Rayner (United States)

Intracameral phenylephrine 1.0%/ketorolac 0.3% (OMIDRIA®) is used during cataract surgery to prevent intraoperative miosis and reduce postoperative pain. Although studied in beagles, no human data exist showing the duration ketorolac remains in the eye postoperatively. A clinical trial measuring ketorolac concentrations in aqueous/vitreous samples necessitated the development of a validation process for acquiring these measurements. Due to limited human aqueous/vitreous humor sample availability, a bioanalytical method was developed and validated to quantify ketorolac levels using human plasma as a surrogate matrix. The developed process involves extracting ketorolac and its internal standard (ketorolac-d5) from plasma as a surrogate for aqueous and vitreous humor using a protein precipitation sample preparation technique, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The validated method can be successfully applied for quantitation of ketorolac over a concentration range of 2.5 ng/mL to 5000 ng/mL. The method met the acceptance criteria with respect to selectivity, specificity, precision, accuracy, linearity, dilution integrity, and stability. The validated method can use plasma as a surrogate matrix for quantitation of ketorolac in aqueous and vitreous humor, thereby eliminating the need to procure human vitreous and aqueous samples for validation prior to initiation of a clinical trial.

The prevention and treatment of cardiovascular diseases (CVD) has largely focused on lowering circulating LDL cholesterol, yet a significant burden of atherosclerotic disease remains even with low LDL. Recently, microRNAs (miRNAs) have emerged as exciting therapeutic targets for CVD. miRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression by degradation or translational inhibition of target mRNAs. A number of miRNAs have been found to modulate all stages of atherosclerosis, particularly those that promote cholesterol efflux from lipid laden macrophages in the vessel wall. However, one of the major challenges of miRNA-based therapy is to achieve tissue-specific, efficient and safe delivery of miRNAs in vivo . Objective: We therefore sought to develop chitosan/miRNA nanoparticles, deliver them to the plaque, and determine if these miRNAs can promote cholesterol efflux to decrease atherosclerosis. Results: We conjugated negatively charged miRNAs with tripolyphosphate (TPP) to support crosslinks between polymeric and nucleic acid units, which were then mixed with varying ratios of chitosan polymer to form nanoparticles that ranged from 150-180nm in size. We next optimized the efficiency of intracellular delivery of different chitosan/miRNA ratios to mouse macrophages (MΦ). We find chitosan nanoparticles can protect as well as transfer exogenous miR-33 to naïve MΦ and reduce mRNA and protein expression of its target gene, ABCA1, confirming that miRNAs delivered via nanoparticle can escape the endosomal system and function in the RISC complex. Because ABCA1 plays a key role in stimulating the efflux of cholesterol from MΦ, we also confirmed that MΦ treated with chitosan/miR-33 nanoparticles exhibited reduced cholesterol efflux to Apolipoprotein A1, further confirming functional delivery of the miRNA. Using this formulation, we have developed a panel of miRNA nanoparticles delivering miRNAs that enhance ABCA1 expression and promote cholesterol efflux. Conclusions: miRNAs can be efficiently delivered to macrophages via nanoparticles where they can function to regulate ABCA1 expression and cholesterol efflux, suggesting that these miRNA-nanoparticles can be used in vivo to target atherosclerotic lesions.

Cellular lipid homeostasis is fundamental to human health. Recent studies indicate that microRNAs (miRNAs) play important roles in the post-transcriptional regulation of lipid metabolism genes. This is illustrated by the discovery, by our group and others, of miR-33 as a central regulator of multiple aspects of lipid metabolism, including cholesterol efflux, fatty acid oxidation, and plasma levels of HDL cholesterol and VLDL triglycerides. In a study of miR-33 antagonism in non-human primates, we identified the most highly de-repressed miR-33 target gene as OSBPL6 (oxysterol binding protein like 6). A member of the OSBP family of cytosolic proteins that bind cholesterol/oxysterols, OSBPL6 contains an ER-targeting FFAT motif as well as a predicted plasma membrane-targeting pleckstrin homology domain, suggesting a putative role for OSBPL6 in vesicular traffic between these compartments. Notably, the OSBPL6 gene resides in a chromosome 2 locus associated with a predisposition to CAD in two major human heart disease studies (Family Heart Study, Framingham Heart Study). We therefore sought to further understand the regulation and function of OSBPL6 in cellular lipid homeostasis. Results We show that OSBPL6 is induced in macrophages and hepatocytes in response to cholesterol loading in vitro, as well as in the livers of LDLR-/- mice and non-human primates fed a high fat diet. Overexpression of miR-33 reduces OSBPL6-3’UTR activity by 40%, and this repression is relieved by mutation of the putative miR-33 binding site in the 3’UTR, confirming that OSBPL6 is a direct target of miR-33. Accordingly, transfection of HEPG2 cells with miR-33 mimic decreases mRNA expression of Osbpl6, but not the related family member Osbpl1. Notably, siRNA-mediated knockdown of OSBPL6 in cholesterol-loaded HEPG2 cells or THP-1 macrophages impairs cholesterol efflux to both apoA-I and HDL, and conversely, OSBPL6 overexpression enhances cholesterol efflux to these acceptors. Conclusion These data identify OSBPL6 as a novel miR-33 target gene in mice and humans that regulates cellular cholesterol trafficking and efflux via the ABC transporters, further highlighting the coordinated regulation by miR-33 of pathways that promote cellular cholesterol clearance.

Despite the success of statins to reduce levels of LDL-cholesterol (LDL-C), cardiovascular disease remains the leading cause of mortality in westernized countries. PCSK9 inhibition is emerging as an exciting new therapy to further lower LDL and holds tremendous promise to reduce the significant residual cardiovascular risk in many individuals. Recent advances from our group and others have shown that microRNAs (miRNAs) have emerged as critical components in the regulation of cholesterol metabolism. In addition to miR-33, which we discovered as a key regulator of metabolic programs including cholesterol and fatty acid homeostasis, we identified two new cholesterol responsive miRNAs, miR-520d-5p (miR-520d) and miR-224. Here we show that miR-520d and miR-224 target the 3’UTR of both PCSK9 and Idol, “accessory” proteins that regulate cell surface expression of the LDL receptor (LDLR). Moreover, miR-520d and miR-224 simultaneously target the 3’UTR of HMGCR, which codes for the limiting enzyme in cholesterol synthesis and is the target of statins. In mouse and human cells, the overexpression of miR-520d inhibits PCSK9, Idol and HMGCR mRNA levels by 30-50% compared to a control miRNA, while overexpression of miR-224 results in an even more potent 75% decrease in PCSK9, Idol and HMGCR mRNA compared to controls. In human hepatocytes (HepG2/Huh7) and mouse peritoneal macrophages, miR-520d and miR-224 overexpression caused a repression of PCSK9, Idol and HMGCR protein levels, and a decrease in PCSK9 protein secretion. Importantly, these miR-224 and miR-520d-induced changes in PCSK9, Idol and HMGCR expression were associated with a concomitant increase in LDLR protein expression, increased cell surface expression of an LDLR-GFP fusion protein and enhanced LDL binding to the cell surface. Notably, these increases in LDLR were above and beyond those achieved with statin treatment. Together, these findings suggest a role of miR-224 and miR-520d in contributing to the post-transcriptional regulation of LDLR expression, and represent the first evidence of miRNA control of the LDLR pathway. This highlights the therapeutic promise of miR-224/miR-520d for reducing LDL cholesterol beyond statin therapy alone.