and compared pharmacokinetics and -dynamics with intravenous, intraperitoneal, and subcutaneous delivery. Intestinally infused AONs were taken up, but resulted in lower plasma levels compared to other delivery routes, although bioavailability could be largely improved by supplementation of sodium caprate. After intestinal infusion, AON levels in all tissues were lower than for other administration routes, as were the ratios of target versus nontarget organ levels, except for diaphragm and heart where comparable levels and ratios were observed. For each administration route, low levels of exon skipping in triceps was observed 3 hours post-AON administration. These data suggest that oral administration of naked 2'-O-methyl phosphorothioate AONs may be feasible, but only when high AON concentrations are used in combination with sodium caprate.

Peptide conjugation of 2'-O-methyl phosphorothioate antisense oligonucleotides enhances cardiac uptake and exon skipping in mdx mice.

Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy.