Inhibition of reactive nitrogen species production in COPD airways: comparison of inhaled corticosteroid and oral theophylline.

BACKGROUND: Reactive nitrogen species (RNS) are thought to be one of the important factors in the pathogenesis of chronic obstructive pulmonary disease (COPD). A study was undertaken to examine the effects of theophylline and fluticasone propionate (FP) on RNS production in subjects with COPD. METHODS: Sixteen COPD subjects participated in the study. Theophylline (400 mg/day orally) or FP (400 mug/day inhalation) were administered for 4 weeks in a randomised crossover manner with a washout period of 4 weeks. Induced sputum was collected at the beginning and end of each treatment period. 3-nitrotyrosine (3-NT), which is a footprint of RNS, was quantified by high performance liquid chromatography with an electrochemical detection method as well as by immunohistochemical staining. RESULTS: Theophylline significantly reduced the level of 3-NT in the sputum supernatant as well as the number of 3-NT positive cells (both p<0.01). FP also reduced 3-NT formation, but the effect was smaller than that of theophylline. Theophylline also significantly reduced the neutrophil cell counts in the sputum (p<0.01), while FP treatment had no effect on the number of inflammatory cells in the sputum, except eosinophils. CONCLUSIONS: Theophylline reduces nitrative stress and neutrophil infiltration in COPD airways to a larger extent than inhaled corticosteroid.

Terminally alkylated heparin. 2. Potent antiproliferative agent for vascular smooth muscle cells.

The antiproliferative activity of alkylated heparin, in which the terminal end of heparin is derivatized with an alkyl group (butyl, octyl, lauryl, stearyl), was examined using vascular smooth muscle cells. The proliferation of cells, which were growth-arrested prior to addition of heparin, was inhibited in proportion to both increase in the chain length of the alkyl group of alkylated heparin and alkylated heparin concentration in the serum-containing medium. The antiproliferative activity of stearyl group derivatized heparin was significantly stronger than that of nonmodified heparin. Little proliferation was observed at high dose (500 microg/mL). Confocal laser scanning microscopic observation indicated that alkylated heparin was accumulated on the cell membranes at an early incubation time, followed by homogeneous distribution of intracellular space. The therapeutic potential of alkylated heparin for preventing restenosis after balloon angioplasty is discussed.

Single low-dose administration of human recombinant hepatocyte growth factor attenuates intimal hyperplasia in a balloon-injured rabbit iliac artery model.

BACKGROUND: Previous studies have shown that repeated systemic administration of human recombinant hepatocyte growth factor (hrHGF) in mg/kg levels modulates the wound-healing process in various diseases. Recently, HGF has been characterized as one of the most potent endothelial-cell-specific growth factors. We tested our hypothesis that local delivery of hrHGF, even at low microg/kg levels (> or =2 orders of magnitude lower than systemically administered doses), might attenuate neointimal hyperplasia in response to vascular injury via accelerated reendothelialization. METHODS AND RESULTS: The iliac artery was denuded in 16 New Zealand White rabbits (3 kg), followed by administration, via a drug delivery catheter, of either hrHGF (10 microg; n = 11) or control vehicle (n=5) over 20 minutes. In pilot studies using this device, the drug permeated into the medial tissues, where it persisted for > or =24 hours. Four weeks after the local delivery of hrHGF, computer-assisted morphometric analysis revealed significant reduction in the intimal area (hrHGF, 0.37+/-0.21 versus control, 0.68+/-0.16 mm(2), mean +/- SD; P<0.05) but no change in the medial area (hrHGF, 1.03+/-0.21 versus control, 1.10+/-0.52 mm(2)). Scanning electron microscopy revealed extensive endothelialization with regular and confluent endothelial cell layer regeneration in the hrHGF-treated vessels. CONCLUSIONS: Accelerated endothelialization after local delivery of hrHGF, a novel and potent endothelial cell mitogen, effectively attenuates neointimal proliferation even after single low-dose administration. This observation could have potential therapeutic implications in the prevention of restenosis after angioplasty.

Epithelial cell kinetics in the inflammatory process of chicken trachea infected with infectious bronchitis virus.

All stages of degeneration and regeneration in chicken tracheal epithelium were studied morphologically following an intratracheal inoculation of infectious bronchitis virus (IBV). Viral antigen was detected in the cytoplasm of tracheal epithelium from 1 to 7 days post-inoculation (d.p.i.) with a peak on 3 d.p.i. At 1 d.p.i., almost all epithelial cells were involved in the degeneration. At this time, labelling index of bromodeoxyuridine (BrdU) in the basal cells showed significantly high value compared with control. At 2 and 3 d.p.i., a great number of basal cells were recognized, but the BrdU labelling index tended to decrease. At 4 and 5 d.p.i., the BrdU labelling index of basal cells significantly decreased than 1 d.p.i., and a few number of regenerated immature ciliated epithelia appeared. At 6 to 11 d.p.i., the ciliated columnar epithelia increased rapidly in number, and returned to the normal appearance except for non-ciliated patch by 13 d.p.i. These results suggested that the tracheal epithelial cells infected with IBV degenerated within 24 hours and proliferating activity of basal cells functioned immediately, and 3 to 4 days later, these basal cells were differentiated to the ciliated epithelia.

