Introduction

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The launch of the first selective 5-hydroxytryptamine (5-HT)_1B/1D receptor agonist sumatriptan (Humphrey and Feniuk, 1991) in the early 1990s has been hailed as the most significant advance in the acute treatment of migraine since the introduction of dihydroergotamine (DHE) more than 50 years ago. Sumatriptan therefore represented a major new therapeutic principle in migraine, selectively activating 5-HT_1B/1D receptor subtypes (previously 5-HT_1-like; Humphrey and Feniuk, 1991). Although the precise mechanism by which sumatriptan alleviates migraine is still not fully elucidated, three distinct pharmacological actions on the vasculature and neurons have been invoked to explain its antimigraine effect. Vasoconstriction of cranial blood vessels (Humphrey and Feniuk, 1991; Ferrari and Saxena, 1993), inhibition of neurogenic inflammation involving reduced vasodilator sensory neuropeptide release from peripheral (dural) trigeminal nerve terminals (Moskowitz, 1992) and/or inhibition of firing of trigeminal neurons (Hoskin et al., 1996) have been proposed. Sumatriptan is an effective acute treatment for migraine (Ferrari, 1998), but it has major limitations: low oral bioavailability (see Goadsby, 1998b), fewer than half of the patients who are treated are pain free at 2 h after drug administration, and about one third of responders experience headache recurrence within 24 to 48 h (Ferrari, 1998). The room for improvement over the clinical effectiveness of sumatriptan is therefore substantial. Thus, newer agents, all of which are tryptamine derivatives, including naratriptan, rizatriptan, and zolmitriptan, have improved oral bioavailabilities (40-74%; Ferrari, 1998; Goadsby, 1998b) but apparently have not superseded sumatriptan in terms of therapeutic effectiveness according to initial reports (Ferrari, 1998; Goadsby, 1998a, b). Indeed, a limited therapeutic response ("ceiling effect") to this class of agents in aborting a migraine attack is currently being unveiled (Ferrari, 1998; Goadsby, 1998a, b).

The rationale for our chemical approach was to take advantage of the superior potency and efficacy characteristics of 5-HT compared with tryptamine at 5-HT_1B/1D receptors because we hypothesized that the magnitude of the intrinsic activity that is produced at 5-HT_1B/1D receptors by selective agonists is a key determinant of therapeutic antimigraine effectiveness. Most, if not all, of the selective 5-HT_1B/1D receptor agonists derived from tryptamine that have been described to date, including sumatriptan, naratriptan, rizatriptan, zolmitriptan, and eletriptan, behave as partial agonists with respect to the endogenous agonist 5-HT (Connor et al., 1997; Martin et al., 1997; Pauwels et al., 1997; Willems et al., 1998). Moreover, it might be considered, on theoretical grounds, that the relatively low intrinsic activity of the currently available 5-HT_1B/1D receptor agonists might limit their therapeutic effectiveness in alleviating migraine. In principle, a high-efficacy agonist will have the advantage over a lower efficacy agonist (i.e., a partial agonist) in producing full responses whatever the extent of receptor reserve in the tissue or organ in question (Kenakin, 1993). Thus, and in contrast to a partial agonist, a high-efficacy 5-HT_1B/1D receptor agonist can be expected to elicit large amplitude responses throughout the entire peripheral and central trigeminovascular system where 5-HT_1B/1D receptors are located. The outcome of our research effort is F 11356 (Perez et al., 1995; Fig. 1), which is presently shown to exert potent and selective actions, and exceptionally high efficacy at 5-HT_1B and 5-HT_1D receptors both in vitro and in vivo in models relevant to the vascular and neurogenic hypotheses in migraine. Because F 11356 is orally active, has a long duration of action, gains access to the brain, and is well tolerated in animals, the drug is endowed with the potential to provide acute therapeutic relief from migraine headache, which is superior to that offered by currently available treatments.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Chemical structure of F 11356, a novel arylpiperazide derivative of serotonin. For details of chemical synthesis, see Perez et al. (1995).

	Materials and Methods

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All experiments were carried out in accordance with the European Communities Counsel Directive of November 24, 1986 (86/609/EEC), and the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (NIH publication no. 85-23, revised 1985) and were approved by the local ethics committee.

