ΑΤ1 receptor (AT1R) antagonists exert their antihypertensive effects by preventing the vasoconstrictive hormone AngII to bind to the AT1 receptor. It has been proposed that these biological effects ...are mediated through a two-step mechanism reaction. In the first step, they are incorporated in the core of the lipid bilayers and in the second step they reach the active site of the receptor through lateral diffusion. In this model, drug/membrane interactions are key elements for the drugs achieving inhibition at the AT1 receptor. In this work, the interactions of the prodrug candesartan cilexetil (TCV-116) with lipid bilayers are studied at molecular detail. Solid-state 13C-CP/MAS, 2D 1H-1H NOESY NMR spectroscopy and in silico calculations are used. TCV-116 and olmesartan, another drug which acts as an AT1R antagonist are compared for their dynamic effects in lipid bilayers using solid-state 2H-NMR. We find a similar localization of TCV-116 compared to other AT1 antagonists in the intermediate polar region. In addition, we can identify specific local interactions. These interactions may be associated in part with the discrete pharmacological profiles observed for different antagonists.
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•Comparative dynamic effects of TCV-116 and olmesartan•Information for topography of TCV-116 and olmesartan in phospholipids•Drug/membrane interactions (use of solid state NMR, in silico calculations)•Cholesterol appears to modify and compete with the properties of AT1R antagonists.
Development of safe and effective drugs requires complete impurity evaluation and, therefore, knowledge about the formation and elimination of impurities is necessary. During impurity profiling of a ...key intermediate during synthesis of candesartan cilexetil (1-(((cyclohexyloxy)carbonyl) oxy)ethyl 1-((2’-(2
-tetrazol-5-yl)-1,1’-biphenyl-4-yl) methyl)-2-ethoxy-1
-benzo
imidazole-7-carboxylate, TCV-116), a novel compound, which had not been reported previously, was observed. Structural elucidation of impurity was achieved by liquid chromatography hyphenated to different high resolution mass analyzers. Based on exact mass measurements and fragmentation pattern, a chloro alkyl carbonate ester analogue of the intermediate was identified. Structure of the impurity was confirmed by mass spectro-metric and NMR analyses of the target substance. Identified impurity could represent a hazard if it is transferred to the final API stage and its presence should be kept below allowed limits. Further investigation could reveal whether bis(1-chloroethyl) carbonate is a precursor to impurity formation. Therefore, synthesis should be regulated so as to minimize impurity production. Analysis of the final product indicated that the amount of impurity did not exceed 50 mg L
, which represents the detection limit, determined according to the signal/noise ratio.
It was reported that angiotensin II stimulates angiogenesis in vivo, and angiotensin-converting enzyme (ACE) inhibitors inhibit angiogenesis. We found that an AT1-receptor (AT1-R) antagonist, ...TCV-116, inhibited tumor growth, tumor-associated angiogenesis, and metastasis in a murine model. Tumor growth of Sarcoma 180 (S-180) cells and of fibrosarcoma (NFSA) cells was strongly inhibited by administration of TCV-116 in the diet at a dose of approximately 100
mg/kg/day. This reduction was accompanied with a marked reduction in tumor-associated angiogenesis. The same treatment also reduced the lung metastasis of intravenously injected Lewis lung carcinoma cells. These effects of TCV-116 were equivalent to those of the ACE inhibitor, lisinopril. In S-180 and NFSA tumor tissues, ACE and AT1a receptor (AT1a-R) mRNAs were expressed when assessed with RT-PCR. AT1b receptor and AT2 receptor, however, were not detected. Immunoreactive AT1-R was detected mainly on the neovascularized vascular endothelial cells in which expression was reduced by TCV-116 and lisinopril. These results suggested that TCV-116 inhibits the angiogenesis, growth, and metastasis of tumors highly dependent on AT1a-R blockade. Blockade of AT1a-R signaling may therefore become an effective novel strategy for tumor chemoprevention.
The results of the EUCLID trial (EURODIAB Controlled Trial of Lisinopril in Insulin-dependent Diabetes Mellitus) highlighted the importance of the renin-angiotensin system in the pathogenesis of ...diabetic retinopathy. Candesartan cilexetil (TCV-116), a potent angiotensin II (AII) receptor antagonist, has beneficial effects on hypertension as well as on heart, renal and cerebrovascular disease. We aimed to evaluate the effectiveness of candesarten cilexetil in ameliorating retinal disorders induced by hyperglycaemia.
We compared retinal vascular endothelial growth factor (VEGF) mRNA expression and the latencies of retinal oscillatory potentials in TCV-116-treated and control groups of stroke-prone spontaneously hypertensive rats with streptozocin (STZ)-induced diabetes.
Retinal VEGF mRNA expression was significantly higher and the latencies of oscillatory potentials were significantly elongated in STZ-treated spontaneously hypertensive rats compared with a non-treated spontaneously hypertensive rat group matched for age. These changes were dependent on hyperglycaemia but independent of hypertension. Treatment with TCV-116 (3 mg/kg) significantly diminished retinal VEGF mRNA expression and the latencies of oscillatory potential peaks, but had no effect on plasma glucose concentrations.
