Serotonin (5-HT) is an important vasoactive product released from platelets undergoing aggregation (1,2). Both platelets and vascular smooth muscles are endowed with 5-HT2A receptors. Their activation causes further aggregation of platelets (3) and constriction of the vascular smooth muscles (4,5). 5-HT2A-receptor antagonists may inhibit both vasoconstriction and formation of thrombi under certain pathophysiologic conditions in which serotonin is released from the activated platelets.
DV-7028 (3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-6,7,8,9-tetrahydro-2H-pyrido[1,2,-a]-1,3,5-triazine-2,4 (3H)-dione maleate; Fig. 1) is a 5-HT2A-receptor antagonist, exhibiting no affinity toward 5-HT1A, 5-HT1B, and 5-HT1D receptors. The affinity of this compound is 14-26 times lower for the 5-HT2C, α1-adrenoceptors, dopamine D2, and histamine H1 receptors compared with 5-HT2A receptors. Structurally DV-7028 is related to ketanserin, the prototype 5-HT2A-receptor antagonist (6).
DV-7028 shows good correlation between its antagonistic effects on 5-HT2 receptors and inhibition of platelet aggregation or smooth-muscle contraction induced by serotonin (6). Thus it has been proposed as a good candidate for antithrombotic therapy in humans.
The aim of this study was to examine the influence of DV-7028 on the circulatory system and to determine the effect of DV-7028 on vascular 5-HT2A receptors, by using different experimental models.
MATERIALS AND METHODS
Male, normotensive Wistar rats (weight 220-260 g) were housed in groups of six to 12, in a room maintained at 20°C on a 12 h light/dark cycle. Food and water were available ad libitum.
Anesthetized and pithed rats
Rats were anesthetized with pentobarbital (40 mg/kg, i.p.), and the right carotid artery was cannulated with a polyethylene tube filled with heparinized (150 IU/ml) saline solution, and the blood pressure was measured via a pressure transducer (Gould-P23 ID, Bialystok, Poland) connected to a Trendscope 8031 cardiomonitor (SandW Medico Teknik Ltd, Bialystok, Poland). To record the heart rate, subcutaneous needle electrodes were connected to the same monitor Trendscope 8031. The trachea was separated and cannula inserted for artificial respiration. The rats were pithed and vagotomized as previously described (7). In brief, after injection of atropine (1.0 mg/kg, i.p.), both vagus nerves were carefully isolated and cut. A pithing rod (0D, 1.5 mm; 19 cm long) was introduced through the left orbit into the spinal cord (C6) to destroy the spinal column completely. The tracheal cannula tubing was immediately connected to the respiratory system (Medipan), and the rats were ventilated with air (10.0 ml/kg, 80 strokes/min). During the experiments, body temperature was maintained at 36.7-37.0°C by using a thermostatically controlled pad. After 15 min of stabilization of blood pressure and heart rate, each rat received NaCl 0.9% (0.2 ml, i.v.) or one i.v. dose of DV-7028 (0.1, 0.3, 1.0 mg/kg) or ketanserin (1.0 mg/kg) or ritanserin (1.0 mg/kg). In another series of experiments, the pressor responses to 5-HT (0.003, 0.01, 0.03, 0.1, 0.3, and 1.0 mg/kg, i.v.) were studied in pithed rats under controlled conditions and 5 min after the i.v. administration of DV-7028 (1, 10, and 100 μg/kg) or ketanserin (100 μg/kg).
Isolated perfused hindlegs of the rat
Perfusion of the hindlegs of the rat was carried out according to Smid and Husek (8). Male Wistar rats were anesthetized with pentobarbital (40 mg/kg, i.p.).