Receptor-Binding Assays. F 11356 and other ligands were examined in vitro using membrane preparations from brain tissue or mammalian cell lines expressing recombinant 5-HT receptors. Radioligand-binding studies were performed as previously described in cell lines that had been transfected with the following receptors or gene products: human (h) 5-HT_1A (HeLa/HA7: Pauwels and Palmier, 1995), h 5-HT_1B and h 5-HT_1D (Cos-7: Pauwels et al., 1995; C6 rat glioma: Pauwels et al., 1996, 1997; Wurch et al., 1997b), rabbit 5-HT_1B (Cos-7: Wurch et al., 1997b), rat 5-HT_1B (Cos-7: Pleus and Bylund, 1992), guinea pig 5-HT_1B (C6 rat glioma: Pauwels et al., 1998b), rat and guinea pig 5-HT_1D (Cos-7: Wurch et al., 1997a), h 5-HT_1E and h 5-HT_1F (Cos-7: Pauwels et al. 1997), h 5-HT_2A (HEK 293: Leysen et al., 1982), h 5-HT_5A (Cos-7: Rees et al., 1994), h 5-HT₆ (Cos-7: Monsma et al., 1993), h 5-HT₇ (Cos-7: To et al., 1995), and rat 5-HT₇ (Cos-7: Shen et al., 1993).

Inhibition of Forskolin-Induced Cyclic AMP (cAMP) Formation. Inhibition of forskolin (100 µM)-induced cAMP formation in C6 glioma cells stably expressing h 5-HT_1B, h 5-HT_1D, or h 5-HT_1A receptors, respectively, by F 11356 and other ligands was investigated as described previously (Pauwels et al. 1996).

Stimulation of Specific [³⁵S]Guanosine-5'-O-(3-thio)triphosphate (GTPS) Radioligand Binding. [³⁵S]GTPS specific binding was determined in the presence or absence of F 11356 and other ligands in C6 glioma cell membranes stably expressing h 5-HT_1B and h 5-HT_1D receptors, as described previously (Pauwels et al. 1997). Maximal stimulation of specific [³⁵S]GTPS binding was expressed as a percentage of the response obtained with 10 µM 5-HT.

Contractile Responses in Rabbit Isolated Saphenous Vein. Rabbit isolated saphenous vein rings with intact endothelium were prepared for isometric tension recording, as previously described (Valentin et al., 1996), in the presence of N-nitro-L-arginine methyl ester (L-NAME, 10 µM) to inhibit endothelial NO synthase. Cumulative concentration-effect curves were determined for F 11356, 5-HT, sumatriptan, and naratriptan in either the presence or absence of the mixed 5-HT_1B/1D receptor antagonist GR 127935 (Clitherow et al., 1994).

Stimulation of Outward K⁺ Current in Guinea Pig Isolated Trigeminal Ganglion Cells. Guinea pig trigeminal ganglion cells were isolated from right and left ganglia as described by Liu and Simon (1994) and were cultured for 12 to 18 h at 37°C in 5% CO₂ in a Dulbecco's modified Eagle's medium containing 10% heat-inactivated DCS. Outward K⁺ currents were studied as described previously (Le Grand et al., 1998a) in trigeminal ganglion cells using the patch-clamp technique in the whole-cell configuration. Cumulative concentration-effect curves were obtained for F 11356 and sumatriptan, with each concentration being applied for 3 min. Steady-state outward currents (I_Kss) were measured at the end of a 3-s test pulse (stimulation frequency = 0.05 Hz) relative to the current before the beginning of the test pulse. Experiments were repeated in either the presence or absence of GR 127935 (0.1 µM).

Hypothermia in Guinea Pigs. Rectal temperature was measured to the nearest 0.1°C in male Dunkin-Hartley guinea pigs (300-400 g; Charles River, France) as described by Skingle et al. (1994). Temperature measurements were taken immediately before the oral administration of F 11356 and other compounds, and at 15 and 30 min and 1, 2, 4, and 8 h after drug or vehicle administration. Drugs were administered in 1% methyl cellulose in distilled water.

