Frovatriptan (VML 251/SB-209509) is a novel high-affinity 5-hydroxytryptamine (5-HT1B/1D)-receptor lig- and (1), which has been shown to be effective as an acute treatment for migraine (2). Frovatriptan constricts rabbit isolated basilar artery (1), increases carotid vascular resistance in anesthetized dogs (3), and constricts isolated human cerebral arteries (4). In contrast to other 5-HT1B/1D-receptor agonists (5-9), frovatriptan has been shown to produce bell-shaped concentration-response relations in human isolated coronary arteries, with relaxation occurring at high concentrations (4). Evidence for these effects also is apparent in vivo, as high doses of frovatriptan can produce a reduction in coronary vascular resistance at doses that produce marked constriction of the carotid vascular bed of anesthetized beagles (3).
Both 5-HT1 and 5-HT2 receptors contribute to mediating the contractile actions of 5-HT in human isolated coronary arteries (10-12). 5-HT1B/1D agonists therefore produce contraction of these vessels but may have a markedly reduced maximal effect compared with 5-HT (5-10), depending on the 5-HT-receptor populations in the individual artery segment (10,11). This profile is consistent with the effects of sumatriptan in humans, in whom a small constriction has been observed (13,14); however, sumatriptan has no effect on myocardial perfusion in healthy volunteers (15). These coronary constrictor effects of sumatriptan are in contrast to the in vivo profile of 5-HT, which has been shown to dilate coronary arteries in patients undergoing quantitative angiography (16,17).
We previously demonstrated that, unlike other 5-HT1B/1D-receptor agonists (5-9), frovatriptan produces bell-shaped concentration-response relations in human isolated coronary arteries. The cardiac effects of frovatriptan were investigated in more detail on canine isolated coronary arteries and on coronary and cardiac function in vivo in anesthetized, open-chest dogs, under physiologic and pathophysiologic conditions, after myocardial infarction.
Rings of beagle left descending coronary artery or circumflex (LCX) artery were set up for recording of isometric tension, as previously reported (18). The effects of frovatriptan were investigated in arteries that were denuded of endothelium. Experiments were performed in the presence of ketanserin, 1 μM, cocaine, 6 μM, prazosin, 1 μM, and ascorbate, 0.2 mM. This protocol was used to rule out activation of 5-HT2 receptors, neuronal uptake, and adrenoceptor activation. Ascorbate was used as an antioxidant.
After determination of initial reactivity to 5-HT, the concentration-response characteristics for frovatriptan were investigated under resting tone. In a further series of experiments, the effects of frovatriptan were studied in beagle isolated coronary arteries precontracted with either the thromboxane mimetic U46619 (6 nM) or sumatriptan (10 μM).
Adult, mongrel, specific-bred dogs (10-17 kg) of either sex were anesthetized with sodium pentobarbitone (35 mg/kg, i.v.) and maintained by continuous infusion (5 mg/kg/h). Depth of anesthesia was continuously monitored, and body temperature maintained at 37°C by a heating blanket. The animals were intubated and artificially respired with room air at a tidal volume of 20 ml/kg and a rate of 12 strokes/min. The right femoral artery and vein were isolated and cannulated for the measurement of arterial blood pressure and drug administration, respectively. Lead II of the ECG was used to determine heart rate.
Cardiac function. The left carotid artery was cannulated with a Millar transducer-tip catheter, which was advanced into the left ventricle to measure left ventricular systolic pressure (LVSP). The output of this transducer was amplified to obtain left ventricular end-diastolic pressure (LVEDP) and differentiated for the measurement of cardiac contractility (dP/dt). The heart was exposed through a left thoracotomy at the fifth intercostal space and suspended in a pericardial cradle. A 2-cm section of the LCX coronary artery was exposed proximal to the first obtuse marginal branch and instrumented proximal to distal as follows: (a) needle cannula (26 gauge) for drug administration (intracoronary), (b) electromagnetic flow probe (model 501; Carolina Medical Instruments, NC, U.S.A.) to measure coronary blood flow (CBF), and (c) an occluder (to provide a similar surgical protocol, as described later). The descending thoracic aorta was isolated and fixed with a flow probe to measure aortic blood flow (ABF). All hemodynamic parameters were recorded on an eight-channel polygraph (Grass Instruments 7D, U.S.A.).
After a stabilization period of ≥60 min after surgery, frovatriptan, sumatriptan, or vehicle (saline/0.9% NaCl, wt/vol) were administered in ascending doses from 0.0001-1.0 mg/kg, either intravenously or into the coronary artery.
