Pitavastatin-Incorporated Nanoparticles for Chronic Limb Threatening Ischemia: A Phase I/IIa Clinical Trial.

AIM: To assess the results of a phase I/IIa open-label dose-escalation clinical trial of 5-day repeated intramuscular administration of pitavastatin-incorporated poly (lactic-co-glycolic acid) nanoparticles (NK-104-NP) in patients with chronic limb threatening ischemia (CLTI). METHODS: NK-104-NP was formulated using an emulsion solvent diffusion method. NK-104-NP at four doses (nanoparticles containing 0.5, 1, 2, and 4 mg of pitavastatin calcium, n=4 patients per dose) was investigated in a dose-escalation manner and administered intramuscularly into the ischemic limbs of 16 patients with CLTI. The safety and therapeutic efficacy of treatment were investigated over a 26-week follow-up period. RESULTS: No cardiovascular or other serious adverse events caused by NK-104-NP were detected during the follow-up period. Improvements in Fontaine and Rutherford classifications were noted in five patients (one, three, and one in the 1-, 2-, and 4-mg dose groups, respectively). Pharmacokinetic parameters including the maximum serum concentration and the area under the blood concentration-time curve increased with pitavastatin treatment in a dose-dependent manner. The area under the curve was slightly increased at day 5 compared with that at day 1 of treatment, although the difference was not statistically significant. CONCLUSIONS: This is the first clinical trial of pitavastatin-incorporated nanoparticles in patients with CLTI. Intramuscular administration of NK-104-NP to the ischemic limbs of patients with CLTI was safe and well tolerated and resulted in improvements in limb function.

Nanoparticle-Mediated Delivery of Pitavastatin to Monocytes/Macrophages Inhibits Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Apoe-/- Mice.

AIM: Abdominal aortic aneurysm (AAA) is a lethal and multifactorial disease. To prevent a rupture and dissection of enlarged AAA, prophylactic surgery and stenting are currently available. There are, however, no medical therapies preventing these complications of AAA. Statin is one of the candidates, but its efficacy on AAA formation/progression remains controversial. We have previously demonstrated that nanoparticles (NPs) incorporating pitavastatin (Pitava-NPs)-clinical trials using these nanoparticles have been already conducted-suppressed progression of atherosclerosis in apolipoprotein E-deficient ( Apoe-/-) mice. Therefore, we have tested a hypothesis that monocytes/macrophages-targeting delivery of pitavastatin prevents the progression of AAA. METHODS: Angiotensin II was intraperitoneally injected by osmotic mini-pumps to induce AAA formation in Apoe-/- mice. NPs consisting of poly(lactic-co-glycolic acid) were used for in vivo delivery of pitavastatin to monocytes/macrophages. RESULTS: Intravenously administered Pitava-NPs (containing 0.012 mg/kg/week pitavastatin) inhibited AAA formation accompanied with reduction of macrophage accumulation and monocyte chemoattractant protein-1 (MCP-1) expression. Ex vivo molecular imaging revealed that Pitava-NPs not only reduced macrophage accumulation but also attenuated matrix metalloproteinase activity in the abdominal aorta, which was underpinned by attenuated elastin degradation. CONCLUSION: These results suggest that Pitava-NPs inhibit AAA formation associated with reduced macrophage accumulation and MCP-1 expression. This clinically feasible nanomedicine could be an innovative therapeutic strategy that prevents devastating complications of AAA.

Nanoparticle-Mediated Simultaneous Targeting of Mitochondrial Injury and Inflammation Attenuates Myocardial Ischemia-Reperfusion Injury.

