Ken Mizuno

Route of Penetration of Topically Instilled Nipradilol into the Ipsilateral Posterior Retina

PURPOSE To investigate how topically instilled nipradilol penetrates the ipsilateral posterior retina-choroid in normal rabbit eyes. METHODS Albino rabbits were used. Topical instillation (1%, 100 microL) or intracameral (0.1%, 100 microL) or sub-Tenon injection (0.1%, 10 microL) of [(14)C]nipradilol was performed in one eye. Ocular and periocular distribution and the concentrations of [(14)C]nipradilol were determined by whole-head autoradiography, the results of which were validated by measurements in isolated tissues. In addition, the unchanged form of nonradiolabeled nipradilol in the posterior retina after topical instillation (1%, 100 microL) was quantified by liquid chromatography-tandem mass spectrometry (LC/MS/MS). RESULTS Autoradiography revealed that the nipradilol concentration after topical instillation was higher in the ipsilateral posterior retina-choroid than on the contralateral side (142.9 ng/g vs. 108.3 ng/g, P = 0.026), and in the periocular tissue around the optic nerve insertion on the ipsilateral side than on the contralateral side (207.1 ng/g vs. 141.1 ng/g, P < 0.001). After intracameral injection, radioactivity was observed only in anterior, but not posterior parts of the eye. Radioactivity was observed only in the ipsilateral posterior retina-choroid and periocular tissues around the optic nerve insertion after sub-Tenon injection. The results in the isolated tissues validated autoradiographic evaluations. CONCLUSIONS These results suggest that diffusion from posterior periocular tissues across the posterior sclera may be the main route for local penetration of the instilled drug to reach the posterior retina-choroid in albino rabbits.

Effects of K-115 (Ripasudil), a novel ROCK inhibitor, on trabecular meshwork and Schlemm’s canal endothelial cells

Ripasudil hydrochloride hydrate (K-115), a specific Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, was the first ophthalmic solution developed for the treatment of glaucoma and ocular hypertension in Japan. Topical administration of K-115 decreased intraocular pressure (IOP) and increased outflow facility in rabbits. This study evaluated the effect of K-115 on monkey trabecular meshwork (TM) cells and Schlemm’s canal endothelial (SCE) cells. K-115 induced retraction and rounding of cell bodies as well as disruption of actin bundles in TM cells. In SCE-cell monolayer permeability studies, K-115 significantly decreased transendothelial electrical resistance (TEER) and increased the transendothelial flux of FITC-dextran. Further, K-115 disrupted cellular localization of ZO-1 expression in SCE-cell monolayers. These results indicate that K-115 decreases IOP by increasing outflow facility in association with the modulation of TM cell behavior and SCE cell permeability in association with disruption of tight junction.

New CETP Inhibitor K-312 Reduces PCSK9 Expression: A Potential Effect on LDL Cholesterol Metabolism

Despite significant reduction of cardiovascular events by statin treatment, substantial residual risk persists, driving emerging needs for the development of new therapies. We identified a novel cholesteryl ester transfer protein (CETP) inhibitor, K-312, that raises HDL and lowers LDL cholesterol levels in animals. K-312 also suppresses hepatocyte expression of proprotein convertase subtilisin/kexin 9 (PCSK9), a molecule that increases LDL cholesterol. We explored the underlying mechanism for the reduction of PCSK9 expression by K-312. K-312 inhibited in vitro human plasma CETP activity (IC50; 0.06 μM). Administration of K-312 to cholesterol-fed New Zealand White rabbits for 18 wk raised HDL cholesterol, decreased LDL cholesterol, and attenuated aortic atherosclerosis. Our search for additional beneficial characteristics of this compound revealed that K-312 decreases PCSK9 expression in human primary hepatocytes and in the human hepatoma cell line HepG2. siRNA silencing of CETP in HepG2 did not compromise the suppression of PCSK9 by K-312, suggesting a mechanism independent of CETP. In HepG2 cells, K-312 treatment decreased the active forms of sterol regulatory element-binding proteins (SREBP-1 and -2) that regulate promoter activity of PCSK9. Chromatin immunoprecipitation assays demonstrated that K-312 decreased the occupancy of SREBP-1 and SREBP-2 on the sterol regulatory element of the PCSK9 promoter. PCSK9 protein levels decreased by K-312 treatment in the circulating blood of cholesterol-fed rabbits, as determined by two independent mass spectrometry approaches, including the recently developed, highly sensitive parallel reaction monitoring method. New CETP inhibitor K-312 decreases LDL cholesterol and PCSK9 levels, serving as a new therapy for dyslipidemia and cardiovascular disease.