The abdominal cavity was opened, and the aorta and inferior vena cava were carefully exposed. Ligatures were applied around the renal vessels, and these were tightened to prevent leakage during perfusion. Double ligatures just below renal arteries were loosely applied around the aorta and vena cava, separately. The upper ligature around the aorta was tightened, and an 18-gauge needle below the ligature was immediately inserted into the vessel and pushed up distally to the iliofemoral bifurcation. The lower ligature was then tightened, securing the needle in place, and the perfusion started by means of a constant-flow roller pump. Next, the upper ligature around the vena cava at the level of the renal veins was tightened, and the vessel was severed distal to the tied ligature to allow unimpeded outflow. The animal thorax was opened, and the pulmonary vessels severed to kill the animal. After 20 min, when the hindleg circulation was totally emptied of blood, a plastic cannula was inserted distally into the vena cava to collect perfusate samples and tied into place. The rat hindlegs were perfused at a constant flow of 7.5-8.0 ml/min by using Tyrode's solution (composition in mM: NaCl, 140; KCl, 2.7; NaHCO3, 11.9; CaCl2, 2.5; MgCl2, 1; NaH2PO4, 0.5; and glucose, 12.0) containing 0.1 mg/ml bovine serum albumin (BSA), pH 7.4 at 37°C, and equilibrated with 95% O2 and 5% CO2. Arterial vasoconstriction was estimated from the increase in perfusion pressure (mm Hg) at a constant flow rate. The perfusing pressure was measured by a Gould P231 D transducer connected to a Trendscope 8031 cardiomonitor (SandW Medico Teknik Ltd.). Each experiment was started with a 30-min perfusion of the Tyrode's/BSA solution through the hindlegs to clear the vessels of the residual blood. Serotonin, 0.1 μm/ml, was injected as a bolus in 0.2 ml Tyrode's solution into the perfusion circuit. DV-7028 (1, 10, and 100 nM and 1 μM), dissolved in perfusate, was continuously perfused through an injection line for 15 min before agonist (serotonin) exposure.
Rat tail artery preparation
The rats were anesthetized with pentobarbital (40 mg/kg, i.p.). The preparation of the rat tail artery was described in detail elsewhere (9). The proximal part of the tail artery was dissected out and placed for 1 h in an isolated organ bath filled with 20 ml Krebs solution. The composition of Krebs solution in mM was KCl, 4.7; NaCl, 110; MgSO4·7H2O, 1.2; KH2PO4, 1.2; NaHCO3, 25; glucose, 1.1; and CaCl2, 3.4. The solution was constantly aerated with O2/CO2 mixture (95:5) at 37°C. The artery segment was maintained under a longitudinal tension of 0.5 g. The isolated blood vessel was perfused with the Krebs solution by using a peristaltic pump (Zalip-ppl 0.5). During the incubation period (1 h), the flow rate was increased from 0.1 to 2 ml/min, so that the initial pressure was maintained at 20-40 mm Hg. The direction of the flow was proximal to distal. Cumulative concentration-response curves were constructed by increasing the concentration of serotonin in a geometric manner (without washing out after a single dose) according to Van Rossum et al. (10).
The final concentrations of serotonin were 0.1, 0.3, 1.0, 3.0, 10, 30, 100, 300, and 1,000 μM. Equilibrium was reached for each dose within half a minute so that, in general, it took ≤4 min to make a complete cumulative concentration-response curve. The effect obtained for every cumulative dose was measured in mm Hg and calculated as a percentage of serotonin concentration over controls. The concentration-response curves to serotonin were constructed in each blood vessel, the first serving as the control, and the second, the test curve, constructed 30 min after incubation with DV-7028 0.01, 0.1, 1, and 10 μM or ketanserin, 1 μM. Concentration ratios for the curves were determined at concentrations of serotonin producing half-maximal responses (EC50) and were used for construction of Schild regressions for estimation of pA2 values (11).
The values are expressed as the mean ± SEM; n represents the number of experiments. For comparison of mean values, Student's t tests for paired or unpaired data were used. When two or more treatment groups were compared with the same control group, Bonferroni's procedure was used. The differences were considered to be significant when p values were <0.05.