Coronary perfusion after myocardial ischemia. In a separate group of animals, the heart was exposed through a left thoracotomy at the fifth intercostal space and suspended in a pericardial cradle. A 2-cm section of the LCX was exposed proximal to the first obtuse marginal branch and instrumented proximal to distal as follows: (a) electromagnetic flow probe (model 501; Carolina Medical Instruments) to measure blood flow; (b) an occluder, and (c) stenosis. All hemodynamic parameters were recorded on an eight-channel polygraph (Grass Instruments 7D).
After a stabilization period of 30 min, saline (0.9% NaCl wt/vol), frovatriptan (0.1 mg/kg), or sumatriptan (1.0 mg/kg), which are comparable doses of 5-HT1B/1D agonist in terms of their effects on carotid resistance, were administered intravenously. Fifteen minutes later, the LCX artery was occluded, reducing the CBF to zero for 60 min. The stenosis was removed 15 min after the release of the occluder, and reperfusion was allowed for 3 h. The dogs were killed by an overdose of saturated KCl. The left ventricle was sliced into six equal sections of 1 cm and placed into a 1.5% solution of triphenyl tetrazolium hydrochloride (TTC) at 37°C for 15 min. The slices were cut and weighed (grams) according to the remaining left ventricle (red tissue) and infarct area (white or brown; 19-21).
In experiments on isolated arteries, the effects of frovatriptan were expressed as a percentage of the initial 5-HT contraction or as a percentage reversal of contraction in experiments in which tone was increased with sumatriptan or U46619. In experiments on anesthetized dog, mean arterial blood pressure (MABP) was calculated as (systolic − diastolic)/3 + diastolic), coronary vascular resistance was calculated as (MABP/mean coronary blood flow, mm Hg · min/ml) and systemic peripheral resistance (SPR) as MABP/mean aortic blood flow (mm Hg · min/ml).
MABP and heart rate were expressed as absolute values. CBF and coronary vascular resistance were expressed as a percentage of Control, which was calculated as the mean of two readings before drug administrations. Statistical significance was assessed by repeated-measures analysis of variance and the Bonferroni t test by using SAS-RA.
Frovatriptan (VML 251/SB 209509 [(+)-6-carboxamido-3-methylamino-1,2,3,4-tetrahydrocarbazole succinate or corresponding camphor sulfonate salts] was synthesized at Smith Kline Beecham Pharmaceuticals. Sumatriptan succinate was extracted from commercially available tablets (Imigran). All drugs for in vivo administration were dissolved in saline (0.9% NaCl wt/vol).
Sodium Pentobarbital Injection (65 mg/ml) was purchased from Anpro Pharmaceuticals, Arcadia, CA, U.S.A. 5-Hydroxytryptamine, prazosin hydrochloride, indomethacin, U-46619, and cocaine were purchased from Sigma (Poole, U.K.). Ketanserin was obtained from Janssen (Beerse, Belgium).
In vitro reactivity
Frovatriptan elicited contractions ≤56 ± 7% of the maximal response to 5-HT (600 nM;18) in beagle isolated coronary artery preparations (Fig. 1). The effects of frovatriptan were complex in the dog isolated artery, as a bell-shaped concentration-response relation was apparent. For the contractile response, the pD2 value was 7.55 ± 0.08 (n = 11). However, high concentrations, >60 μM of frovatriptan produced smaller responses.
In artery rings precontracted with U46619, a similar bell-shaped concentration response also was observed in dog coronary artery (Fig. 2). Under these conditions, concentrations of frovatriptan (>2 μM) produced concentration-related full reversal of the contractile response to U46619 (Fig. 2).
In arteries precontracted with sumatriptan (10 μM), no further contraction was observed with increasing concentrations of frovatriptan. However, marked reversal of precontraction was observed at concentrations >6 μM frovatriptan (Fig. 2).