Angiopoietin Like Protein 2 (ANGPTL2) Promotes Adipose Tissue Macrophage and T lymphocyte Accumulation and Leads to Insulin Resistance

Objectives Angiopoietin-like protein 2 (ANGPTL2), a recently identified pro-inflammatory cytokine, is mainly secreted from the adipose tissue. This study aimed to explore the role of ANGPTL2 in adipose tissue inflammation and macrophage activation in a mouse model of diabetes. Methodology/Principal Findings Adenovirus mediated lacZ (Ad-LacZ) or human ANGPTL2 (Ad-ANGPTL2) was delivered via tail vein in diabetic db/db mice. Ad-ANGPTL2 treatment for 2 weeks impaired both glucose tolerance and insulin sensitivity as compared to Ad-LacZ treatment. Ad-ANGPTL2 treatment significantly induced pro-inflammatory gene expression in white adipose tissue. We also isolated stromal vascular fraction from epididymal fat pad and analyzed adipose tissue macrophage and T lymphocyte populations by flow cytometry. Ad-ANGPTL2 treated mice had more adipose tissue macrophages (F4/80+CD11b+) and a larger M1 macrophage subpopulation (F4/80+CD11b+CD11c+). Moreover, Ad-ANGPTL2 treatment increased a CD8-positive T cell population in adipose tissue, which preceded increased macrophage accumulation. Consistent with our in vivo results, recombinant human ANGPTL2 protein treatment increased mRNA levels of pro-inflammatory gene products and production of TNF-α protein in the human macrophage-like cell line THP-1. Furthermore, Ad-ANGPTL2 treatment induced lipid accumulation and increased fatty acid synthesis, lipid metabolism related gene expression in mouse liver. Conclusion ANGPTL2 treatment promotes macrophage accumulation and activation. These results suggest potential mechanisms for insulin resistance.

Dimerization of sortilin regulates its trafficking to extracellular vesicles

Extracellular vesicles (EVs) play a critical role in intercellular communication by transferring microRNAs, lipids, and proteins to neighboring cells. Sortilin, a sorting receptor that directs target proteins to the secretory or endocytic compartments of cells, is found in both EVs and cells. In many human diseases, including cancer and cardiovascular disorders, sortilin expression levels are atypically high. To elucidate the relationship between cardiovascular disease, particularly vascular calcification, and sortilin expression levels, we explored the trafficking of sortilin in both the intracellular and extracellular milieu. We previously demonstrated that sortilin promotes vascular calcification via its trafficking of tissue-nonspecific alkaline phosphatase to EVs. Although recent reports have noted that sortilin is regulated by multiple post-translational modifications, the precise mechanisms of sortilin trafficking still need to be determined. Here, we show that sortilin forms homodimers with an intermolecular disulfide bond at the cysteine 783 (Cys783) residue, and because Cys783 can be palmitoylated, it could be shared via palmitoylation and an intermolecular disulfide bond. Formation of this intermolecular disulfide bond leads to trafficking of sortilin to EVs by preventing palmitoylation, which further promotes sortilin trafficking to the Golgi apparatus. Moreover, we found that sortilin-derived propeptide decreased sortilin homodimers within EVs. In conclusion, sortilin is transported to EVs via the formation of homodimers with an intermolecular disulfide bond, which is endogenously regulated by its own propeptide. Therefore, we propose that inhibiting dimerization of sortilin acts as a new therapeutic strategy for the treatment of EV-associated diseases, including vascular calcification and cancer.

