Pharmacokinetics of low doses of colchicine in the leukocytes of Japanese healthy individuals.

The venerable drug colchicine has garnered significant recent attention due to its endorsement by the United States Food and Drug Administration as an anti-inflammatory medication for cardiovascular diseases. However, the administration of this drug at its minimal available dose of 0.5 mg has been associated with certain adverse reactions. Once colchicine is administered, the drug disappears from blood in a short time and distributes in the leukocytes for a certain period of time that elicits anti-inflammatory effect. Consequently, an in-depth comprehension of the pharmacokinetics of lower dosages within leukocytes assumes important for its broader application in routine clinical contexts. In this study, we present a comprehensive analysis of the pharmacological disposition of colchicine in the plasma, polymorphonuclear leukocytes, and mononuclear leukocytes among healthy Japanese male subjects, following both single and multiple oral administrations of 0.5 mg and 0.25 mg doses of colchicine. Our investigation reveals that colchicine persists within leukocyte populations even when administered at reduced dosages. The findings herein hold promise for mitigating the adverse effects associated with its use in the treatment of inflammatory cardiovascular disorders.

Effects of Low-Dose Colchicine on Serum High-Sensitivity C-Reactive Protein Level in Coronary Artery Disease Patients with Type 2 Diabetes Mellitus and Enhanced Inflammatory Response Protocol for a Randomized, Double-Blind, Placebo-Controlled, Phase 2, Dose-Finding Study.

Although cardiovascular mortality in Japan is lower than in other industrialized countries, clinical outcomes in coronary artery disease (CAD) patients with type 2 diabetes mellitus (T2DM) remain poor despite multiple evidence-based drug therapies and interventions. We assumed that part of residual risk in these patients may be attributable to enhanced inflammation, which can be inhibited presumably by colchicine. However, dose-responsiveness of anti-inflammatory effect of colchicine has not been elucidated. Therefore, we designed a multicenter, randomized, double-blinded, parallel-group study to explore the dose-dependent effects of low-dose colchicine on serum high-sensitivity C-reactive protein (hs-CRP) concentration and safety in CAD patients with T2DM and enhanced inflammatory response as a phase 2 study. Enhanced inflammatory response was defined as peripheral white-blood cell count ≥7,000/µL. Patients (N = 63) will be randomly assigned to two doses of colchicine 0.25 mg/day, 0.5 mg/day, or placebo in a 1:1:1 ratio once daily for 12 weeks. Changes in serum hs-CRP levels will be evaluated as the primary endpoint, and changes in flow-mediated vasodilation and plasma myeloperoxidase levels will be evaluated as secondary endpoints. The results of this study will contribute to the development of a protocol for a planned future phase 3 trial to estimate the reduction in CAD. The present study describes the rationale, design, and methods of the trial.

Effect of short-term colchicine treatment on endothelial function in patients with coronary artery disease.

BACKGROUND: Inflammation is associated with endothelial dysfunction and plays an important role in the pathogenesis and development of cardiovascular diseases. It has been shown that colchicine, an anti-inflammatory drug, improves the cardiovascular outcome in patients with cardiovascular disease. The purpose of this study was to evaluate the short-term effect of low-dose colchicine on endothelial function in patients with coronary artery disease (CAD). METHODS: This was a double-blind, randomized, placebo-controlled, crossover-within-subject clinical trial. A total of 28 patients with CAD received low-dose colchicine (0.5 mg/day) or a placebo for 7 days with a washout period of at least 14 days. Flow-mediated vasodilation (FMD) and serum concentrations of high-sensitivity C-reactive protein (hs-CRP) were measured after the 7-day treatment with colchicine or the placebo. RESULTS: The serum concentration of hs-CRP was significantly decreased after administration of colchicine compared with that after administration of the placebo [median (interquartile range): 0.04 (0.02-0.08) mg/dL vs. 0.07 (0.04-0.11) mg/dL, P = 0.003], while there was no significant difference in FMD between the treatments [median (interquartile range): 3.1% (1.5-5.3%) vs. 3.3% (1.9-5.2%), P = 0.384]. Colchicine, however, significantly improved FMD in coronary artery disease patients with white blood cell (WBC) counts of ≥7500 WBC/mm3 [median (interquartile range): 3.3% (2.1-6.6%) vs. 2.0% (1.4-3.8%), P = 0.043]. CONCLUSIONS: Administration of low-dose colchicine did not improve endothelial function in patients with CAD, but exploratory analysis suggested that endothelial function is significantly improved in patients with leukocyte activation.

Renin-Angiotensin System Inhibitors Can Prevent Intravenous Lipid Infusion-Induced Myocardial Microvascular Dysfunction and Leukocyte Activation.

BACKGROUND: Levels of triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to endothelial dysfunction through renin-angiotensin system (RAS) activation and oxidative stress. The present study investigated how systemic FFA loading affected myocardial microcirculation during hyperemia via RAS.Methods and Results:Eight healthy men received candesartan, perindopril, or a placebo for 2 days in a double-blind crossover design, and then myocardial microcirculation during hyperemia induced by a 2-h infusion of lipid/heparin was assessed using dipyridamole stress-myocardial contrast echocardiography (MCE). Leukocyte activity and hemorheology were also assessed ex vivo using a microchannel flow analyzer, serum levels of oxidative stress markers, and IκB-α expression in mononuclear cells. Serum FFA elevation by the infusion of lipid/heparin significantly decreased myocardial capillary blood velocity and myocardial blood flow during hyperemia. Both candesartan and perindopril significantly prevented the FFA-induced decrease in capillary blood velocity and myocardial blood flow during hyperemia. Systemic FFA loading also caused an increase in the number of adherent leukocytes and prolonged the whole blood passage time. These effects were blocked completely by candesartan and partially by perindopril. Both agents prevented the FFA-induced enhancement of oxidative stress and IκB-α degradation in mononuclear cells. CONCLUSIONS: Both candesartan and perindopril can prevent FFA-induced myocardial microcirculatory dysfunction during hyperemia via modulation of leukocyte activation and microvascular endothelial function.

Effects of Oral Antidiabetic Drugs on Changes in the Liver-to-Spleen Ratio on Computed Tomography and Inflammatory Biomarkers in Patients With Type 2 Diabetes and Nonalcoholic Fatty Liver Disease.

PURPOSE: Oral antidiabetic drugs (OADs) such as pioglitazone and metformin have beneficial effects in patients with nonalcoholic steatohepatitis. We prospectively assessed the effects of OADs on nonalcoholic fatty liver disease (NAFLD) in 886 men with type 2 diabetes mellitus and in a murine model of NAFLD. METHODS: Patients were randomized to receive pioglitazone, metformin, sitagliptin, or a non-OAD (control) for 6 months. All the patients received dietary and exercise guidance once a month during this study. Changes in the liver-to-spleen ratio on computed tomography (CT) and NAFLD-related parameters were measured from baseline to the end of treatment. FINDINGS: The liver/spleen ratio improved significantly in the pioglitazone and metformin groups compared with the control group (both P < 0.01), but not in the sitagliptin group (P = 0.73). The mean changes from baseline were -3.464 ± 10.156%, 19.236 ± 9.896%, 4.783 ± 1.467%, and 1.328 ± 0.802% in the control, pioglitazone, metformin, and sitagliptin groups, respectively. Multivariable analysis showed that the liver/spleen ratio was strongly correlated with high-sensitivity C-reactive protein concentration in the pioglitazone group (F = 9.973; P < 0.01) and abdominal visceral fat volume in the metformin group (F = 6.049; P < 0.05). CONCLUSIONS: Pioglitazone elicited the greatest improvements in features of NAFLD in type 2 diabetes mellitus. (Trial Registration: www.isrctn.org/, ISRCTN33414972, http://www.isrctn.org/).

Peroxidized unsaturated fatty acids stimulate Toll-like receptor 4 signaling in endothelial cells.

AIM: Although unsaturated fatty acids are assumed to be protective against inflammatory disorders that include a pathway involving Toll-like receptor 4 (TLR4) activation, they might actually be toxic because of their high susceptibility to lipid peroxidation. Here we studied the effects of peroxidized unsaturated fatty acids on the TLR4-nuclear factor (NF)-κB pathway in endothelial cells. MAIN METHODS: Confluent cultured endothelial cells from bovine aorta were incubated for 1h with fatty acids integrated into phosphatidylcholine vesicles. Lipopolysaccharide (LPS) or phosphatidylcholine vesicles without fatty acids were also applied as a positive control or a control for fatty acid groups, respectively. Activation of TLR4 and downstream signaling was assessed by membrane fractionation and Western blotting or immunofluorescent staining. KEY FINDINGS: In the same way as LPS, application of sufficiently peroxidized unsaturated fatty acids like oleic acid or docosahexaenoic acid, acutely caused TLR4 translocation to caveolae/raft membranes, leading to activation of NF-κB signaling in endothelial cells. In contrast, saturated fatty acids did not show such effects. Applying well-peroxidized unsaturated fatty acids, but not saturated fatty acids, acutely activates the TLR4/NF-κB pathway. SIGNIFICANCE: Peroxidation of unsaturated fatty acid is essential for the acute activation of TLR4 by the fatty acids that follow the same pathway as the activation by LPS. Unsaturated fatty acids have been assumed to be protective against inflammatory disorders, and drugs containing unsaturated fatty acids are now developed and provided. Our result suggests that, for inflammatory disorders involving TLR4 signaling, using unsaturated fatty acids as anti-inflammatory drugs may cause contrary effects.

Dihydropyridine calcium channel blockers inhibit non-esterified-fatty-acid-induced endothelial and rheological dysfunction.