DV-7028 (3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-6,7,8,9-tetrahydro-2H-pyrido[1,2,-a]-1,3,5-triazine-2,4(3H)-dione) maleate was obtained from Daiichi Pharmaceutical Co., Ltd., Kitakasai, Japan, ketanserin tartrate and ritanserin (Janssen, Beerse, Belgium), 5-hydroxytryptamine hydrochloride and vasopressin (Sigma Chemical Co., St. Louis, MO, U.S.A.), atropine sulfate (Polfa, Warsaw, Poland), and pentobarbital (Biowed, Pulawy, Poland).
Effects of DV-7028, ketanserin, and ritanserin on mean blood pressure and heart rate in anesthetized rats
Intravenous administration of DV-7028 in a dose of 0.3 and 1.0 mg/kg resulted in a decrease in mean blood pressure (ΔMBP, −17.6 ± 3.8 mm Hg; n = 5; p < 0.001; and −33.0 ± 4.6; n = 8; p < 0.001; in 5 min, respectively) and a decrease in heart rate (ΔHR, −19.2 ± 4.9 beats/min; n = 5; p < 0.001; and −57.6 ± 9.4; n = 8 in 10 min; p < 0.001). This effect was most pronounced in the first 25 min after injection of DV-7028 (p < 0.01-0.001). DV-7028 in a dose of 0.1 mg/kg did not alter the parameters studied (Fig. 2a and b).
Ketanserin exhibited similar potency to DV-7028 in the rat cardiovascular system (Fig. 3a and b); given intravenously, in a dose of 1 mg/kg, ketanserin significantly reduced the MBP (ΔMBP, −30.2 ± 3.7 mm Hg; n = 5; p < 0.001 in 5 min) and slowed the heart rate (ΔHR, −59.0 ± 8.4 beats/min; n = 5; p < 0.001 in 5 min). In contrast, ritanserin, in a dose of 1 mg/kg, did not change significantly the MBP in anesthetized rats, but reduced the HR, ΔHR, −12.8 ± 2.4 beats/min; n = 4; p < 0.05 in 10 min (in the control group, ΔHR, 4.8 ± 2.2 beats/min; n = 10; in 10 min).
Effects of DV-7028 on the mean blood pressure and heart rate in anesthetized rats, in vagotomized anesthetized rats, and in pithed rats
DV-7028, in a dose of 1 mg/kg administered i.v., significantly reduced the MBP and slowed the HR in normotensive anesthetized rats (Fig. 3 a and b). This hypotensive effect was less pronounced in vagotomized anesthetized rats (ΔMBP, −19.0 ± 4.3 mm Hg in 5 min, −11.8 ± 3.4 in 20 min, and −8.0 ± 2.8 in 25 min; p < 0.05; n = 5). Bradycardia was significantly attenuated after i.v. administration of DV-7028, 1 mg/kg, to vagotomized anesthetized rats (ΔHR, −11.8 ± 6.0 beats/min in 5 min; n = 5; p < 0.001). The MBP in pithed rats was 48.2 ± 3.1 mm Hg, whereas in the control group of anesthetized rats, it was 98.2 ± 6.5 mm Hg. Because of the low baseline value of the MBP in pithed rats, they were given vasopressin intravenously (via the femoral vein) in a dose of 0.04-0.4 iU/kg/h, and then their new "baseline" MBP was adjusted to 96.6 ± 3.9 mm Hg. Intravenous administration of DV-7028 in a dose of 1 mg/kg affected neither the MBP nor the HR in pithed rats (Fig. 4 a and b).
Effects of DV-7028 and ketanserin on the increase in the mean blood pressure elicited by intravenous administration of serotonin in pithed normotensive rats
Intravenous administration of serotonin (0.003-1 mg/kg) induced a dose-dependent increase in BP in pithed rats (Fig. 5a and b). DV-7028 inhibited the pressor effect of serotonin in pithed rats in a dose-dependent manner (0.01 mg/kg, i.v.; p < 0.01; n = 6; and 0.1 mg/kg, i.v.; p > 0.001; n = 5), shifting the dose-response curve for serotonin to the right but in a not quite parallel fashion. Inhibition by ketanserin (0.1 mg/kg, i.v.) of the pressor effect to serotonin (1 mg/kg, i.v.) in pithed rats was approximately twofold more pronounced (p < 0.001) than by DV-7028 (0.1 mg/kg, i.v.; Fig. 5b).
