Benidipine Hydrochloride Research Articles

Effects of Benidipine Hydrochloride on Cerebrovascular Lesions in Salt-Loaded Stroke-Prone Spontaneously Hypertensive Rats: Evaluation by Magnetic Resonance Imaging

We determined possible protective effects of benidipine hydrochloride (benidipine), a dihydropyridine calcium antagonist, on cerebrovascular lesions in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). The animals were orally treated with benidipine at 1, 3 and 10 mg/kg daily for 7 weeks, and their neurological symptoms, body weight changes, systolic blood pressure and cerebrovascular lesions on magnetic resonance imaging (MRI) were determined at various time points of treatment. Moreover, the brains of the rats that showed cerebrovascular lesions on MRI in the course of treatment or completed 7-week treatment were examined histopathologically. Control rats presented such symptoms as sedation, ataxia and aggressiveness, while their MRI analysis revealed high signals over wide areas from the occipital to frontal cortex and from the corpus callosum to external capsule. These high signal areas corresponded in location to edematous or softening lesions revealed by the histopathological observation. Treatment with benidipine at 3 and 10 mg/kg ameliorated neurological symptoms, significantly suppressing cerebrovascular damages on MRI. Benidipine at 3 mg/kg significantly decreased blood pressure for the first four weeks but it did not thereafter. These findings demonstrate that benidipine can protect salt-loaded SHRSP from cerebrovascular injury as assessed by MRI.

Dose-dependent Effect of Benidipine in Patients with mild moderate Hypertension

Background and Objectives:This study was designed to evaluate the appropriate dose and dose-dependent effect of benidipine hydrochloride, a Ca-channel blocker, in patients with mild–moderate essential hypertension. Material and Methods:Benidipine was administered in 2 mg, 4 mg and 8 mg once daily with 1 month interval in 41 hypertensive patients with diastolic blood pressure over 90 mmHg and systolic blood pressure from 140 to 210 mmHg. Blood pressure, heart rate, subjective symptoms and adverse effects were checked every 4 weeks after benidipine administration. Laboratory examinations were performed before and after benidipine administration. Results:The dose-dependent, antihypertensive effect of benidipine was evaluated in 41 patients. The blood pressure significantly reduced from 166±15 mmHg/103±7 mmHg to 13815 mmHg/88±11 mmHg at 12 weeks administration of benidipine and overall effective rate was 95%. The systolic and diastolic blood pressure was reduced significantly in proportion to dose of benidipine (p<0.0001). Antihypertensive effect was prominent at 4mg of benidipine. The heart rate was not affected by benidipine. No significant laboratory changes were observed. Conclusion:Benidipine has a dose-dependent effect in the treatment of mild-moderate hypertension, and the dosage to be needed may be 4mg or more for sufficient antihypertensive effect. (Korean Circulation J 2000;30(5):586-591)

Effects of a Novel Calcium Antagonist, Benidipine Hydrochloride, on Platelet Responsiveness to Mental Stress in Patients with Essential Hypertension

The effects of a novel calcium antagonist, benidipine hydrochloride, on responses of platelets to mental stress were evaluated in nine patients with essential hypertension. Before and 12 weeks after the monotherapy with benidipine (2-4 mg/day), platelet aggregability and plasma beta-thromboglobulin were determined during rest and after a 10-min arithmetic stress. Before the treatment, arithmetic stress significantly increased platelet aggregability in response to adenosine diphosphate (ADP) and plasma beta-thromboglobulin level. Blood pressure, pulse rate, and plasma catecholamines also increased during arithmetic stress. The treatment with benidipine did not affect resting values of platelet functions, but attenuated significantly stress-induced alterations in primary aggregation to 1.0 microM ADP (34 +/- 4% to 40 +/- 3% before treatment vs. 32 +/- 2% to 34 +/- 3% after benidipine), ADP threshold for biphasic aggregation (2.2 +/- 0.4 to 1.8 +/- 0.3 microM before treatment vs. 2.2 +/- 0.3 to 2.2 +/- 0.4 microM after benidipine) and plasma beta-thromboglobulin level (74 +/- 16 to 104 +/- 15 ng/ml before treatment vs. 60 +/- 10 to 52 +/- 8 ng/ml after benidipine; p < 0.05 for Stress x Treatment interactions in all values). The pretreatment elevations in blood pressure and sympathetic activity with stress were not modified by benidipine treatment. In conclusion, the monotherapy with benidipine did not affect platelet function during the resting condition, but significantly suppressed the platelet activation induced by arithmetic stress in patients with essential hypertension.