Background The opening of mitochondrial permeability transition pore and inflammation cooperatively progress myocardial ischemia-reperfusion (IR) injury, which hampers therapeutic effects of primary reperfusion therapy for acute myocardial infarction. We examined the therapeutic effects of nanoparticle-mediated medicine that simultaneously targets mitochondrial permeability transition pore and inflammation during IR injury. Methods and Results We used mice lacking cyclophilin D (CypD, a key molecule for mitochondrial permeability transition pore opening) and C-C chemokine receptor 2 and found that CypD contributes to the progression of myocardial IR injury at early time point (30-45 minutes) after reperfusion, whereas C-C chemokine receptor 2 contributes to IR injury at later time point (45-60 minutes) after reperfusion. Double deficiency of CypD and C-C chemokine receptor 2 enhanced cardioprotection compared with single deficiency regardless of the durations of ischemia. Deletion of C-C chemokine receptor 2, but not deletion of CypD, decreased the recruitment of Ly-6Chigh monocytes after myocardial IR injury. In CypD-knockout mice, administration of interleukin-1ß blocking antibody reduced the recruitment of these monocytes. Combined administration of polymeric nanoparticles composed of poly-lactic/glycolic acid and encapsulating nanoparticles containing cyclosporine A or pitavastatin, which inhibit mitochondrial permeability transition pore opening and monocyte-mediated inflammation, respectively, augmented the cardioprotection as compared with single administration of nanoparticles containing cyclosporine A or pitavastatin after myocardial IR injury. Conclusions Nanoparticle-mediated simultaneous targeting of mitochondrial injury and inflammation could be a novel therapeutic strategy for the treatment of myocardial IR injury.

Nanoparticle-Mediated Delivery of 2-Deoxy-D-Glucose Induces Antitumor Immunity and Cytotoxicity in Liver Tumors in Mice.

BACKGROUND & AIMS: Immune checkpoint inhibitors have shed light on the importance of antitumor immunity as a therapeutic strategy for hepatocellular carcinoma (HCC). The altered glucose metabolism known as the Warburg effect recently has gained attention as a cancer immune-resistance mechanism. Considering glycolysis inhibitors as therapeutic agents, their specific delivery to cancer cells is critical not to induce adverse effects. Thus, we investigated antitumor effects of a glycolysis inhibitor, consisting of 2-deoxy-D-glucose (2DG)-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (2DG-PLGA-NPs), against hepatocellular carcinoma in mice. METHODS: The antitumor effects of 2DG-PLGA-NPs were examined using hepatoma cell lines, xenograft tumors, and hepatocarcinogenic and syngeneic mouse models. RESULTS: The 2DG-PLGA-NPs induced cytotoxic effects and antitumor immunity through enhanced T-cell trafficking. In addition, 2DG-PLGA-NPs induced decreased lactate production and increased interferon-γ-positive T cells in liver tumors. Human CD8+ T cells cocultured with 2DG-PLGA-NP-treated Huh7 cells showed their increased interferon-γ production and glucose uptake compared with the CD8+ T cells co-cultured with PLGA-NP-treated Huh7 cells. Chemotaxis of CD8+ T cells was suppressed by lactate and enhanced by glucose. Interferon-γ enhanced CD8+ T-cell chemotaxis in both an autocrine and paracrine manner. Notably, the 2DG-PLGA-NPs augmented chemokine (CXCL9/CXCL10) production in liver tumors via interferon-γ-Janus kinase-signal transducers and activator of transcription pathway and 5' adenosine monophosphate-activated protein kinase-mediated suppression of histone H3 lysine 27 trimethylation. These 2DG-PLGA-NPs not only amplified antitumor effects induced by sorafenib or an anti-programmed death-1 antibody, but also suppressed anti-programmed death-1-resistant tumors. CONCLUSIONS: The newly developed 2DG-PLGA-NPs showed antitumor immunity and cytotoxicity in liver tumors in mice, suggesting the potential of 2DG-PLGA-NPs for future clinical applications.

Simultaneous targeting of mitochondria and monocytes enhances neuroprotection against ischemia-reperfusion injury.

Ischemia-reperfusion injury impairs the efficacy of reperfusion therapy after ischemic stroke. Cyclophilin D (CypD)-mediated openings of mitochondrial permeability transition pore (mPTP) and subsequent monocyte-mediated inflammation are considered as major mechanisms of reperfusion injury. However, no medical therapies are currently available. Therefore, we have tested a hypothesis that simultaneous targeting of mPTP and inflammation confers substantial neuroprotection after cerebral ischemia-reperfusion. To address this point, we prepared CypD knockout mice, C-C chemokine receptor 2 (CCR2) knockout mice and CypD/CCR2 double knockout mice. These mice were subjected to 60 min transient cerebral ischemia by occluding middle cerebral arteries. Neurological deficits evaluated 3 days after reperfusion were significantly attenuated in CypD/CCR2 double knockout mice as compared to wild-type mice and other single knockout mice. Then, we have prepared polymeric nanoparticles containing cyclosporine A (CsA-NPs) and pitavastatin (Pitava-NPs), targeting mPTP opening and inflammation, respectively. Simultaneous administration of CsA-NP and Pitava-NP at the time of reperfusion also decreased infarct size and attenuated neurological deficits as compared to control nanoparticles and single administration of CsA-NPs or Pitava-NPs. These results indicate that simultaneous targeting of the mPTP opening and monocyte-mediated inflammation could be a novel strategy for better neurological outcomes in patients with ischemic stroke.