Influence of DV-7028 on the pressor response to serotonin in the perfused hindlegs of the rat
Under conditions of constant flow rate of Tyrode's buffer, administration of 0.1 mM serotonin (directly to the rat in the abdominal aorta) resulted in an increase in perfusion pressure by 168.4 ± 13.8 mm Hg (i.e., 100.0 ± 8.2%; n = 10; in the control group; Fig. 6a). Administration of DV-7028, 1 nM, 10 nM, 100 nM, and 1 μM, to the perfusing solution 30 min before and during the injection of serotonin induced a statistically significant concentration-dependent decrease in the pressor response to serotonin (98.3 ± 9.9%; n = 4; NS; 70.0 ± 6.5%; n = 6; p < 0.05; 27.4 ± 4.3; n = 6; p < 0.001 and 5.7 ± 0.9; n = 4; p < 0.001; respectively). Figure 6b shows the concentration-inhibition curve for DV-7028 in the perfused hindlegs of the rat. The concentration of DV-7028 producing half-maximal inhibition (IC50) was 0.032 ± 0.004 μM.
Effects of DV-7028 and ketanserin on the pressor response of the perfused rat tail artery to serotonin
Figure 7 shows the cumulative concentration-response curves for serotonin obtained in the rat tail artery and the effects of increasing concentrations (0.01, 0.1, 1.0, and 10 μM) of DV-7028. The EC50 values, defined as the negative logarithm of the serotonin concentration producing 50% of the maximal contraction was 11.4 μM. DV-7028 (0.1 μM) shifted the concentration-response curve for serotonin to the right, in a not quite parallel fashion, by approximately eightfold (EC50, 79.4 μM;Fig. 7a). The estimated pA2 value for DV-7028 was 7.92 ± 0.13, slope 0.94 ± 0.11 (Fig. 7b). DV-7028 (1 μM) and ketanserin (1 μM) were found to be similarly potent antagonists on 5-HT2 receptors in this experimental model of the isolated rat artery (Fig. 7c).
As outlined in the Introduction, the aim of our study was to examine the effects of DV-7028 on 5-HT2A receptors in the cardiovascular system of the rat. We demonstrated that DV-7028, given intravenously in a single dose, caused a significant dose-dependent decrease in the MBP and in the HR in anesthetized normotensive rats. Our data did not correspond with those of Shibano et al. (6), who administered DV-7028 orally in a dose 10 times higher than we did. The authors failed to observe a significant decline in systolic blood pressure in conscious, spontaneously hypertensive rats. This difference is difficult to explain. One might suggest that it is attributable to the different route of drug administration (oral vs. intravenous), the different strain of rats, the possible effect of pentobarbital anesthesia or the method of BP measurement (direct vs. undirect), or a combination of these. As found by other authors (12,13), a hypotensive effect was demonstrated after administration of ketanserin, a classic but less selective 5-HT2A-receptor antagonist, whereas ritanserin, a more selective 5-HT2-receptor antagonist (13), did not alter the MBP but decreased the HR. It is well documented in binding studies that ketanserin, besides its high affinity for 5-HT2A receptors at low concentrations, also possesses a high affinity for α1-adrenoceptors and histamine H1 receptors (14). Thus at least in rats, α1-adrenoceptors are involved in the hypotensive action of ketanserin (12), whereas ritanserin lacks affinity for adrenoceptors and is devoid of hypotensive effects in this species (15). It might be speculated that interaction with α1-adrenoceptors also plays an essential role in the cardiovascular changes observed after DV-7028 administration. Shibano et al. (6), in receptor-binding assays, showed that DV-7028 exhibited a high affinity for the 5-HT2 receptors with a Ki value of 22 nM and a low affinity for the adrenergic α1-receptors (Ki = 417 nM). In the same experiment, ketanserin bound to the 5-HT2 receptors with a Ki value of 2.9 nM and to adrenergic α1-receptors with Ki values of 34 nM. Thus the selectivity of DV-7028 for the 5-HT2 receptors, compared with the adrenergic α1-receptors, is greater than that of ketanserin.