Vasodilating effect of benidipine hydrochloride in the renal and hindquarter vascular regions (supplied by terminal aorta) of spontaneously hypertensive rats

One of two Ca antagonists, benidipine (3–30 μg/kg) or nifedipine (30–600 g/kg), was administered in a bolus injection through the jugular vein, and the changes in mean arterial pressure (MAP), renal flow (RF), and hindquarter flow (HQF) in conscious spontaneously hypertensive rats (SHRs) and normotensive control rats (NCRs). Renal vascular resistance (RR) and hindquarter resistance (HQR) were calculated as MAP divided by RF and HQF, respectively. When a high dose was administered to decrease the blood pressure by about 20%, the RR was significantly lower with benidipine than with nifedipine. The decrease in HQR was not significantly different between benidipine and nifedipine. When a low dose was administered to decrease the blood pressure by about 7%, the decrease in RR was not significantly different between benidipine and nifedipine, but the HQR was significantly lower with benidipine than with nifedipine. In the NCRs, no pharmacological properties were significantly different between these two Ca antagonists.

Enantiomer-enantiomer interaction of a calcium channel antagonist, benidipine hydrochloride, during liver metabolism in the rat.

Benidipine hydrochloride is a dihydropyridine calcium antagonist and it is used clinically as a racemate which consists of two optical isomers. The antihypertensive activity of the (+)-alpha isomer is 30-100-fold more potent than that of the (-)-alpha isomer. In rats, after oral administration, plasma concentrations of the (+)-alpha isomer were higher than those after oral administration of the (-)-alpha isomer. In addition, the sum of the plasma concentrations after separate oral administration of each isomer was lower than the plasma concentrations after oral administration of the racemate at a dose equivalent to that of two isomers. The metabolic velocity of the (+)-alpha isomer in male rat liver microsomes was lower than that of the (-)-alpha isomer in the expected liver concentration during absorption, which is consistent with the in vivo results. In rat liver microsomes, each isomer competitively inhibited the metabolism of the other, indicating a metabolic enantiomer-enantiomer interaction of benidipine in the rat.

Alteration of muscle fiber composition linking to insulin resistance and hypertension in fructose-fed rats

The aim of this study was to examine the role of muscle fiber composition in insulin resistance and the effect of a calcium channel antagonist on insulin sensitivity in fructose-induced insulin resistant and hypertensive rats. Six-week-old male Sprague-Dawley rats were fed either normal rat chow (control) or fructose-rich diet (FFR). For the last 2 weeks of a 6-week period of either diet, the rats were treated, by gavage, with gum arabic solution (control or FFR) or a dihydropyridine calcium channel antagonist, benidipine hydrochloride (3 mg/kg/day: FFR + Ca), then the euglycemic hyperinsulinemic glucose clamp technique was performed to evaluate insulin sensitivity. Blood pressure was measured weekly for 6 weeks. At the end of the glucose clamp, the soleus muscle was dissected out for determination of muscle fiber composition by ATPase methods. Blood pressure was elevated at 2 weeks after the start of fructose-rich chow feeding and persisted thereafter throughout the study. Blood pressure at the glucose clamp in the FFR was significantly higher than that in the control group (142 ± 2 v 155 ± 2 mm Hg, P < .01) and the calcium antagonist significantly lowered blood pressure of FFR (136 ± 6 mm Hg for FFR + Ca, P < .05). The average rate of glucose infusion during glucose clamp, as a measure of insulin sensitivity (M value), was significantly lower in the FFR than in the control (15.4 ± 0.4 v 10.9 ± 0.6 mg/kg/min, P < .01). The calcium channel antagonist partially improved the M value compared to that of FFR (13.4 ± 0.7 mg/kg/min in FFR + Ca, P < .01 compared to FFR, P < .05 compared to control). The composite ratio of type I fiber in soleus muscle was significantly decreased in FFR compared to control (81.7 ± 1.5% v 75.0 ± 1.7%, P < .01), and the composite ratio of type I fiber in rats treated with the calcium channel antagonist (FFR + Ca) recovered to the control level (79.9 ± 1.1%, P < .05 compared to FFR). The M value was significantly correlated with the compositions of type I and type II fibers (for type I fibers, r = 0.80, P < .01; for type II fibers, r = −0.81, P < .01). These results suggest that fiber composition of skeletal muscle links insulin resistance and that a calcium channel antagonist may modulate muscle fiber composition in hypertensive animal model, fructose-fed rats.