Additive Intraocular Pressure-Lowering Effects of Ripasudil with Glaucoma Therapeutic Agents in Rabbits and Monkeys

Ripasudil hydrochloride hydrate (K-115), a specific Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, is developed for the treatment of glaucoma and ocular hypertension. Topical administration of ripasudil decreases intraocular pressure (IOP) by increasing conventional outflow through the trabeculae to Schlemms canal, which is different from existing agents that suppress aqueous humor production or promote uveoscleral outflow. In this study, we demonstrated that ripasudil significantly lowered IOP in combined regimens with other glaucoma therapeutic agents in rabbits and monkeys. Ripasudil showed additional effects on maximum IOP lowering or prolonged the duration of IOP-lowering effects with combined administration of timolol, nipradilol, brimonidine, brinzolamide, latanoprost, latanoprost/timolol fixed combination, and dorzolamide/timolol fixed combination. These results indicate that facilitation of conventional outflow by ripasudil provides additive IOP-lowering effect with other classes of antiglaucoma agents. Ripasudil is expected to have substantial utility in combined regimens with existing agents for glaucoma treatment.

Design, synthesis and pharmacology of aortic-selective acyl-CoA: Cholesterol O-acyltransferase (ACAT/SOAT) inhibitors

We describe our molecular design of aortic-selective acyl-coenzyme A:cholesterol O-acyltransferase (ACAT, also abbreviated as SOAT) inhibitors, their structure-activity relationships (SARs) and their pharmacokinetic (PK) and pharmacological profiles. The connection of two weak ligands-N-(2,6-diisopropylphenyl)acetamide (50% inhibitory concentration [IC50] = 8.6 μM) and 2-(methylthio)benzo[d]oxazole (IC50 = 31 μM)-via a linker comprising a 6 methylene group chains yielded a highly potent molecule, 9-(benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)nonanamide (3h) that exhibited high potency (IC50 = 0.004 μM) toward aortic ACAT. This head-to-tail design made it possible to markedly enhance the activity to 2150- to 7750-fold and to discriminate the isoform-selectivity based on the double-induced fit mechanism. At doses of 1 and 3 mg/kg, 3h significantly decreased the lipid-accumulation areas in the aortic arch to 74 and 69%, respectively without reducing the plasma total cholesterol level in high fat- and cholesterol-fed F1B hamsters. Here, we demonstrate the antiatherosclerotic effect of 3hin vivo via its direct action on aortic ACAT and its powerful modulator of cholesterol level. This molecule is a potential therapeutic agent for the treatment of diseases involving ACAT-1 overexpression.

Influence of peripheral iridectomy on intravitreous penetration of topical nipradilol.

Purpose. Penetration of drug from the anterior chamber to the vitreous is substantial in aphakic eyes, but negligible in normal phakic eyes. The purpose of this study is to investigate the effect of the presence of peripheral iridectomy (PI) which bypasses iris-lens diaphragm on the drug penetration from the anterior chamber to the vitreous. Methods. Twelve Japanese White rabbits underwent PI in a randomly chosen eye and the same procedures except removal of the peripheral iris in the contralateral eye. Nine weeks after the procedure, topical instillation of 20µl of 1% nipradilol into the both eyes was repeated three times at five-minute intervals, and two hours later the animals were sacrificed and the both eyes were enucleated. Concentrations of nipradilol in the aqueous and vitreous were determined using a high-performance liquid chromatography. Results. The concentrations of nipradilol were significantly greater in the eyes with PI than those in the contralateral control eyes in the aqueous (5636 ± 1688 vs 2835 ± 663 ng/g, mean ± standard error, n = 12, p = 0.0028) and in the anterior vitreous (11.9 ± 2.5 vs 5.6 ± 1.0 ng/g, p = 0.0047), while not in the posterior vitreous or in the posterior retina-choroid. The ratios of the nipradilol concentrations in the anterior or posterior vitreous to that in the aqueous were not significantly different between the both eyes. Conclusions. The presence of PI had little effect on the penetration of topically instilled nipradilol from the anterior chamber to vitreous.