In vivo reactivity
Coronary and cardiac function. Average resting mean arterial blood pressure was between 83 and 121 mm Hg in experimental groups with resting heart rate between 132 and 178 beats/min (Table 1). Other resting haemodynamic and cardiac variables in experimental groups are shown in Table 1. Administration of either vehicle or frovatriptan by intravenous or intracoronary routes had little effect on MABP, heart rate, ABF, SPR, or cardiac contractility. No overall consistent response was observed with sumatriptan; however, low doses of sumatriptan (0.0003 mg/kg), administered via the intravenous route, produced a small but significant increase in MABP to 111.6 ± 2.8% compared with vehicle value of 101.0 ± 2.5% and small (<10%) decrease in heart rate at 0.03 mg/kg. Intracoronary artery administration of sumatriptan (0.0003 mg/kg) produced an increase in SPR to 120.5 ± 8.2% compared with vehicle response of 97.8 ± 5.4%, whereas higher doses had no consistent effect. This dose of sumatriptan also produced a significant increase in CBF and decrease in coronary vascular resistance. Intravenous administration of sumatriptan also produced a dose-related reduction in LVEDP, with a reduction to 41.7 ± 25% of control values observed at 1 mg/kg (Fig. 3). However, administration of sumatriptan by the intracoronary route had no effect (Fig. 4). Administration of vehicle or frovatriptan by either route had little effect on LVEDP or LVSP (Figs. 3 and 4). The highest dose of frovatriptan produced a reduction in coronary vascular resistance to 78.0 ± 7.0% of control values; however, this was not significantly different from vehicle (91.9 ± 7.2%) by using repeated-measures analysis between the three treatment groups.
Coronary perfusion after myocardial infarct. Mean arterial blood pressure was 116 ± 6 mm Hg (n = 6), 121 ± 4 mm Hg (n = 5), and 120 ± 7 mm Hg (n = 5) in saline-, sumatriptan-, and frovatriptan-treated animals, respectively. Heart rate (beats/min) was 165 ± 7, 163 ± 2, and 171 ± 5 in these groups of animals. MABP and heart rate did not significantly change during coronary artery occlusion or reperfusion throughout the duration of the experiment. Respective resting CBFs and coronary vascular resistance (in parentheses) values were similar in all groups and were 19.3 ± 7.3 ml/min (6.7 ± 1.1 mm Hg · min/ml), 23.1 ± 2.0 ml/min (5.48 ± 0.53 mm Hg · min/ml), and 15.6 ± 2.1 ml/min (8.3 ± 1.1 mm Hg · min/ml) in saline-, sumatriptan-, and frovatriptan-treated animals, respectively.
This protocol produced an infarct of 12.97 ± 3.78% of the left ventricle in vehicle-treated animals (n = 6). Sumatriptan or frovatriptan had no significant effect on lesion size, with lesions of 17.17 ± 2.74% (n = 5) and 12.06 ± 2.58% (n = 5), respectively.
CBF was reduced to zero in all three treatment groups during ischemia. During the reperfusion period, CBF values were assessed immediately after reperfusion and at 60-min intervals. CBF tended to increase over resting values in the control group, with a corresponding tendency for a decreased coronary vascular resistance (Fig. 5). CBF and vascular resistance in animals treated with frovatriptan were similar to those in the control group. However, sumatriptan produced a significant decrease in CBF, which was reflected in the increased coronary vascular resistance throughout the reperfusion period.
This study demonstrates that frovatriptan produces effects in canine arteries similar to those previously observed in human isolated coronary arteries (4) and confirms the dog as a useful model for humans. Frovatriptan produces marked bell-shaped concentration-response curves, with concentrations of frovatriptan between 6 nM and 6 μM producing contraction. This profile of response is similar to that observed for 5-HT (18,22) or for 5-car-boxamidotryptamine (5-CT; 18), which are also potent relaxant agents in the coronary vasculature (18,22,23). Activation of smooth-muscle 5-HT7 receptors may mediate these effects because a 5-HT7-receptor ligand has been shown to inhibit 5-HT-induced relaxation and enhance 5-HT-induced contraction in the dog (23). It is interesting to note that frovatriptan has a modest affinity (pKi, 6.7) for the human cloned 5-HT7 receptor (1), although it is ≥50-fold selective for human cloned 5-HT1B (pKi, 8.6) or 5-HT1D (pKi, 8.4) relative to 5-HT7 receptors (1). It is therefore possible that activation of the 5-HT7 receptor may play a role in the relaxant activity of frovatriptan in coronary arteries, although this hypothesis would require further study.
In human arteries, there is some evidence that activation of thromboxane receptors enhances the contractile activity of 5-HT1B/1D-receptor agonists (24,25). In canine arteries, sumatriptan produces further contraction and no relaxation (26). This study clearly shows that frovatriptan can produce relaxation of precontracted dog coronary arteries in vitro, effects that may limit contraction of the coronary vasculature.