Arteriolar and venular vasodilating properties of benidipine hydrochloride, a 1,4-dihydropyridine Ca2+ antagonist with long-lasting action, assessed in rat mesenteric microcirculation.

To obtain direct and visible evidence of the arteriolar vasodilating action in vivo of benidipine hydrochloride, a long-lasting and light-resistant Ca2+ antagonist of the 1,4-dihydropyridine type, we continuously recorded changes in the diameter of mesenteric arterioles (10-40 microm) and venules (20-40 microm) in anesthetized agent-injected Wistar rats by means of digital image processing combined with videomicroscopy. Benidipine injected intravenously brought about a depressor response, and this response persisted much longer than that induced by nifedipine. Benidipine produced a dose-dependent arteriolar vasodilation and a decrease in the blood-flow velocity. It also relaxed the venules during the depressor response, which relaxation was more prominent 1-2 h after the administration. In pithed rats, both pressor response and arteriolar constriction induced by norepinephrine were prevented by benidipine. Benidipine also inhibited adenosine diphosphate (ADP)-induced interruption of arteriolar blood flow. These results suggest that benidipine produced vasodilation in vivo at both the arteriolar and venular levels. The ability of benidipine to prevent microcirculatory disturbance and to produce arteriolar and venular vasodilation seem to account for its long-lasting Ca2+-antagonistic antihypertensive action.

Effect of benidipine hydrochloride, a dihydropyridine-type calcium antagonist, on the function of mouse osteoblastic cells.

The effects of benidipine hydrochloride (BD), a dihydropyridine-type calcium antagonist, on the growth and alkaline phosphatase (ALPase) activity were studied in cultures of mouse osteoblastic cells. BD (0.1-10 nM) increased the ALPase activity of osteoblastic cells and reduced cell proliferation incubated for 48 h. This reduction of cell growth did not appear to be due to cell death. On the other hand, nifedipine and amlodipine (1 nM) had no effect. BD (0.1-10 nM) enhanced the effects of 1,25(OH)2D3 on osteoblastic cells, the decreased cell growth and increased ALPase activity. These findings suggest that BD regulates the growth and differentiation of osteoblasts and stimulates the function of these cells as well as 1,25(OH)2D3.

Diversity in the Renal Hemodynamic Effects of Dihydropyridine Calcium Blockers in Spontaneously Hypertensive Rats

Effects of dihydropyridine (DHP) Ca2+ channel blocker (CaB) on glomerular hemodynamics are controversial. We examined the effects of two DHP derivatives, benidipine hydrochloride (BDP) and nifedipine (NDP), on glomerular hemodynamics by an in vivo micropuncture method by using spontaneously hypertensive rats (SHRs). Systemic bolus infusion of BDP (4 microg/kg) or NDP (250 microg/kg) elicited comparable decreases in mean arterial blood pressure (MAP). The proximal stop-flow pressure (Psf), an indicator of glomerular capillary pressure, revealed significant decreases in BDP but nonsignificant increases in NDP. To minimize the influence of MAP or other systemic events, we monitored Psf during perinephron infusion of CaB and observed significant increases in Psf during 10(-3) M NDP perfusion and nonsignificant changes with 10(-3) M BDP. Moreover, the stability of Psf during alteration of renal perfusion pressure was significantly impaired in the nephron treated with topical NDP. These findings support the notion that CaB has diverse effects on glomerular hemodynamics, and such effects may in part be the result of different pharmacologic actions on the renal microvasculature.

Effects of benidipine hydrochloride on antioxidant enzyme activity in stroke-prone spontaneous hypertensive rats (SHR-SP).