Calcium channel blocking and vasodilating actions of the novel dihydropyridine derivative AE0047.

1. The pharmacological characteristics of AE0047, a newly synthesized dihydropyridine (DHP) derivative, were investigated in vitro. 2. In bovine aortic membrane, AE0047 and other DHP calcium channel blockers (nitrendipine, nicardipine) displayed concentration-dependent antagonism to specific [3H]-PN200-110 binding sites with the following values for inhibition constants (Ki) obtained: 20.8 +/- 8.9, 12.3 +/- 4.5 and 3.9 +/- 1.0 nmol/L for AE0047, nitrendipine and nicardipine, respectively. 3. In guinea-pig ventricular myocytes, AE0047 blocked the L-type calcium current, with values for the dissociation constant (Kd) and Hill coefficient of 11.4 +/- 5.7 nmol/L and 0.852 +/- 0.061, respectively, indicating in the terms of Hill's hypothesis that one drug molecule blocks one calcium channel molecule. 4. In rat aorta, AE0047 inhibited 45Ca uptake induced by high K+ (100 mmol/L) by 55%. 5. AE0047 and nitrendipine concentration dependently relaxed rat aortic strips contracted with 30 mmol/L KCl. The response to nitrendipine reached a plateau within 60 min and disappeared after drug washing. Interestingly, AE0047 required 5 h or more to produce a plateau of response, with no effect of drug washing. This confirmed the slow onset and long duration of its vasodilating action. 6. With AE0047, tissue content in rat aorta increased more slowly than with nitrendipine and release of AE0047 from tissue was also slower. 7. The data suggest that AE0047 is incorporated slowly into smooth muscle membranes, approaches receptors slowly through the membrane bilayer and accumulates in the membrane because of its high lipophilicity, resulting in an anti-hypertensive action that is slow in onset and of long duration.

Possible mechanism of action of AE0047, a calcium antagonist, on triglyceride metabolism.

We evaluated the effect of AE0047, a dihydropyridine-type calcium antagonist, on the plasma lipid levels of obese Zucker rats. In rats treated orally with 3 to 10 mg/kg/day AE0047 for 7 days, plasma triglyceride (TG) and TG-rich lipoprotein levels dose-dependently decreased, whereas those of high-density lipoprotein cholesterol increased. Total cholesterol and low-density lipoprotein levels did not change. To elucidate the mechanism by which AE0047 decreases plasma TG levels, we examined the effect of AE0047 on the synthesis and secretion of TG-rich lipoproteins in human intestinal cell line Caco-2, as well as on the association and degradation of very low density lipoprotein (VLDL) in human hepatoblastoma cells HepG2. When Caco-2 cells were grown on a membrane filter and 14C-oleic acid was added to the apical side, 10(-5) and 10(-6) M AE0047 inhibited basolateral secretion of 14C-TG. AE0047 also suppressed the basolateral secretion of apolipoprotein B. In HepG2 cells, AE0047 increased the cellular uptake of 125I-VLDL. These results suggested that AE0047 decreased plasma TG level by the inhibition of intestinal chylomicron secretion and the enhancement of hepatic uptake of VLDL. AE0047 may be beneficial for the treatment of hypertensive patients with hypertriglyceridemia to reduce the risk factors of coronary heart disease.

Inhibition of cholesterol biosynthesis by squalene epoxidase inhibitor avoids apoptotic cell death in L6 myoblasts.

The relationship between the inhibition of cholesterol biosynthesis and occurrence of myopathy was studied in L6 myoblasts using two lines of cholesterol biosynthesis inhibitors, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (simvastatin) and squalene epoxidase inhibitors (TU-2078 and NB-598). All inhibitors completely inhibited the cholesterol synthesis in L6 myoblasts at doses of 1 and 3 microM. Simvastatin (3 microM) inhibited the fusion reaction of L6 myoblasts followed by the severe cellular damage. The myoblasts also had failed actin fiber formation and creatinine phosphokinase (CPK) production. Additionally, this agent also caused apoptotic cell death in differentiated L6 muscle fiber, indicating that skeletal myopathy by HMG-CoA reductase inhibitors seems to occur not only in differentiating immature myoblasts but also in matured skeletal myotubes. In contrast, TU-2078 and NB-598 had no effect on the fusion reaction of differentiating myoblasts or on the cellular viability of muscle fiber at 3 microM, enough to completely inhibit cholesterol biosynthesis. It is conceivable that the mevalonate depletion and subsequent failure of ras farnesylation induced by simvastatin might cause the defects in differentiation and maintenance of the muscle fiber. Squalene epoxidase inhibitors did not show this adverse effect presumably because of the enzyme inhibition downstream of farnesyl synthesis. The present findings suggest the safe use of squalene epoxidase inhibitors in lipid-lowering therapy.