Macrophage (Drp1) Dynamin-Related Protein 1 Accelerates Intimal Thickening After Vascular Injury.

OBJECTIVE: Mitochondria consistently change their morphology in a process regulated by proteins, including Drp1 (dynamin-related protein 1), a protein promoting mitochondrial fission. Drp1 is involved in the mechanisms underlying various cardiovascular diseases, such as myocardial ischemia/reperfusion injury, heart failure, and pulmonary arterial hypertension. However, its role in macrophages, which promote various vascular diseases, is poorly understood. We therefore tested our hypothesis that macrophage Drp1 promotes vascular remodeling after injury. METHOD AND RESULTS: To explore the selective role of macrophage Drp1, we created macrophage-selective Drp1-deficient mice and performed femoral arterial wire injury. In these mice, intimal thickening and negative remodeling were attenuated at 4 weeks after injury when compared with control mice. Deletion of macrophage Drp1 also attenuated the macrophage accumulation and cell proliferation in the injured arteries. Gain- and loss-of-function experiments using cultured macrophages indicated that Drp1 induces the expression of molecules associated with inflammatory macrophages. Morphologically, mitochondrial fission was induced in inflammatory macrophages, whereas mitochondrial fusion was induced in less inflammatory/reparative macrophages. Pharmacological inhibition or knockdown of Drp1 decreased the mitochondrial reactive oxygen species and chemotactic activity in cultured macrophages. Co-culture experiments of macrophages with vascular smooth muscle cells indicated that deletion of macrophage Drp1 suppresses growth and migration of vascular smooth muscle cells induced by macrophage-derived soluble factors. CONCLUSIONS: Macrophage Drp1 accelerates intimal thickening after vascular injury by promoting macrophage-mediated inflammation. Macrophage Drp1 may be a potential therapeutic target of vascular diseases.

Therapeutic Arteriogenesis/Angiogenesis for Peripheral Arterial Disease by Nanoparticle-Mediated Delivery of Pitavastatin into Vascular Endothelial Cells.

Two decades have passed since therapeutic angiogenesis was proposed to promote reparative collateral growth as an alternative therapy for ischemic diseases in patients for whom neither surgical revascularization nor endovascular therapy was suitable. When therapeutic angiogenesis first began, local administration was conducted using recombinant growth factor proteins or gene-encoding growth factors for endothelial cells. Since then, autologous stem cells and endothelial progenitor cell transplantation therapy have been developed. Although many clinical trials have been performed on patients, most therapies have not yet become standard treatments. We have developed a nanoparticle (NP)-mediated, drug-targeting delivery system using bioabsorbable poly-lactic/glycolic acid (PLGA) NPs. In several animal models, pitavastatin-incorporated (Pitava)-NPs showed significant therapeutic effects on critical limb ischemia. Because PLGA NPs are delivered selectively to vascular endothelial cells after intramuscular administration, it is suggested that therapeutic angiogenesis/arteriogenesis plays an important role in the mechanism by which Pitava-NPs exert beneficial therapeutic effects. To translate this to clinical medicine, we have performed studies and produced Pitava-NPs in compliance with good laboratory practice/good manufacturing practice regulations, and completed a phase I/II clinical trial, reporting the safety and efficacy of Pitava-NP intramuscular injection for patients with critical limb ischemia. This review will focus on therapeutic angiogenesis/arteriogenesis for peripheral arterial disease induced by Pitava-NPs.

Nanoparticle incorporating Toll-like receptor 4 inhibitor attenuates myocardial ischaemia-reperfusion injury by inhibiting monocyte-mediated inflammation in mice.