Effects of benidipine hydrochloride or triple therapy (hydralazine, reserpine, and hydrochlorothiazide) on renal cortical and medullary intrinsic antioxidant enzyme (AOE) activity were evaluated in stroke-prone spontaneously hypertensive rats (SHR-SP) as an animal model for human essential hypertension with cerebral stroke. This study showed a significant decrease of renal intrinsic glutathione peroxidase (GSH-Px) activity in untreated SHR-SP. Renal GSH-Px activity in untreated SHR-SP was significantly lower than that in Wister Kyoto rats (WKY) as a normotensive reference strain. GSH-Px activity in SHR-SP was significantly improved after benidipine hydrochloride therapy. Levels of urinary albumin excretion or creatinine clearance (Ccr) in SHR-SP were also improved after the therapy. Glomerular sclerosis index was slightly improved in SHR-SP treated with benidipine hydrochloride according to light microscopic analysis. It appears that hypertension may influence the renal intrinsic GSH-Px activity, albuminuria, and Ccr in SHR-SP. Thus it is indicated that control of blood pressure may improve the GSH-Px activity in SHR-SP.

Solid Dispersions of Benidipine Hydrochloride. I. Preparations Using Different Solvent Systems and Dissolution Properties.

The solid dispersion technique has been widely utilized to improve the dissolution profile and oral bioavailability of water insoluble drugs. Benidipine hydrochloride, a calcium-antagonist, is a slightly soluble compound in a weak acid, so that its bioavailability is liable to be influenced by the gastric acidity of patients. In the preparation of solid dispersions by the solvent removal process, organic solvents are used as agents to intimately mix a drug or drugs and carrier molecules. Benidipine hydrochloride is also poorly soluble in commonly used organic solvents. The application of the solvent removal prosess for preparing solid dispersions of the drug makes it necessary to enhance the organic solubility of the drug. Two kinds of solvent systems have been investigated : one in an organic solution of Eudragit[○!R] E-100 (OSE) and the other in binary solvent mixtures (BSM). In the OSE, the presence of Eudragit[○!R] E-100 in dichloromethane resulted in an appreciable increase in drug solubility. However, in ethanol, this solubilization effect of the polymer was completely inhibited. In the BSM, enhanced solubility of the drug was obtained in ethanol-dichloromethane mixtures. Further, the addition of polyvinylpyrrolidone (PVP) or hydroxypropylmethyl-cellulose (HPMC) to this system did not deposit the dissolved drug. In these two solvent systems, solubilization of benidipine hydrochloride was presumed to be caused by intermolecular interactions. It enabled the preparation of solid dispersions of benidipine hydrochloride with Eudragit[○!R] E-100, PVP or HPMC. Especially, when dispersed in PVP or HPMC, the dissolution rate of the drug improved remarkably in a weak acid (pH 6.0).

Solid Dispersions of Benidipine Hydrochloride. II. Investigation of the Interactions among Drug, Polymer and Solvent in Preparations.

The solubilization mechanism of benidipine hydrochloride in two kinds of organic solvent systems has been investigated : one in an organic solution of Eudragit[○!R] E-100 (OSE), and the other in binary solvent mixtures (BSM) of alchol-cosolvent. In the OSE, the organic solubility of the drug was increased in proportion to the polymer concentration. Fourier transform infrared spectroscopy clearly demonstrated the existence of hydrogen bonds between dimethylamino groups of the polymer and piperidine NH+ of the drug. The ultraviolet absorption maximum of the drug in the 350 nm region blue-shifted in dichloromethane or chloroform with polymer, whereas this shift was not found in ethanol or acetonitrile with the polymer. In the BSM, the logarithmic maximum solubility of the drug in ethanol-cosolvent mixtures was linearly related to the π* (dipolarity-polarizability) value of the cosolvent. The initial slope of the drug solubility curve in alcohol-dichloromethane mixtures was in good correlation with the α (hydrogen-bond donor acidity) value alcohol. Therefore, it was understood that the solubilization of benidipine hydrochloride in the OSE resulted form the drug-polymer complexation with the intermolecular hydrogen bonding and dipolar interactions, and the solubilization effect in the BSM arose from the enhancement of the proton donor ability of alcohol molecules by the dipolar interactions with the cosolvent.

Suppressive effects of benidipine on the development of hypertension and renal lesions in salt-loaded stroke-prone spontaneously hypertensive rats.