Carotid Hemodynamics in Anesthetized Pigs. Male Landrace pigs (19-25 kg; M. Gaec, Soreze, France) were anesthetized with azaperone (3 mg/kg i.m.) followed by sodium pentobarbital (25 mg/kg i.v. bolus and 10-20 mg/kg/h infusion) and then intubated and artificially ventilated (Alpha100, Minerve, Esternay, France) with a mixture of room air and oxygen (0.5-1 liter/min). Blood gases and arterial pressure were maintained within physiological limits. Heparin (500 IU/kg i.v.) was also administered. Before thoracotomy, catheters were placed in the inferior vena cava via the left saphenous vein for drug/vehicle administration and in the right saphenous vein for anesthesia. Femoral arteries were cannulated (7F; Vygon, Ecouen, France) for aortic blood pressure measurements (Statham P10EZ transducers) or for arterial blood gas determinations (ABL-510; Radiometer, Copenhagen, Denmark). Blood flow was measured in left and right common carotid arteries and left anterior descending coronary artery by means of appropriately sized flow probes connected to a pulsed Doppler flow amplifier (VF1; Crystal Biotech, Northboro, MA). Body temperature was maintained at 37-38°C (by means of a thermostated insulating blanket), and sterile saline was infused i.v. throughout the experiment to compensate fluid loss (0.5-1 liter total volume). Parameters were digitized and analyzed on line by computer using interactive software (Dataflow; Crystal Biotech). Calculated parameters were as follows: total carotid vascular resistance was derived from mean arterial pressure divided by total carotid blood flow, and coronary vascular resistance was derived from coronary perfusion pressure divided by left anterior descending coronary blood flow. Coronary perfusion pressure was determined as the difference between diastolic aortic pressure and left ventricular end-diastolic pressure.

Oral Activity in Conscious Dogs. Four male beagle dogs (18-25 kg; CEDS, Toucy, France) were sedated with a mixture of fentanyl and fluanizone (Hypnorm, 0.1 ml/kg i.m.), received atropine (0.02 mg/kg i.m.), and were anesthetized with sodium pentobarbital (20-25 mg/kg i.v.). After intubation, anesthesia was maintained under artificial ventilation with isoflurane (1-2%) in oxygen (2-4 liters/min) and room air (5-6 liters/min). Under sterile conditions, a ventral neck incision was made, the left carotid artery was isolated, and an appropriately sized pulsed Doppler flow probe (2.8-3.2 mm; Crystal Biotech) was placed around the vessel and secured. Cables were externalized under the neck skin between the scapulae, in an anchor button system (Bioseb, Chaville, France), to maintain sterility. After surgery, each dog was treated with buprenorphine (0.02 mg/kg s.c.) and penicillin-procaine (Duphaphen-LA; 0.2 ml/kg i.m.) for 3 days for analgesic and antibiotic coverage, respectively. During the postoperative period, dogs were trained to lie quietly in a cage. Experiments were conducted 2 to 3 weeks after surgery, and external wires were protected by a nylon vest (Phymep, Paris, France) that the dogs had previously been trained to wear. The four-limb electrocardiogram was derived from lead II and was used for computerized calculation of heart rate. Carotid blood flow and electrocardiographic signals were digitized and recorded on line using interactive software (Dataflow; Crystal Biotech).

In Vivo Receptor Selectivity Studies. Behavioral studies were performed in rats in which lower-lip retraction, flat body posture, and forepaw treading were quantified as described previously (Kleven et al., 1995; n = 6 rats/group).

Further Cardiovascular Studies. Studies of contractile responses in canine isolated coronary arteries were performed as described previously (Valentin et al., 1998). Effects in isolated, Langendorff-perfused guinea pig hearts were assessed as described by Le Grand et al. (1998b). Effects on action potentials recorded in isolated guinea pig papillary muscle were evaluated as described by Le Grand et al. (1995).

Data Analysis. Data are presented as mean ± S.E.M. Concentration- and dose-response curves were fitted using the Marquardt algorithm by means of appropriate software (Origin; Microcal, Northhampton, MA), which gave geometric EC₅₀ or ED₅₀ values with 95% confidence intervals. Statistical analysis was performed on absolute values by ANOVA with or without repeated measurements followed by the Dunnett's post hoc test, as appropriate (Sigmastat; Jandel GmbH, Erkrath, Germany; or Statview; Abacus Concepts Inc., Berkeley, CA), unless stated otherwise. p < .05 was considered statistically significant.

Drugs and Vehicles. F 11356 hydrochloride, sumatriptan hydrochloride, rizatriptan hemisulfate, zolmitriptan base, naratriptan base, GR 127935 dihydrochloride, and (+)-flesinoxan hydrochloride were synthesized by the Divisions of Medicinal Chemistry IV and Analytical Chemistry at the Centre de Recherche Pierre Fabre.