AIMS: Myocardial ischaemia-reperfusion (IR) injury hampers the therapeutic effect of revascularization in patients with acute myocardial infarction (AMI). Innate immunity for damage-associated protein patterns promotes the process of IR injury; however, the blockade of Toll-like receptor 4 (TLR4) in myocardial IR injury has not been translated into clinical practice. Therefore, we aimed to examine whether the nanoparticle-mediated administration of TAK-242, a chemical inhibitor of TLR4, attenuates myocardial IR injury in a clinically feasible protocol in a mouse model. METHODS AND RESULTS: We have prepared poly-(lactic-co-glycolic acid) nanoparticles containing TAK-242 (TAK-242-NP). TAK-242-NP significantly enhanced the drug delivery to monocytes/macrophages in the spleen, blood, and the heart in mice. Intravenous administration of TAK-242-NP (containing 1.0 or 3.0 mg/kg TAK-242) at the time of reperfusion decreased the infarct size, but the TAK-242 solution did not even when administered at a dosage of 10.0 mg/kg. TAK-242-NP inhibited the recruitment of Ly-6Chigh monocytes to the heart, which was accompanied by decreased circulating HMGB1, and NF-κB activation and cytokine expressions in the heart. TAK-242-NP did not decrease the infarct size further in TLR4-deficient mice, confirming the TLR4-specific mechanism in the effects of TAK-242-NP. Furthermore, TAK-242-NP did not decrease the infarct size further in CCR2-deficient mice, suggesting that monocyte/macrophage-mediated inflammation is the primary therapeutic target of TAK-242-NP. CONCLUSION: The nanoparticle-mediated delivery of TAK-242-NP represent a novel and clinical feasible strategy in patients undergone coronary revascularization for AMI by regulating TLR4-dependent monocytes/macrophages-mediated inflammation.

Peroxisome proliferator-activated receptor-gamma targeting nanomedicine promotes cardiac healing after acute myocardial infarction by skewing monocyte/macrophage polarization in preclinical animal models.

Aims: Monocyte-mediated inflammation is a major mechanism underlying myocardial ischaemia-reperfusion (IR) injury and the healing process after acute myocardial infarction (AMI). However, no definitive anti-inflammatory therapies have been developed for clinical use. Pioglitazone, a peroxisome proliferator-activated receptor-gamma (PPARγ) agonist, has unique anti-inflammatory effects on monocytes/macrophages. Here, we tested the hypothesis that nanoparticle (NP)-mediated targeting of pioglitazone to monocytes/macrophages ameliorates IR injury and cardiac remodelling in preclinical animal models. Methods and results: We formulated poly (lactic acid/glycolic acid) NPs containing pioglitazone (pioglitazone-NPs). In a mouse IR model, these NPs were delivered predominantly to circulating monocytes and macrophages in the IR heart. Intravenous treatment with pioglitazone-NPs at the time of reperfusion attenuated IR injury. This effect was abrogated by pre-treatment with the PPARγ antagonist GW9662. In contrast, treatment with a pioglitazone solution had no therapeutic effects on IR injury. Pioglitazone-NPs inhibited Ly6Chigh inflammatory monocyte recruitment as well as inflammatory gene expression in the IR hearts. In a mouse myocardial infarction model, intravenous treatment with pioglitazone-NPs for three consecutive days, starting 6 h after left anterior descending artery ligation, attenuated cardiac remodelling by reducing macrophage recruitment and polarizing macrophages towards the pro-healing M2 phenotype. Furthermore, pioglitazone-NPs significantly decreased mortality after MI. Finally, in a conscious porcine model of myocardial IR, pioglitazone-NPs induced cardioprotection from reperfused infarction, thus providing pre-clinical proof of concept. Conclusion: NP-mediated targeting of pioglitazone to inflammatory monocytes protected the heart from IR injury and cardiac remodelling by antagonizing monocyte/macrophage-mediated acute inflammation and promoting cardiac healing after AMI.

Nanoparticle-Mediated Targeting of Pitavastatin to Small Pulmonary Arteries and Leukocytes by Intravenous Administration Attenuates the Progression of Monocrotaline-Induced Established Pulmonary Arterial Hypertension in Rats.