Synthesis and pharmacological evaluation of N-(6-functionalized-amino-3-pyridyl)-N'-bicycloalkyl-N''-cyanoguanidine s as antihypertensive agents.

A series of amino acid conjugates of N-(6-amino-3-pyridyl)-N'-[exo-bicyclo[2.2.1]hept-2-yl]-N''- cyanoguanidine (4) were prepared and evaluated as antihypertensive agents. The parent compound 4 showed potent potassium channel-opening and antihypertensive activities, but with undesirable changes of the urinary balance of electrolytes. However, alanine and histidine congeners (9,19) reduced this undesirable side effect of 4 through improved pharmacokinetics without loss of antihypertensive activity. They also provided additional information on the structural requirements for pinacidil-type potassium channel openers.

A possible mechanism of action of a new potassium channel opener, AL0671, on lipid metabolism in obese Zucker rats.

Antihypertensive drugs are expected to have a lipid-lowering effect for use in treating ischemic heart disease. We evaluated the effect of (+)-N-(6-amino-3-pyridil)-N'-[(1S,2R,4R)-bicyclo-[2.2.1]hept-2-yl] -N"- cyanoguanidine hydrochloride (AL0671), a newly synthesized cyanoguanidine-derivative potassium channel opener, on serum lipid and lipoprotein levels in obese Zucker rats, a genetically engineered model of type IV hyperlipidemia. AL0671 dose-dependently decreased systolic blood pressure in obese Zucker rats. Serial administration (for 1 or 2 weeks) of AL0671 (5 mg/kg/day) significantly decreased serum total triglyceride, chylomicron and very-low-density lipoprotein levels with increasing high-density lipoprotein cholesterol, whereas low-density lipoprotein levels did not change. AL0671 (5 mg/kg/day) increased lipoprotein lipase activities 4-fold and hepatic triglyceride lipase activities 3-fold in postheparin plasma. Another urea-derivative compound, AL0674, whose potassium channel-opening activity is diminished, did not affect serum lipid and lipoprotein levels. These results suggested that AL0671 activates both lipoprotein lipase and hepatic triglyceride lipase activities through its potassium channel-opening activity followed by decreasing triglyceride-rich lipoproteins in genetically obese hyperlipemic rats. Therefore, AL0671 might be beneficial in the treatment of hypertensive patients with hypertriglyceridemia (probably with insulin resistance).

Novel potassium-channel openers: preparation and pharmacological evaluation of racemic and optically active N-(6-amino-3-pyridyl)-N'-bicycloalkyl-N"-cyanoguanidine derivatives.

The previous paper reported on the synthesis and pharmacological evaluation of N-(6-amino-3-pyridyl)-N'-bicycloalkyl-N"-cyanoguanidine derivatives, from among which three compounds were selected as potent potassium-channel openers. In the present study, selected compounds were tested for antagonism of potassium-induced contraction of rat aorta, hypotensive activity in normotensive rats, and diuretic activity in spontaneously hypertensive rats. This led to further evaluation of compound (+/-)-10 and selection of (+)-N-(6-amino-3-pyridyl)-N'- [(1S,2R,4R)-bicyclo- [2.2.1]hept-2-yl]-N"-cyanoguanidine ((+)-10) (AL0670) for development as an antihypertensive agent. Although AL0670 is regarded as a pinacidil-type K(+)-channel opener, it showed different pharmacological and conformational profiles from pinacidil.

Novel potassium channel openers: synthesis and pharmacological evaluation of new N-(substituted-3-pyridyl)-N'-alkylthioureas and related compounds.

This report describes the synthesis and pharmacological evaluation of a series of novel potassium channel openers related to the pinacidil-type compounds. Thioureas, cyanoguanidines, and pyridine N-oxides were systematically evaluated for their effects on both the inhibition of spontaneous mechanical activity in rat portal vein (in vitro) and their antihypertensive activity (in vivo), and the structure-activity relationship for this series of compounds was discussed. Good correlation between in vitro and iv antihypertensive activity was observed for these compounds. Among them, cyanoguanidines bearing a conformationally rigid unit such as a norbornyl group generally possessed potent activity in both in vitro and in vivo studies. Especially, N-(6-amino-3-pyridyl)-N'-cyano-N"-(1-methyl-2-norbornyl)guanidine (23d) was identified as a more potent potassium channel opener in vitro (EC100 = 3 x 10(-8) M) than pinacidil (EC100 = 10(-7) M).