Circulating NEFAs (non-esterified fatty acids) from adipose tissue lipolysis lead to endothelial dysfunction and insulin resistance in patients with the metabolic syndrome or Type 2 diabetes mellitus. The aim of the present study was to test the hypothesis that DHP (dihydropyridine) CCBs (calcium channel blockers) prevent NEFA-induced endothelial and haemorheological dysfunction independently of their antihypertensive properties. Using a double-blind cross-over study design, nifedipine, amlodipine, diltiazem or placebo were administered to eight healthy subjects for 2 days before each study day. On the study days, the following were assessed before and after the infusion of lipid and heparin to raise serum NEFAs: endothelial function, by measuring FBF (forearm blood flow) responses to ACh (acetylcholine); leucocyte activation, by ex vivo measurement of plasma MPO (myeloperoxidase) levels, adherent leucocyte numbers and whole blood transit time through microchannels; and oxidative stress, by determining plasma levels of d-ROMs (derivatives of reactive oxygen metabolites). Effects of the CCBs on NF-κB (nuclear factor κB) p65 phospholylation stimulated by NEFAs were assessed in cultured monocytic cells in vitro. Elevated NEFAs reduced the responses to ACh and significantly increased whole blood transit time, adherent leucocyte numbers and d-ROMs. Nifedipine and amlodipine, but not diltiazem, prevented NEFA-induced endothelial dysfunction, leucocyte activation and enhancement of oxidative stress without affecting BP (blood pressure), whereas all these drugs prevented NEFA-induced p65 activation in vitro. These results suggest that DHP CCBs, independent of their antihypertensive properties in humans, prevent NEFA-induced endothelial and haemorheological dysfunction through inhibition of NEFA-induced leucocyte activation, although the sensitivity to drugs of leucocyte Ca2+ channels may differ among cells.

Aldosterone enhances ligand-stimulated nitric oxide production in endothelial cells.

Chronic and acute actions of aldosterone have been shown recently to directly affect the cardiovascular system. However, it is unclear whether the acute effects of aldosterone on vasculature are constrictive or dilatory. Here, to clarify the nongenomic effects of aldosterone on endothelial function, we examined the effects of aldosterone on nitric oxide (NO) production in cultured endothelial cells (ECs) and on vascular tone. The intracellular NO production of bovine aortic ECs loaded with DAF-2 was determined using confocal microscopy. Accumulated NO in the culture medium was quantified by a microplate reader using membrane-impermeable DAF-2. Phosphorylation of endothelial NO synthase (eNOS) at Ser(1179) was assessed by Western blotting. Changes in intracellular Ca(2+) ([Ca(2+)](i)) were determined by confocal microscopy in ECs doubly loaded with fluo-4 and Fura Red. The effects of aldosterone, acetylcholine (ACh), and other signaling molecules on the tension of phenylephrine (PE)-contracted aortas of Sprague-Dawley rats were examined in an ex vivo organ bath chamber system. Short-term pre-exposure to aldosterone (1 x 10(-7) mol/L) enhanced ATP-induced NO production in ECs with increased phosphorylation of eNOS at Ser(1179). These effects were blocked by eplerenone, a mineralocorticoid receptor (MR) antagonist, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Notably, aldosterone alone did not affect ATP-induced [Ca(2+)](i) changes or the Ser(1179) phosphorylation. Similarly, aldosterone (1 x 10(-8) to 1 x 10(-7) mol/L) did not affect the tone of rat aortas pre-contracted by PE, but enhanced ACh-induced vasorelaxation, which was again reversed by eplerenone or LY29400. In contrast, sodium nitroprusside-induced vasorelaxation in endothelium-denuded aortas was not affected by aldosterone. Thus, aldosterone acutely enhances ligand-mediated endothelial NO production by eplerenone-sensitive mechanisms involving a PI3K that may synergize Ca(2+)-dependent eNOS phosphorylation at Ser(1179).

Subcortical Ca2+ waves sneaking under the plasma membrane in endothelial cells.

Subplasmalemmal Ca2+, dynamically equilibrated with extracellular Ca2+, affects numerous signaling molecules, effectors, and events within this restricted space. We demonstrated the presence of a novel Ca2+ wave propagating beneath the plasma membrane in response to acute elevation of extracellular [Ca2+], by targeting a Ca2+ sensor, cameleon, to the endothelial plasmalemma. These subcortical waves, spatially distinct from classical cytosolic Ca2+ waves, originated in localized regions and propagated throughout the subplasmalemma. Translocation of an expressed GFP fused with a PH domain of PLC from the plasma membrane to the cytosol accompanied these subcortical waves, and U73122 attenuated not only the GFP-PH translocation, but also the peak amplitude of the subcortical Ca2+ waves; this finding suggests the involvement of local IP3 production through PLC-mediated PIP2 hydrolysis in the initiation of these waves. Changes in NO production as well as PKCbeta-GFP translocation from the cytosol to the plasma membrane, but not of GFP-PLA2 to perinuclear endomembranes, were associated with the subplasmalemmal Ca2+ changes. Thus, extracellular Ca2+ maintains the basal PLC activity of the plasma membrane, is involved in the initiation of compartmentalized subcortical Ca2+ waves, and regulates Ca2+-dependent signaling molecules residing in or translocated to the plasma membrane. The full text of this article is available online at http://circres.ahajournals.org.