Statins are known to improve pulmonary arterial hypertension (PAH) by their anti-inflammatory and anti-proliferative effects in animal models. However, recent clinical studies have reported that clinically approved statin doses failed to improve clinical outcomes in patients with PAH. We therefore hypothesized that nanoparticle (NP) -mediated targeting of pitavastatin could attenuate the progression of established PAH.We induced PAH by subcutaneously injecting monocrotaline (MCT) in Sprague-Dawley rats. On day 14 after the MCT injection, animals that displayed established PAH on echocardiography were included. On day 17, they were randomly assigned to the following 5 groups: daily intravenous administration of (1) vehicle, (2) fluorescein-isothiocyanate-NP, (3) pitavastatin, (4) pitavastatin-NP, or (5) oral sildenafil. Intravenous NP was selectively delivered to small pulmonary arteries and circulating CD11b-positive leukocytes. On day 21, pitavastatin-NP attenuated the progression of PAH at lower doses than pitavastatin alone. This was associated with the inhibition of monocyte-mediated inflammation, proliferation, and remodeling of the pulmonary arteries. Interestingly, sildenafil attenuated the development of PAH, but had no effects on inflammation or remodeling of the pulmonary arteries. In separate experiments, only treatment with pitavastatin-NP reduced the mortality rate at day 35.NP-mediated targeting of pitavastatin to small pulmonary arteries and leukocytes attenuated the progression of established MCT-induced PAH and improved survival. Therapeutically, pitavastatin-NP was associated with anti-inflammatory and anti-proliferative effects on small pulmonary arteries, which was completely distinct from the vasodilatory effect of sildenafil. Pitavastatin-NP can be a novel therapeutic modality for PAH.

Safety, Tolerability, and Pharmacokinetics of NK-104-NP.

Pulmonary hypertension (PH) is a disease with poor prognosis, caused by the obstruction/stenosis of small pulmonary arteries. Statin is known to have vasodilating and anti-inflammatory property and is considered to be a candidate of therapeutic agents for the treatment of PH, but its efficacy has not been verified in clinical trials. We have formulated pitavastatin incorporating nanoparticles composed of poly (lactic-co-glycolic acid) (NK-104-NP) to improve drug delivery to the pulmonary arteries and evaluated their safety and pharmacokinetics in healthy volunteers. To accomplish this purpose, phase I clinical trials were conducted. In the single intravenous administration regimen, 40 healthy subjects were enrolled and PK (pharmacokinetic) parameters in 4 groups (1, 2, 4, and 8 mg as pitavastatin calcium) were as follows: 1.00 hour after the administration, the plasma concentration of pitavastatin reached Cmax (the maximum drug concentration) in all groups. Cmax, AUC0-t (area under the curve from time 0 to the last measurable concentration) and AUC0-∞ (area under the curve from time 0 extrapolated to infinite time) were increased in a dose-dependent manner. Population pharmacokinetic analysis based on these results indicated no accumulation of pitavastatin after repeated administration of NK-104-NP for 7 days. In this 7-day administration trial, the mean Cmax and AUC0-∞ of pitavastatin were not significantly different between days 1 and 7, suggesting that pitavastatin is unlikely to accumulate after repeated administration. In these trials, three adverse events (AEs) were reported, but they were resolved without any complications and judged to have no causal relationships with NK-104-NP. These results indicate that the innovative nanotechnology-based medicine NK-104-NP exhibited dose-dependent pharmacokinetics and was well tolerated with no significant AEs in healthy volunteers.

Lipid-Lowering Therapy With Ezetimibe Decreases Spontaneous Atherothrombotic Occlusions in a Rabbit Model of Plaque Erosion: A Role of Serum Oxysterols.

OBJECTIVE: Plaque erosion is increasing its importance as one of the mechanisms of acute coronary syndromes in this statin era. However, the clinical efficacy of currently used lipid-lowering agents in the prevention of thrombotic complications associated with plaque erosion has not been clarified. Therefore, we examined the therapeutic effects of ezetimibe or rosuvastatin monotherapy on spontaneous atherothrombotic occlusion. APPROACH AND RESULTS: Femoral arteries of Japanese white rabbits, fed a high-cholesterol diet, were injured by balloon catheter, and then angiotensin II was continuously administrated. In 94% of these arteries, spontaneous thrombotic occlusions were observed after 5 weeks (median) of balloon injury. Histochemical analyses indicated that the injured arteries had similar pathological features to human plaque erosions; (1) spontaneous thrombotic occlusion, (2) lack of endothelial cells, and (3) tissue factor expression in vascular smooth muscle cells. Ezetimibe (1.0 mg/kg per day), but not rosuvastatin (0.6 mg/kg per day), significantly decreased thrombotic occlusion of arteries accompanied with accelerated re-endothelialization and the decreases of serum oxysterols despite the comparable on-treatment serum cholesterol levels. The 7-ketocholesterol inhibited the migration of human umbilical vein endothelial cells. Both 7-ketocholesterol and 27-hydroxycholesterol increased tissue factor expression in cultured rat vascular smooth muscle cells. Tissue factor expression was also induced by serum from vehicle- or rosuvastatin-treated rabbits, but the induction was attenuated with serum from ezetimibe-treated rabbits. CONCLUSIONS: We have established a novel rabbit model of spontaneous atherothromobotic occlusion without plaque rupture that is feasible to test the therapeutic effects of various pharmacotherapies. Ezetimibe may decrease atherothrombotic complications after superficial plaque erosion by reducing serum oxysterols.