Most smooth-muscle organs constrict when exposed to serotonin, but their individual receptor types and sensitivity differ (16). The remarkable relation between binding affinities and substance potencies in the in vitro pharmacologic test using rat caudal arteries was observed (17). In this experimental model, the increase in perfusion pressure in response to serotonin results from stimulation of 5-HT2A receptors in the arterial wall (18). In our study, DV-7028 significantly inhibited the vasoconstrictor effect of serotonin, and a concentration-dependent, almost parallel, shift to the right of the serotonin concentration-response curve was recorded. On this basis, we think that the inhibitory effect of DV-7028 occurs through the serotonin 5-HT2A receptor rather than α1-adrenoceptor blockade in this experimental model.
Similarly, in pithed rats, serotonin administered intravenously caused dose-dependent vasoconstriction and increase in BP via 5-HT2A receptors (19,20). Therefore it was of interest to evaluate the antagonistic properties of DV-7028 against vascular 5-HT2A receptors in pithed rats. We demonstrated that DV-7028, when given in high doses, inhibited or even abolished the pressor effects of serotonin in pithed rats. Statistically significant antagonistic effects on the 5-HT2A receptors were demonstrated after injection of DV-7028 in a dose of 0.01 mg/kg. Vascular 5-HT2A-receptor antagonistic properties of DV-7028 were observed in doses that caused neither hypotension nor bradycardia in anesthetized rats. By using the model of the isolated perfused hindlegs of the rat, we showed that DV-7028, 10 nM, attenuated the increase in perfusion pressure elicited by serotonin, whereas a dose of 1 μM abolished the pressor effect of serotonin.
Vasospasm and thrombogenesis lead to most cardiovascular disorders in which platelets play a key role (3). In fact, platelets activated at the interface with an endothelial injury accelerate the local formation of thrombin and release a variety of endogenous products including thromboxane-A2, prostaglandin endoperoxides, adenosine diphosphate (ADP), and serotonin. Accumulation of serotonin at sites of vascular endothelial injury and cardiovascular stenosis mediates platelet aggregation, vasoconstriction, and arterial thrombosis (21). Both platelets and vascular smooth muscle have 5-HT2A receptors. DV-7028 inhibited platelet aggregation induced by a combination of serotonin and collagen, suggesting that the compound could prevent the activation of 5-HT2A receptors in blood platelets (6).
In vagotomized rats, intravenous administration of DV-7028 resulted in a decrease of the MBP. However, this effect was less prominent than that seen in nonvagotomized rats. Moreover, bradycardia induced by intravenous administration of DV-7028 was almost completely abolished by vagotomy. Similar effects in the rat circulatory system were observed by Fozard (12) after administration of ketanserin, a classic antagonist of 5HT2A receptors. In other experiments, Malyszko et al. (22) demonstrated that DV-7028 can modify serotonin turnover in the rat brain. In addition, regional differences regarding the suppression or enhancement of serotonin metabolism in the brain of stressed (foot-shocked) rats given DV-7028 were found. In pithed rats, bradycardia and hypotension was not demonstrated after DV-7028 administration. Therefore it may be suggested that the observed effects seen after a single intravenous dose of DV-7028 are not due to the direct vascular site of action. Nevertheless this speculation remains to be further elucidated.
In conclusion, these data clearly demonstrate that DV-7028 is a potent 5-HT2A-receptor antagonist in the rat cardiovascular system. It can provide a useful tool for the therapy of some clinical disturbances accompanied by circulatory diseases or hypercoagulation or both. In addition, when applied in high doses, it causes bradycardia and hypotension via an unknown site and mechanism.
Acknowledgment: This work was supported by the State Committee for Scientific Research, grant 4 P05A 041 08.
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