Frovatriptan had no effect on coronary or cardiac function after intravenous or intracoronary artery administration in mongrel dogs. A trend for reduced coronary vascular resistance and increased CBF was observed at a dose of 1 mg/kg frovatriptan. The magnitude of the changes was similar to that observed in beagle dogs (3) but did not reach statistical significance after analysis of variance for repeated measures. Sumatriptan also had little overall effect on coronary flow or resistance, but low doses produced a significant increase in coronary flow and reduction in resistance. These effects were not dose related; however, a high-affinity relaxant action of sumatriptan was observed in isolated guinea pig heart (27).
Intravenous administration of sumatriptan produced a dose-related decrease in LVEDP with no effect on contractility (dP/dt), LVSP, or SPR. These effects may not be due to a local effect on the coronary vascular bed, as intracoronary administration of sumatriptan had no effect on these parameters. In patients, sumatriptan has been shown to produce coronary artery constriction and to increase systemic and pulmonary vascular resistance (13,14) and LVEDP with no change in cardiac contractility (28). Similar effects have been noted in chloralose-anaesthetized dogs after administration of low doses of sumatriptan (10-30 μg/kg; 29). In this study, sumatriptan produced modest (10-15%) increases in both total peripheral resistance and LVEDP.
In patients after administration of sumatriptan, an increased LVEDP was suggested to be the result of an increased afterload due to venoconstriction (28). In this study, no effect was found on cardiac output, although increases in pulmonary artery pressure and right atrial pressure were reported (28). Our results may suggest that in anesthetized animals, in which no change in SPR occurs, additional mechanisms contribute to a decrease in LVEDP. It would seem likely that this is mediated by a reduced afterload, although the precise nature of this response is not clear. As this effect was manifested only by intravenous administration of sumatriptan, these effects may reflect changes in systemic vascular beds and may be attributed to the pulmonary circulation. In patients, sumatriptan increased systemic and pulmonary vascular resistance (13,14,28) with a net effect of increasing afterload and LVEDP (28). In pentobarbitone-anesthetized dogs, no increase in SVR was noted. However, the observed decrease in LVEDP would be consistent with an increased pulmonary vascular resistance and reduction of left ventricular afterload. As these experiments were performed in open-chest dogs, lack of control of thoracic pressure may have an effect on the overall response to sumatriptan on pulmonary artery pressure and regional flow. This warrants further investigation. It is not known whether these effects of sumatriptan are produced by activation of the 5-HT1B/1D receptor, but frovatriptan had no effect on these parameters, consistent with its functional selectivity toward the cerebral vascular bed. It should be noted that the observed changes in LVEDP are small in absolute terms (∼3-4 mm Hg) and may have little consequence on normal physiologic processes.
Preliminary studies suggested that sumatriptan may increase the extent of experimentally induced myocardial infarction in the dog (30). In our experiments, in a standard model of infarction, we evaluated sumatriptan and a comparable dose of frovatriptan in terms of producing a 50% increase in carotid vascular resistance in the open-chest dog (3). Neither sumatriptan or frovatriptan had any effect on infarct size. However, we observed a reduction in postinfarction CBF after administration of sumatriptan for ≤3 h. These observations are consistent with those of Ellis et al. (30), who noted an ∼50% reduction in collateral flow to the ischemic region after sumatriptan administration. We showed that sumatriptan also produces a decrease in LVEDP in open-chest dogs, and it seems likely that this effect may contribute to the reduction in CBF after myocardial infarct. The consequences of changes in LVEDP may therefore become apparent in disease models. Overall, these results are consistent with the observed hemodynamic effects of sumatriptan in patients, although the effects of sumatriptan on pulmonary hemodynamics in open-chest dogs would require further verification.
In contrast to the effects of sumatriptan, frovatriptan (0.1 mg/kg) had no effect on CBF after myocardial infarction and was without effect on LVEDP ≤1 mg/kg. These results show a clear separation of effect of frovatriptan and sumatriptan on cardiac function in dogs.
In conclusion, collectively these data show that frovatriptan has no major effect on coronary or cardiac function under physiologic or pathophysiologic conditions. In contrast, sumatriptan produces marked changes in LVEDP when administered systemically and reduces CBF after myocardial infarct. These data emphasize a distinct pharmacologic profile for frovatriptan and its selectivity for the cerebral vascular bed.
Acknowledgment: We thank Steve Parker for his comments and careful review of the manuscript.
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