BubR1 insufficiency impairs angiogenesis in aging and in experimental critical limb ischemic mice.

OBJECTIVES: Budding uninhibited by benzimidazole-related 1 (BubR1), a cell cycle-related protein, is an essential component of the spindle checkpoint that regulates cell division. Mice in which BubR1 expression is reduced to 10% of the normal level display the phenotypic features of progeria. However, the role of BubR1 in vascular diseases and angiogenesis remains unknown. To investigate the influence of BubR1 on angiogenesis, we generated a low-null-BubR1-expressing (BubR1L/-) mouse strain with reduced BubR1 expression as low as 15% of the normal level without any abnormalities in appearance. METHODS: To elucidate the role of BubR1 in angiogenesis, we used a hind limb ischemia model induced in BubR1L/- mice and age-matched wild-type (WT) littermates. To evaluate the pathologic influence of BubR1 on angiogenesis, we measured the blood flow before and after hind limb ischemia surgery, and the expression of typical angiogenic factors in vivo and in vitro. RESULTS: In WT mice, blood flow in the ischemic left limb gradually recovered to approximately 80%, 14 days after surgery. Conversely, in the BubR1L/- group, blood flow in the left ischemic limb recovered to at most 30% (14 days after surgery, P < .01; immediately after the operation, and 5 and 9 days after surgery, P < .05). In adductor and calf muscles from BubR1L/- mice, regenerated muscle bundles, granulation tissue, and inflammatory cell invasion were more evident than in calf muscles from WT mice at 14 days after surgery. All WT mice at 14 days after surgery had complete limb salvage, but loss of limbs was observed in approximately 70% of BubR1L/- mice (P < .05). The vascular endothelial growth factor protein increase in ischemic hind limb muscles was lower in BubR1L/- mice compared with WT mice (P < .05), and vascular endothelial growth factor levels in human aortic smooth muscle cells treated with BubR1 knockdown siRNA were lower compared with scramble siRNA under hypoxic conditions (P < .01). HIF1α protein levels in the muscles after hind limb ischemia surgery were also significantly lower in BubR1L/- mice compared with WT mice (P < .05). CONCLUSIONS: BubR1 insufficiency impairs angiogenesis and results in limb loss in ischemic hind limbs. BubR1 may be a crucial angiogenic factor and might be beneficial for the treatment of limb ischemia.

Nanoparticle-Mediated Delivery of Pitavastatin to Monocytes/Macrophages Inhibits Left Ventricular Remodeling After Acute Myocardial Infarction by Inhibiting Monocyte-Mediated Inflammation.

Left ventricular (LV) remodeling after myocardial infarction (MI) causes heart failure. Although medical therapies including angiotensin converting enzyme inhibitors show inhibitory effects on post-infarct LV remodeling, the prognosis of patients with post-infarct heart failure is still poor. Accumulating evidence suggests that an inflammatory response is implicated in the process of post-infarct LV remodeling. Therefore, we hypothesized that anti-inflammatory therapy by nanoparticle-mediated monocyte/macrophage-targeting delivery of pitavastatin may protect the heart from post-infarct LV remodeling.Male C57BL/6 mice were subjected to permanent coronary ligation and pitavastatin-incorporating nanoparticles (Pitavastatin-NPs) were intravenously injected for 3 to 5 consecutive days. Pitavastatin-NPs were delivered to CD11b+ monocytes/macrophages, but not to cardiomyocytes. Treatment with Pitavastatin-NPs after establishment of MI attenuated post-infarct LV remodeling accompanied by a reduction of monocytes/macrophages in the heart, whereas pitavastatin solution treatment did not. Pitavastatin-NPs inhibited mobilization of monocytes from the spleen after MI. In mice after splenectomy, Pitavastatin-NPs still decreased the number of monocytes/macrophages in the infarcted heart and inhibited post-infarct LV remodeling.Nanoparticle-mediated delivery of pitavastatin to monocytes/macrophages may be a novel therapeutic strategy to protect the heart from post-infarct LV remodeling. Inhibition of monocyte mobilization from the bone marrow is one of the major mechanisms by which Pitavastatin-NPs attenuated post-infarct LV remodeling.