	Results

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Affinity, Potency, Efficacy, and Selectivity of F 11356 at Human and Nonhuman 5-HT_1B and 5-HT_1D Receptors. F 11356 had subnanomolar affinity for cloned human, guinea pig, rabbit, and rat 5-HT_1B and 5-HT_1D receptors, with pK_i values ranging from 9.3 to 10.3 (Table 1). F 11356 had 50 times less binding affinity for the human and rat 5-HT_1A receptor (pK_i = 7.6-7.8; Table 1) and low affinity for other 5-HT receptor subtypes, including the h 5-HT_1F-binding site (Table 1). Thus, F 11356 had high affinity at and high selectivity for 5-HT_1B and 5-HT_1D receptors. F 11356 did not distinguish between 5-HT_1B and 5-HT_1D receptors, possessing equivalent affinity at these sites (Table 1).

View this table: [in this window] [in a new window]   TABLE 2 Inhibition of forskolin-induced cAMP formation and enhancement of specific [35S]GTPS binding in membranes of C6 glioma cells stably expressing h 5-HT1B or h 5-HT1D receptors Inhibition of forskolin (100 µM)-induced cAMP accumulation and enhancement of specific [35S]GTPS binding was determined as described in the text. pD2 values correspond to the negative logarithm of the geometric drug concentration that produces 50% of its maximal response and are expressed with 95% CI. pD2 app. corresponds to apparent pD2 values. n corresponds to the number of independent experiments. N.D., not determined.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Maximal enhancement of specific [35S]GTPS binding in C6 glioma cells stably expressing h 5-HT1B (top) and 5-HT1D receptors (bottom), as described by Pauwels et al. (1997). F 11356 produced equivalent maximum responses (Emax) to 5-HT at both receptors, which was not the case for the other drugs investigated. Data were normalized to 5-HT = 100% enhancement of specific [35S]GTPS binding. For relative potencies, refer to Table 2. Data are mean ± S.E.M. *p < .01 versus 5-HT (Mann-Whitney U test). Data for sumatriptan, zolmitriptan, rizatriptan, naratriptan, and DHE are from Pauwels et al. (1997).

In Vitro Vascular and Neuronal Actions of F 11356. In isolated rabbit saphenous vein rings prepared for isometric tension recording as described previously (Valentin et al., 1996), F 11356 was equipotent to 5-HT and more potent than naratriptan, rizatriptan, zolmitriptan, and sumatriptan in producing contractile responses (Table 3). Furthermore, like naratriptan, F 11356, produced equivalent maximal responses to 5-HT, whereas superior maximal responses compared with 5-HT were elicited by sumatriptan, rizatriptan, and zolmitriptan. Contractile responses evoked by F 11356 were antagonized by GR 127935 in a noncompetitive manner, with a pIC₅₀ value of 9.3 (8.6-10.1), and were abolished by 0.1 µM.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   A, increases in outward steady-state (ss) hyperpolarizing K+ current (IK) evoked by F 11356 () and sumatriptan () in isolated guinea pig trigeminal ganglion cells. Note greater potency and amplitude of responses elicited by F 11356. *p < .05 compared with sumatriptan (ANOVA with repeated measures followed by Student's t test). B, maximum increases in IKss evoked by F 11356 and sumatriptan in the presence or absence of GR 127935 (0.1 µM) or EGTA (5 mM). *p < .05 compared with vehicle controls (ANOVA with repeated measures followed by Student's t test). Data are mean ± S.E.M. See Materials and Methods for further details.