1. The effects of benidipine hydrochloride (benidipine), a long-acting dihydropyridine calcium antagonist, on the development of hypertension and renal lesions were examined in stroke-prone spontaneously hypertensive rats (SHRSP). 2. SHRSP were fed with 8% NaCl diet for 3 weeks from 13 weeks of age, and benidipine (1 or 3 mg/kg per day) was orally administered during the same period. 3. The high salt diet accelerated an increase in urinary excretions of total protein and albumin, and caused marked arteriole, glomerular and tubular lesions in the kidney. 4. Benidipine significantly inhibited these changes, and also suppressed the elevation of blood pressure in salt-loaded SHRSP. 5. These results reveal that benidipine has protective effects against the development of hypertension and the progression of renal lesions induced by the high salt diet in SHRSP.

Efficacy of Benidipine Hydrochloride on Myocardial Ischemia and Reperfusion

The efficacy of benidipine hydrochloride in preventing myocardial ischemia and reperfusion injury was evaluated in isolated rabbit hearts (n = 28). Isovolumic left ventricular function, coronary flow, creatine phosphokinase (CPK) release, and myocardial water content were measured after ischemia during both normothermia (37°C; Group I) and hypothermia (23°C; Group II). After baseline measurements, hearts were induced to arrest by chilled cardioplegic solution. Each group was divided into two subgroups, depending upon whether benidipine hydrochloride (10-9 mole/liter) was added in the cardioplegia (A, without benidipine; B, with benidipine). After 30 min of ischemia for Group I and 180 min for Group II (which added another cardioplegia every 30 min), hearts were reperfused. Measurements the same as those at baseline were carried out every 15 or 30 min for up to 60 min. Benidipine-treated hearts started beating in a shorter time than did control hearts (Group I-B, 38.7 ± 3.7 sec vs Group I-A, 59.9 ± 5.6; Group II-B, 36.7 ± 2.0 vs Group II-A, 47.8 ± 3.3). The percentage of recovery of left ventricular developed pressure after 60 min of reperfusion was significantly better in benidipine groups (P < 0.05). With respect to changes in coronary flow and CPK release after reperfusion, benidipine groups were preserved extremely well. We conclude that the addition of benidipine hydrochloride to cardioplegic solution significantly improves ventricular function after myocardial ischemia and reperfusion.

The protective effect of benidipine hydrochloride against hypoxic myocardial injury of cultured mouse fetal heart cells

The protective effect of benidipine hydrochloride, a 1,4-dihydropyridine calcium antagonist, was investigated in cultured mouse fetal heart cells under the hypoxic condition (PO2, < or = 20 mmHg). The preparation of cultured fetal mouse heart muscle cells was subjected to the hypoxic condition for 24 hr. After the hypoxic treatment for 24 hr, the spontaneously beating rate of cultured heart cells was reduced subsequently and the beating almost ceased. Protection against cell injury was assessed by cell viability (NR assay), LDH release and cellular ATP levels. Under the hypoxic condition, cell viability and cellular ATP levels were markedly decreased, and LDH release was increased. Benidipine hydrochloride, at concentrations higher than 1-10 nM, strongly protected against the decreases in cell viability and cellular ATP levels as well as the increase in LDH release. The potency of the protective effect of benidipine hydrochloride was greater than those of the other tested 1,4-dihydropyridine calcium antagonists (nisoldipine, nitrendipine and nifedipine). These results suggest that benidipine hydrochloride exerts protective actions against hypoxic myocardial injury of cultured mouse fetal heart cells.

Effects of benidipine hydrochloride on contractions induced by some contractile agents in isolated cerebral and mesenteric arteries of dogs.