Nanoparticle-mediated drug delivery system for atherosclerotic cardiovascular disease.

Administration of drugs and other therapeutic agents has been the central strategy of contemporary medicine for cardiovascular disease. The use of drug delivery systems (DDS) includes micelles, liposomes, polymeric nanoparticles, dendrimers, carbon nanotubes, and crystalline metals. Nano-DDS modify in vivo drug kinetics, depending on (patho)physiological mechanisms such as retard excretion, vascular permeability, and incorporation by mononuclear phagocyte systems, which constitute the 'passive-targeting' property of nano-DDS. These properties of nano-DDS are applicable to inflammatory diseases including atherosclerosis. Atherosclerotic plaque destabilization and rupture account for the majority of acute myocardial infarction, for which inflammatory monocytes and macrophages play critical roles. In our experience, polymeric nanoparticles have been delivered to inflammatory monocytes and macrophages in an atherosclerotic mouse model. Nano-DDS loaded with pioglitazone reduced Ly6Chigh inflammatory monocytes and increased Ly6Clow non-inflammatory monocytes in the peripheral blood, and induced M2 macrophage-associated genes in the aorta. Pioglitazone-nanoparticles finally stabilized atherosclerotic plaques assessed by a decrease in the number of buried fibrous caps in the plaque. Application of nano-DDS is a unique and promising approach to prevent life-threatening cardiovascular events including acute myocardial infarction by regulating inflammation in the cardiovascular system.

Marked augmentation of PLGA nanoparticle-induced metabolically beneficial impact of γ-oryzanol on fuel dyshomeostasis in genetically obese-diabetic ob/ob mice.

Our previous works demonstrated that brown rice-specific bioactive substance, γ-oryzanol acts as a chaperone, attenuates exaggerated endoplasmic reticulum (ER) stress in brain hypothalamus and pancreatic islets, thereby ameliorating metabolic derangement in high fat diet (HFD)-induced obese diabetic mice. However, extremely low absorption efficiency from intestine of γ-oryzanol is a tough obstacle for the clinical application. Therefore, in this study, to overcome extremely low bioavailability of γ-oryzanol with super-high lipophilicity, we encapsulated γ-oryzanol in polymer poly (DL-lactide-co-glycolide) (PLGA) nanoparticles (Nano-Orz), and evaluated its metabolically beneficial impact in genetically obese-diabetic ob/ob mice, the best-known severest diabetic model in mice. To our surprise, Nano-Orz markedly ameliorated fuel metabolism with an unexpected magnitude (∼1000-fold lower dose) compared with regular γ-oryzanol. Furthermore, such a conspicuous impact was achievable by its administration once every 2 weeks. Besides the excellent impact on dysfunction of hypothalamus and pancreatic islets, Nano-Orz markedly decreased ER stress and inflammation in liver and adipose tissue. Collectively, nanotechnology-based developments of functional foods oriented toward γ-oryzanol shed light on the novel approach for the treatment of a variety of metabolic diseases in humans.

Anti-inflammatory Nanomedicine for Cardiovascular Disease.

Coronary artery disease, in the development of which inflammation mediated by innate immune cells plays a critical role, is one of the leading causes of death worldwide. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are a widely used lipid-lowering drug that has lipid-independent vasculoprotective effects, such as improvement of endothelial dysfunction, antioxidant properties, and inhibitory effects on inflammation. Despite recent advances in lipid-lowering therapy, clinical trials of statins suggest that anti-inflammatory therapy beyond lipid-lowering therapy is indispensible to further reduce cardiovascular events. One possible therapeutic option to the residual risk is to directly intervene in the inflammatory process by utilizing a nanotechnology-based drug delivery system (nano-DDS). Various nano-sized materials are currently developed as DDS, including micelles, liposomes, polymeric nanoparticles, dendrimers, carbon nanotubes, and metallic nanoparticles. The application of nano-DDS to coronary artery disease is a feasible strategy since the inflammatory milieu enhances incorporation of nano-sized materials into mononuclear phagocytic system and permeability of target lesions, which confers nano-DDS on "passive-targeting" property. Recently, we have developed a polymeric nanoparticle-incorporating statin to maximize its anti-inflammatory property. This statin nanoparticle has been tested in various disease models, including plaque destabilization and rupture, myocardial ischemia-reperfusion injury, and ventricular remodeling after acute myocardial infarction, and its clinical application is in progress. In this review, we present current development of DDS and future perspective on the application of anti-inflammatory nanomedicine to treat life-threatening cardiovascular diseases.