In Vivo Activity of F 11356 at Carotid Arterial 5-HT_1B/1D Receptors. Baseline values of hemodynamic parameters in anesthetized pigs are shown in Table 4. F 11356, administered as cumulative i.v. infusions to anesthetized pigs, induced a pronounced, dose-related reduction in total carotid blood flow [ED₅₀ = 0.53 (0.42-0.67) µg/kg i.v.]. F 11356 simultaneously evoked large magnitude increases in total carotid vascular resistance (Fig. 4A). Mean systemic arterial pressure was increased by F 11356 only at the dose of 10 µg/kg (maximum change, 17 ± 5%, p < .05 compared with vehicle, Fig. 4A). Sumatriptan failed to significantly decrease total carotid blood flow or to affect mean arterial pressure (p = NS) and produced a bell-shaped dose-response curve of increases in total carotid vascular resistance (Fig. 4B). Naratriptan significantly reduced total carotid blood flow only at the dose of 40 µg/kg (p < .05 compared with vehicle) and, like sumatriptan, produced a bell-shaped dose-response curve of increases in total carotid vascular resistance and mean arterial pressure (Fig. 4C). Zolmitriptan and rizatriptan dose-dependently and moderately reduced total carotid blood flow [ED₅₀ = 5.6 (2.8-11.2) and 19.8 (6.8-57.0), respectively]. Although zolmitriptan produced dose-dependent, moderate increases in total carotid vascular resistance (Fig. 4D), rizatriptan significantly (p < .05) increased total carotid vascular resistance at 40 µg/kg but not at other doses (Fig. 4E). Zolmitriptan produced slight increases in mean arterial pressure at 10 and 40 µg/kg (Fig. 4D), whereas rizatriptan was without effect (Fig. 4E). Near-maximal reductions in total carotid blood flow (48 ± 4%; p < .05 compared with vehicle) and increased total carotid vascular resistance (99 ± 8%; p < .05 compared with vehicle) with F 11356 were only slowly reversible, being maintained for at least 60 min after the infusion of F 11356 was stopped (Fig. 5). As illustrated in Fig. 5, increases in total carotid vascular resistance had returned to near-control values 60 min after the infusion of sumatriptan, rizatriptan, naratriptan, and zolmitriptan was stopped, which is in contrast to that observed with F 11356. The drugs and vehicle investigated had no significant effects on heart rate or coronary vascular resistance (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Comparison of increases in total carotid vascular resistance () and mean arterial pressure () elicited by F 11356 (A), sumatriptan (B), naratriptan (C), zolmitriptan (D), and rizatriptan (E) in anesthetized pigs. Note different abscissa in A (greater potency of F 11356) and bell-shaped dose-response curve in B and C. *p < .05 compared with vehicle (ANOVA with repeated measures followed by Dunnett's test).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effects of F 11356, naratriptan, zolmitriptan, sumatriptan, rizatriptan, and corresponding vehicles on total carotid vascular resistance in anesthetized pigs 60 min after drug/vehicle infusion was stopped. Increases in total carotid vascular resistance remained at near-maximal values 60 min after F 11356 infusion was stopped. Data are mean ± S.E.M. *p < .05 compared with corresponding vehicle controls (one-way ANOVA with repeated measures followed by Dunnett's test).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effects of orally administered F 11356 (0.63 mg/kg, , and 2.5 mg/kg, ) and placebo (empty gelatin capsules, ) on unilateral carotid blood flow (A) and heart rate (B) in conscious dogs. Data are mean ± S.E.M. Dose-dependent and long-lasting (more than 12 h at 2.5 mg/kg) reductions in carotid blood flow were observed with F 11356 in the absence of changes in heart rate, behavior, or clinical signs. At this dose, maximum values were attained 30 min after F 11356 administration. *p < .05 compared with placebo (one-way ANOVA with repeated measures followed by Dunnett's test).

Hypothermia in Guinea Pigs. Orally administered F 11356 evoked hypothermic responses in guinea pigs and was found to be more potent than sumatriptan, zolmitriptan, naratriptan, rizatriptan, and DHE (Table 6). Relatively high doses (up to 40 mg/kg) of sumatriptan and DHE failed to produce hypothermic responses of sufficient magnitude to determine ED₅₀ values.

In Vivo Receptor Selectivity of F 11356. The in vitro procedures described earlier indicated that F 11356 had affinity for 5-HT_1A receptors that is 50-fold lower than that for 5-HT_1B/1D receptors. In rats, characteristic behavioral effects of 5-HT_1A receptor agonists such as lower-lip retraction, flat body posture, or forepaw treading (Kleven et al., 1995) were not observed after a high dose of F 11356 (40 mg/kg p.o.; data not given).

Further Cardiovascular Studies. The studies performed in anesthetized pigs and conscious dogs described earlier demonstrate that F 11356, at the pharmacological doses investigated, is devoid of any major hemodynamic side effects. The following studies have been carried out to extend its cardiovascular profile: Contractile responses evoked by F 11356 were compared with those of DHE, naratriptan, and sumatriptan in the canine isolated coronary artery as described previously (Valentin et al., 1998; n = 15-17 rings/group) in the absence of endothelium. All four agonists contracted coronary arteries, with a rank order of potency (pD₂ values) of DHE [6.9 (5.3-7.9)]  naratriptan [6.8 (5.7-7.3)]  F 11356 [6.7 (5.7-7.3)] > sumatriptan [4.8 (3.6-5.6)]. Rank order of intrinsic activity (E_max ± S.E.M.) was sumatriptan (2.5 ± 0.6 mN) > naratriptan (1.7 ± 0.6 mN) > F 11356 (1.0 ± 0.4 mN) > DHE (0.6 ± 0.2 mN). No significant differences were noted between E_max values. However, E_max values produced by all four drugs were substantially lower than those evoked by an approximate EC₅₀ concentration (10 nM) of the thromboxane A₂ analog U 46619 (27.5 ± 3.9 mN), indicating relatively weak contractile responses induced by F 11356, sumatriptan, naratriptan, and DHE.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

