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.

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 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.

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.

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.

Ezetimibe reduced hepatic steatosis induced by dietary oxysterols in nonhuman primates.

Oxidized cholesterol (oxysterols) plays an important and multifaceted role in lipid metabolism. Here we examined whether dietary oxysterols accelerate hepatic lipid accumulation and inflammation in nonhuman primates. We also examined the effect of the Niemann-Pick C1-like1 inhibitor, ezetimibe (Ez). Macaca fascicularis (5-year-old males) were fed either regular cholesterol + high-fat diet (control-HFD) or oxysterols + high-fat diet (ox-HFD; with 0.015% of oxysterols cholesterol) for 24 weeks. Compared with control-HFD, ox-HFD did not affect plasma lipid levels, but it did affect hepatic lipid levels [total cholesterol, 40.9 mg·g-1 (ox-HFD) versus 3.2 (control-HFD) mg·g-1; triglycerides, 28.0 (ox-HFD) versus 5.7 (control-HFD) mg·g-1]. Ox-HFD increased lipid accumulation as well as recruitment of inflammatory cells when compared to control-HFD. We then examined the effects of Ez, 0.2 mg·kg-1·day-1 for 12 weeks. In addition to a significant reduction in dyslipidemia, Ez alleviated biochemical and pathological aspects of steatosis. Dietary oxysterols aggravate steatosis in nonhuman primates. Treatment with Ez may be a novel therapeutic approach to NAFLD by alleviating dyslipidemia.

A Translational Study of a New Therapeutic Approach for Acute Myocardial Infarction: Nanoparticle-Mediated Delivery of Pitavastatin into Reperfused Myocardium Reduces Ischemia-Reperfusion Injury in a Preclinical Porcine Model.

BACKGROUND: There is an unmet need to develop an innovative cardioprotective modality for acute myocardial infarction, for which interventional reperfusion therapy is hampered by ischemia-reperfusion (IR) injury. We recently reported that bioabsorbable poly(lactic acid/glycolic acid) (PLGA) nanoparticle-mediated treatment with pitavastatin (pitavastatin-NP) exerts a cardioprotective effect in a rat IR injury model by activating the PI3K-Akt pathway and inhibiting inflammation. To obtain preclinical proof-of-concept evidence, in this study, we examined the effect of pitavastatin-NP on myocardial IR injury in conscious and anesthetized pig models. METHODS AND RESULTS: Eighty-four Bama mini-pigs were surgically implanted with a pneumatic cuff occluder at the left circumflex coronary artery (LCx) and telemetry transmitters to continuously monitor electrocardiogram as well as to monitor arterial blood pressure and heart rate. The LCx was occluded for 60 minutes, followed by 24 hours of reperfusion under conscious conditions. Intravenous administration of pitavastatin-NP containing ≥ 8 mg/body of pitavastatin 5 minutes before reperfusion significantly reduced infarct size; by contrast, pitavastatin alone (8 mg/body) showed no therapeutic effects. Pitavastatin-NP produced anti-apoptotic effects on cultured cardiomyocytes in vitro. Cardiac magnetic resonance imaging performed 4 weeks after IR injury revealed that pitavastatin-NP reduced the extent of left ventricle remodeling. Importantly, pitavastatin-NP exerted no significant effects on blood pressure, heart rate, or serum biochemistry. Exploratory examinations in anesthetized pigs showed pharmacokinetic analysis and the effects of pitavastatin-NP on no-reflow phenomenon. CONCLUSIONS: NP-mediated delivery of pitavastatin to IR-injured myocardium exerts cardioprotective effects on IR injury without apparent adverse side effects in a preclinical conscious pig model. Thus, pitavastatin-NP represents a novel therapeutic modality for IR injury in acute myocardial infarction.

Nanoparticle-Mediated Delivery of Irbesartan Induces Cardioprotection from Myocardial Ischemia-Reperfusion Injury by Antagonizing Monocyte-Mediated Inflammation.