Mutant CCL2 protein coating mitigates wear particle-induced bone loss in a murine continuous polyethylene infusion model.

Wear particle-induced osteolysis limits the long-term survivorship of total joint replacement (TJR). Monocyte/macrophages are the key cells of this adverse reaction. Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) is the most important chemokine regulating trafficking of monocyte/macrophages in particle-induced inflammation. 7ND recombinant protein is a mutant of CCL2 that inhibits CCL2 signaling. We have recently developed a layer-by-layer (LBL) coating platform on implant surfaces that can release biologically active 7ND. In this study, we investigated the effect of 7ND on wear particle-induced bone loss using the murine continuous polyethylene (PE) particle infusion model with 7ND coating of a titanium rod as a local drug delivery device. PE particles were infused into hollow titanium rods with or without 7ND coating implanted in the distal femur for 4 weeks. Specific groups were also injected with RAW 264.7 as the reporter macrophages. Wear particle-induced bone loss and the effects of 7ND were evaluated by microCT, immunohistochemical staining, and bioluminescence imaging. Local delivery of 7ND using the LBL coating decreased systemic macrophage recruitment, the number of osteoclasts and wear particle-induced bone loss. The development of a novel orthopaedic implant coating with anti-CCL2 protein may be a promising strategy to mitigate peri-prosthetic osteolysis.

Ezetimibe in Combination With Statins Ameliorates Endothelial Dysfunction in Coronary Arteries After Stenting: The CuVIC Trial (Effect of Cholesterol Absorption Inhibitor Usage on Target Vessel Dysfunction After Coronary Stenting), a Multicenter Randomized Controlled Trial.

OBJECTIVES: We sought to investigate whether treatment with ezetimibe in combination with statins improves coronary endothelial function in target vessels in coronary artery disease patients after coronary stenting. APPROACH AND RESULTS: We conducted a multicenter, prospective, randomized, open-label, blinded-end point trial among 11 cardiovascular treatment centers. From 2011 to 2013, 260 coronary artery disease patients who underwent coronary stenting were randomly allocated to 2 arms (statin monotherapy, S versus ezetimibe [10 mg/d]+statin combinational therapy, E+S). We defined target vessel dysfunction as the primary composite outcome, which comprised target vessel failure during treatment and at the 6- to 8-month follow-up coronary angiography and coronary endothelial dysfunction determined via intracoronary acetylcholine testing performed in cases without target vessel failure at the follow-up coronary angiography. Coadministration of ezetimibe with statins further lowered low-density lipoprotein cholesterol levels (83±23 mg/dL in S versus 67±23 mg/dL in E+S; P<0.0001), with significant decreases in oxidized low-density lipoprotein and oxysterol levels. Among patients without target vessel failure, 46 out of 89 patients (52%) in the S arm and 34 out of 96 patients (35%) in the E+S arm were found to have coronary endothelial dysfunction (P=0.0256), and the incidence of target vessel dysfunction at follow-up was significantly decreased in the E+S arm (69/112 (62%) in S versus 47/109 (43%) in E+S; P=0.0059). A post hoc analysis of post-treatment low-density lipoprotein cholesterol-matched subgroups revealed that the incidence of both target vessel dysfunction and coronary endothelial dysfunction significantly decreased in the E+S arm, with significant reductions in oxysterol levels. CONCLUSIONS: The CuVIC trial (Effect of Cholesterol Absorption Inhibitor Usage on Target Vessel Dysfunction after Coronary Stenting) has shown that ezetimibe with statins, compared with statin monotherapy, improves functional prognoses, ameliorating endothelial dysfunction in stented coronary arteries, and was associated with larger decreases in oxysterol levels.