The effects of benidipine on contractions induced by contractile agents were studied in isolated dog basilar, middle cerebral and mesenteric arteries and compared with those of nicardipine. KCl (20-80 mM), U-46619 (3 x 10(-10)-3 x 10(-6)M) and 5-hydroxytryptamine (5-HT) (10(-9)-3 x 10(-6)M) produced concentration-dependent contractions in the three arteries. Benidipine (10(-11)-8 x 10(-9)M) inhibited non-competitively the KCl-induced contraction in the basilar, middle cerebral and mesenteric arteries with a pD'2 of 9.1, 9.7 and 8.5, respectively. There was a significant difference between the pD'2 values in the cerebral and mesenteric arteries. Benidipine (10(-6)-3 x 10(-5)M) attenuated significantly the contraction produced by both U-46619 and 5-HT in the three arteries, the pD'2 being 4.4-4.9. Nicardipine inhibited the contraction induced by the three contractile agents in the same manner as benidipine. The contraction caused by 70 mM KCl in the cerebral and mesenteric arteries was reduced by 33-38 and 18%, respectively following treatment with 0.25 mM Ca2+ solution. Furthermore, pretreatment with a Ca(2+)-free solution containing 1 mM EGTA inhibited KCl-induced contractions in the basilar, middle cerebral and mesenteric arteries by 98.7, 95.6 and 92.1%, respectively. It also attenuated the contractions produced by both 10(-6) M U-46619 and 3 x 10(-6) M 5-HT in the cerebral and mesenteric arteries by 56-61 and 34-39%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of benidipine hydrochloride on cardiac hypertrophy and coronary flow reserve in renovascular hypertensive rats

Effects of benidipine hydrochloride on cardiac hypertrophy and coronary flow reserve in renovascular hypertensive rats

Effects of benidipine hydrochloride (Coniel), a new calcium antagonist, on the cardiac output, regional blood flow and vascular resistance in conscious, spontaneously hypertensive rats.

Benidipine hydrochloride is a calcium antagonist with a 1,4-dihydropyridine derivative structure, and exhibits long-lasting antihypertensive effects by inhibiting the voltage-dependent Ca2+ channels. This study was undertaken to examine the effect of benidipine on central haemodynamics and regional blood flow (RBF) after intravenous administration of benidipine in conscious, spontaneously hypertensive rats. The microsphere method was used to measure cardiac output and RBF before and after the drug administration, using microspheres labelled with 57Co and 51Cr. Thirty minutes after the intravenous administration of benidipine (3 micrograms kg-1), the mean arterial pressure fell by 15% without significantly increasing the heart rate. The cardiac output increased by 41% and the systemic resistance decreased by 39%. Benidipine significantly increased RBF by 37, 35, and 22% in kidney, heart, and small intestine, respectively, and decreased vascular resistance by 38, 38, and 32%, respectively. We concluded that benidipine reduced blood pressure by increasing RBF in the kidney and heart, while keeping RBF in other organs at a normal level. These results will provide a fundamental basis in support of the clinical benefits of benidipine for hypertensive patients, particularly those with renal failure.

P-221 - Anti-proliferative effects of benidipine hydrochloride in porcine cultured vascular smooth muscle cells and in the rats subjected to balloon-catheter-induced endothelial denudation.

P-221 - Anti-proliferative effects of benidipine hydrochloride in porcine cultured vascular smooth muscle cells and in the rats subjected to balloon-catheter-induced endothelial denudation.

Anti-proliferative effects of benidipine hydrochloride in porcine cultured vascular smooth muscle cells and in rats subjected to balloon catheter-induced endothelial denudation.

Using the [3H]thymidine incorporation technique, the anti-proliferative effects of benidipine, a long-acting calcium antagonist, on porcine cultured vascular smooth muscle cells (VSMCs) were determined and compared with those of other calcium antagonists. Benidipine inhibited serum-stimulated [3H]thymidine incorporation into VSMCs (IC50, 0.2 microM), and this inhibitory effect was significantly more potent than that of nitrendipine, felodipine, nisoldipine, manidipine, amlodipine, nifedipine, verapamil and diltiazem. When growth-arrested cells were stimulated with platelet-derived growth factor followed by insulin, benidipine, administered with either stimulation, inhibited [3H]thymidine incorporation into VSMCs. This suggests that it acts in both the G0/G1 and G1/S phases. In another series of experiments, the anti-proliferative effect in vivo was investigated using rats subjected to balloon catheter-induced endothelial denudation of the aorta. Benidipine (5 mg/kg, p.o., b.i.d.) significantly reduced the incorporation of [3H]thymidine into aortic DNA 48 h after balloon injury, whereas it did not affect incorporation into bone marrow, suggesting that it inhibits arterial DNA synthesis. From our results, benidipine was shown to exert antiproliferative effects on VSMCs in vivo as well as in vitro. The drug may be useful for the treatment of vascular proliferative diseases such as restenosis following percutaneous transluminal angioplasty and atherosclerosis.