Myocardial ischemia-reperfusion (IR) injury limits the therapeutic effect of early reperfusion therapy for acute myocardial infarction (AMI), in which the recruitment of inflammatory monocytes plays a causative role. Here we develop bioabsorbable poly-lactic/glycolic acid (PLGA) nanoparticles incorporating irbesartan, an angiotensin II type 1 receptor blocker with a peroxisome proliferator-activated receptor (PPAR)γ agonistic effect (irbesartan-NP). In a mouse model of IR injury, intravenous PLGA nanoparticles distribute to the IR myocardium and monocytes in the blood and in the IR heart. Single intravenous treatment at the time of reperfusion with irbesartan-NP (3.0 mg kg(-1) irbesartan), but not with control nanoparticles or irbesartan solution (3.0 mg kg(-1)), inhibits the recruitment of inflammatory monocytes to the IR heart, and reduces the infarct size via PPARγ-dependent anti-inflammatory mechanisms, and ameliorates left ventricular remodeling 21 days after IR. Irbesartan-NP is a novel approach to treat myocardial IR injury in patients with AMI.

Nanoparticle-Mediated Delivery of Mitochondrial Division Inhibitor 1 to the Myocardium Protects the Heart From Ischemia-Reperfusion Injury Through Inhibition of Mitochondria Outer Membrane Permeabilization: A New Therapeutic Modality for Acute Myocardial Infarction.

BACKGROUND: Mitochondria-mediated cell death plays a critical role in myocardial ischemia-reperfusion (IR) injury. We hypothesized that nanoparticle-mediated drug delivery of mitochondrial division inhibitor 1 (Mdivi1) protects hearts from IR injury through inhibition of mitochondria outer membrane permeabilization (MOMP), which causes mitochondrial-mediated cell death. METHODS AND RESULTS: We formulated poly (lactic-co-glycolic acid) nanoparticles containing Mdivi1 (Mdivi1-NP). We recently demonstrated that these nanoparticles could be successfully delivered to the cytosol and mitochondria of cardiomyocytes under H2O2-induced oxidative stress that mimicked IR injury. Pretreatment with Mdivi1-NP ameliorated H2O2-induced cell death in rat neonatal cardiomyocytes more potently than Mdivi1 alone, as indicated by a lower estimated half-maximal effective concentration and greater maximal effect on cell survival. Mdivi1-NP treatment of Langendorff-perfused mouse hearts through the coronary arteries at the time of reperfusion reduced infarct size after IR injury more effectively than Mdivi1 alone. Mdivi1-NP treatment also inhibited Drp1-mediated Bax translocation to the mitochondria and subsequent cytochrome c leakage into the cytosol, namely, MOMP, in mouse IR hearts. MOMP inhibition was also observed in cyclophilin D knockout (CypD-KO) mice, which lack the mitochondrial permeability transition pore (MPTP) opening. Intravenous Mdivi1-NP treatment in vivo at the time of reperfusion reduced IR injury in wild-type and CypD-KO mice, but not Bax-KO mice. CONCLUSIONS: Mdivi1-NP treatment reduced IR injury through inhibition of MOMP, even in the absence of a CypD/MPTP opening. Thus, nanoparticle-mediated drug delivery of Mdivi1 may be a novel treatment strategy for IR injury.

Nanoparticle-Mediated Targeting of Cyclosporine A Enhances Cardioprotection Against Ischemia-Reperfusion Injury Through Inhibition of Mitochondrial Permeability Transition Pore Opening.

Myocardial ischemia-reperfusion (IR) injury limits the therapeutic effects of early reperfusion therapy for acute myocardial infarction (MI), in which mitochondrial permeability transition pore (mPTP) opening plays a critical role. Our aim was to determine whether poly-lactic/glycolic acid (PLGA) nanoparticle-mediated mitochondrial targeting of a molecule that inhibits mPTP opening, cyclosporine A (CsA), enhances CsA-induced cardioprotection. In an in vivo murine IR model, intravenously injected PLGA nanoparticles were located at the IR myocardium mitochondria. Treatment with nanoparticles incorporated with CsA (CsA-NP) at the onset of reperfusion enhanced cardioprotection against IR injury by CsA alone (as indicated by the reduced MI size at a lower CsA concentration) through the inhibition of mPTP opening. Left ventricular remodeling was ameliorated 28 days after IR, but the treatment did not affect inflammatory monocyte recruitment to the IR heart. In cultured rat cardiomyocytes in vitro, mitochondrial PLGA nanoparticle-targeting was observed after the addition of hydrogen peroxide, which represents oxidative stress during IR, and was prevented by CsA. CsA-NP can be developed as an effective mPTP opening inhibitor and may protect organs from IR injury.