These results suggest that TCV-116 is effective in preventing the development of diabetic retinopathy already in the early stages.
Vascular injury activates various kinds of genes, including proto-oncogenes, growth factors, and extracellular matrix proteins. However, the significance of activation of these genes in neointimal ...formation is poorly understood. Angiotensin II type 1 (AT1) receptor antagonist is shown to prevent neointimal formation after vascular injury, although the mechanism is unclear. To understand the molecular mechanism of vascular thickening, we examined the effects of AT1 receptor blockade on the gene expression of proto-oncogenes, transforming growth factor-beta 1 (TGF-beta 1), and extracellular matrix proteins after vascular injury.
Endothelial denudation of the left common carotid artery in Sprague-Dawley rats was performed with a Fogarty 2F balloon catheter. TCV-116 (10 mg.kg-1.d-1), a selective nonpeptide AT1 receptor antagonist, or vehicle was administered orally to rats from 1 day before to 14 days after balloon injury. Injured left and uninjured right common carotid arteries were removed from rats at 1, 6, and 24 hours and 3, 7, and 14 days after balloon injury. Tissue mRNA levels were measured with Northern blot analysis using specific cDNA probes and corrected for 18S ribosomal RNA value. Arterial mRNAs for c-fos, c-jun, jun B, jun D, and Egr-1 increased significantly at 1 hour after balloon injury and decreased rapidly. At 6 hours, ornithine decarboxylase (ODC) mRNA expression reached the maximal levels. TGF-beta 1 and fibronectin mRNA levels started to increase at 6 hours after injury and remained enhanced until 7 days after injury. On the other hand, collagen types I, III, and IV and laminin mRNA levels were not significantly increased over 7 days. Treatment with TCV-116 significantly inhibited the induction of mRNAs for c-fos, c-jun, Egr-1, ODC, and fibronectin in injured artery, whereas the increase in TGF-beta 1 gene expression after injury was not prevented by TCV-116. Immunohistological studies indicated that TCV-116 decreased not only the intimal thickening but also the amount of these extracellular matrix proteins in the intima.
The results indicate that AT1 receptor blockade inhibits the induction of immediate-early genes, ODC, and fibronectin in rat injured artery. Thus, inhibition of intimal thickening by AT1 receptor blockade may be mediated at least in part by suppression of multiple genes related to cell growth and migration in the very early phase after vascular injury.
The effects of chronic treatment with an angiotensin II receptor antagonist, candesartan cilexetil (TCV-116, 0.1, 1, 10 mg/kg), and an angiotensin converting enzyme inhibitor, enalapril maleate ...(enalapril, 10 mg/kg), on the development of end-organ damage were examined in stroke-prone spontaneously hypertensive rats (SHRSP). The control SHRSP developed severe hypertension with stroke signs and increased urinary protein excretion. TCV-116 (0.1 mg/kg) reduced the stroke incidence and urinary protein excretion without affecting the blood pressure. TCV-116 (1 and 10 mg/kg) and enalapril reduced blood pressure, the stroke incidence, the urinary indices and left ventricular weight. Circulating renin-angiotensin system (RAS) and renal renin mRNA expression were significantly accelerated or tended to be accelerated in the control SHRSP with end-organ damages. A low dose of TCV-116 tended to reduce the RAS indices in plasma by improving the damages, whereas a high dose (10 mg/kg) increased them by the reflexes with blocking RAS. The present results indicate that chronic All blockade reduces the increase in blood pressure, end-organ damages and RAS related to the damages in SHRSP.
Second Department of Internal Medicine, Yamaguchi University School
of Medicine, Ube, Yamaguchi 755, Japan
The goal of this study was to examine the
effect of an angiotensin II type 1 (AT 1 ...)-receptor antagonist
(TCV-116) on left ventricular (LV) geometry and function during the
development of pressure-overload LV hypertrophy. A low (LD; 0.3 mg · kg 1 · day 1 ) or a high (HD; 3.0 mg · kg 1 · day 1 )
dose of TCV-116 was administered to abdominal aortic-banded rats over 4 wk, and hemodynamics and morphology were then evaluated. In both LD and
HD groups, peak LV pressures were decreased to a similar extent
compared with the vehicle-treated group but stayed at higher levels
than in the sham-operated group. In the LD group, both end-diastolic
wall thickness (3.08 ± 0.14 mm) and myocyte width (13.3 ± 0.1 µm) decreased compared with those in the vehicle-treated group (3.67 ± 0.19 mm and 15.3 ± 0.1 µm, respectively; both
P < 0.05). In the HD group, myocyte
length was further decreased (HD: 82.6 ± 2.6, LD: 94.1 ± 2.9 µm; P < 0.05) in association with a reduction in LV midwall radius (HD: 3.36 ± 0.12, LD: 3.60 ± 0.14 mm; P < 0.05) and peak midwall
fiber stress (HD: 69 ± 8, LD: 83 ± 10 × 10 3
dyn/cm 2 ;
P < 0.05). There was no significant
difference in cardiac output among all groups. The
AT 1 -receptor antagonist TCV-116
induced an inhibition of the development of pressure-overload
hypertrophy. Morphologically, not only the width but also the length of
myocytes was attenuated with TCV-116, leading to a reduction of midwall radius and hence wall stress, which in turn may contribute to a
preservation of cardiac output.
pressure overload; TCV-116; pressure-volume relation
Angiotensin-converting enzyme inhibitors (ACE-I) and specific nonpeptide angiotensin II (ANG II) receptor antagonists have been used extensively to treat a variety of cardiovascular disorders in ...experimental animals and humans. Despite their widespread use, only a limited amount of data has been published regarding the effect that renin-angiotensin system (RAS) blockade may have on ANG II receptors, and very often this information is contradictory. The present study was designed to investigate whether changes in plasma ANG II levels induced by RAS blockade could alter glomerular ANG II receptor characteristics. Captopril was employed as an ACE-I with losartan and TCV-116, two AT
1 receptor antagonists of different chemical structure. Two experimental protocols were established. Protocol 1 contained 3 experimental groups: controls (Sprague–Dawley rats, 250–300 g BW), and animals treated with either captopril (0.5 g/l via drinking water) or losartan (10 mg/kg BW p.o.). In protocol 2, the animals were treated as in protocol 1 except that losartan was replaced by TCV-116 (1 mg/kg BW p.o.). At the end of treatment (3 days), all groups were killed by decapitation, blood was collected for plasma renin activity (PRA) measurement, and hearts and kidneys were excised. ANG II receptors were assessed by radioligand binding assays on membrane preparations of purified glomeruli, by displacement of
125I–Sar
1, Ile
8–ANG II with specific nonpeptide antagonists of AT
1 (losartan) and AT
2 (PD 123319) receptor subtypes. RAS blockade by either ACE-I or AT
1 antagonists increased PRA. The binding assays showed that renal glomeruli from treated rats and controls expressed a single population (AT
1) of ANG II receptors. The density of glomerular AT
1 receptors was not modulated by captopril, but was significantly lower in animals treated with either losartan (
B
max: 854±169 vs. 379±79 fmol/mg protein and
K
d: 59±6 vs. 45±6 nM for controls and losartan, respectively) or TCV-116 (480±72 vs. 188±16 fmol/mg protein and
K
d: 45±9 vs. 37±18 nM for controls and TCV-116, respectively) than in their controls. No changes in receptor affinity (
K
d) were detected. Previous membrane “acid-wash” did not modify the results. We conclude that short-term RAS blockade by AT
1 antagonists, but not by ACE-I, induces true downregulation of renal glomerular ANG II receptors. No AT
2 receptor subtype was detected.
Vascular hypertrophy is considered to be an adaptive response to increased arterial wall stress in hypertension. Although there are several reports concerning the effect of angiotensin II inhibition ...on the development of vascular hypertrophy, little information is available as to its effect on vascular hypertrophy in parallel with the evaluation of arterial wall characteristics. The goal of this study was to evaluate the effect of angiotensin II type 1 receptor antagonist TCV-116 on pressure overloadinduced vascular hypertrophy in parallel with the assessment of aortic impedance.Low dose (LD; 0.3 mg/kg/day) or high dose (HD; 3.0 mg/kg/day) of TCV-116 was administered to abdominal aortic-banded rats over 4 weeks; then hemodynamics and morphology were evaluated. In both the LD and HD groups, blood pressures were decreased to a similar extent compared with those of the vehicle-treated group (
P .05). Left ventricular (LV) weight and LV weight/body weight ratio was inhibited in both TCV-116-treated groups (
P.05), whereas the media cross-sectional area of the aorta was inhibited only in the HD group (
P .05). After the treatment of TCV-116 (LD, HD), total systemic resistance was decreased compared with the vehicle-treated group (
P .05), but there was no significant difference between the TCV-116-treated groups. In contrast, the first harmonic of the impedance modulus revealed the decrease only in the HD group (
P < .05).TCV-116 attenuated the development of pressure overload LV hypertrophy and vascular hypertrophy as well; however, the dose of TCV-116 required for the inhibition of vascular hypertrophy was significantly higher than that for LV hypertrophy. Vascular hypertrophy may be less pressure dependent than cardiac hypertrophy. On chronic addition of high dose of TCV-116, arterial wave reflection was decreased in association with the attenuation of vascular hypertrophy.
Candesartan cilexetil (TCV-116), an angiotensin II receptor antagonist, was administered orally to male F344/Jcl and Crj:CD (SD) rats at 1000 mg kg
−1 day
−1 for 1–28 days, and the possible mechanism ...for the anemia induced by TCV-116 was investigated. In the TCV-116 group, the erythrocyte count, hematocrit value and hemoglobin concentration were decreased by 7–8% as compared with the values in the control group after dosing for 28 days. The plasma and renal erythropoietin levels, the reticulocyte count in the peripheral blood and the erythroid cell count upon bone marrow examination were decreased on day 7, but there were no accompanying histopathological renal lesions. Renal blood flow was increased, and mean blood pressure was decreased after TCV-116. These results suggest that the primary cause of the anemia induced by TCV-116 treatment is the increase in renal blood flow followed by a decrease in erythropoietin production.
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