F 11356 is a novel arylpiperazide 5-HT derivative that has been designed to take advantage of the superior potency and efficacy characteristics of 5-HT compared with tryptamine at 5-HT_1B/1D receptors. Indeed, the most striking feature of the pharmacological profile of F 11356, besides high potency and selectivity, is the uniquely high level of intrinsic activity exerted by the compound at 5-HT_1B/1D receptors.

Radioligand Binding and Cellular Assays. F 11356 has subnanomolar affinity for recombinant human and nonhuman 5-HT_1B and 5-HT_1D receptors, does not distinguish between the two subtypes, and displays high selectivity over other 5-HT-binding sites, including 5-HT_1F sites. F 11356 has low or no detectable affinity for a number of major neurotransmitter, autacoid, ion channel, or reuptake binding sites (unpublished observations). Moderate (submicromolar) affinity was shown for F 11356 at 5-HT_1A receptors, and the intact cell cAMP assay yielded similarly weak 5-HT_1A receptor agonist activity for F 11356. The radioligand-binding profile of F 11356 is one of a high-affinity, high-selectivity 5-HT_1B/1D receptor ligand.

In Vitro Activity in Vascular and Neuronal Models. 5-HT_1B receptors have been shown to mediate vasoconstriction (Hamel et al., 1993; Valentin et al., 1996), and any involvement of 5-HT_1D receptors in mediating vasoconstriction has not been thus far demonstrated. The isolated rabbit saphenous vein is a well established preparation in which potency and efficacy characteristics of 5-HT_1B/1D receptor agonists have been determined (Martin and MacLennan, 1990). F 11356 induced 5-HT_1B receptor-mediated contractions of isolated rabbit saphenous vein rings with a similar potency and maximal response as those of 5-HT. Naratriptan, sumatriptan, rizatriptan, and zolmitriptan were less potent than F 11356. Although naratriptan produced equivalent maximum responses to F 11356 and 5-HT, those generated by rizatriptan, sumatriptan, and zolmitriptan were of greater magnitude. As shown for sumatriptan (Valentin et al., 1996), a component of the contractile responses evoked by rizatriptan and zolmitriptan is dependent on an intact endothelium, suggesting the release of an endothelium-derived contraction factor (Valentin et al., 1996). However, this does not appear to apply to F 11356 or naratriptan because both produced equivalent maximum responses to 5-HT. In agreement with this finding, no significant differences in maximum responses were noted between F 11356, 5-HT, rizatriptan, sumatriptan, zolmitriptan, and naratriptan in the absence of endothelium (unpublished observations). Sensitivity of contractile responses evoked by F 11356 to the mixed 5-HT_1B/1D receptor antagonist GR 127935 (Clitherow et al., 1994) supports the involvement of 5-HT_1B receptors in mediating these responses, as described previously for 5-HT, 5-carboxamidotryptamine, and sumatriptan (Valentin et al., 1996). The greater potency of F 11356 and 5-HT compared with that of the tryptamine derivatives investigated is also due in part to the high-efficacy characteristics of the former two compounds at the saphenous vein 5-HT_1B receptor. These data provide further confirmation of the superior potency of F 11356 at 5-HT_1B receptors compared with the tryptamine derivatives investigated and similar potency of F 11356 as that of 5-HT. These data also demonstrate that F 11356 is active at the vascular level, which is in line with the vascular hypothesis of migraine (Ferrari and Saxena, 1993).

In Vivo Pharmacological Profile of F 11356. 5-HT_1B/1D receptor agonists elicit vasoconstriction in the carotid vascular bed of dogs (Feniuk et al., 1989) and pigs (Den Boer et al., 1991). This pharmacological activity is considered to be of importance in the migraine abortive effects of 5-HT_1B/1D (previously 5-HT_1-like) receptor agonists (Feniuk et al., 1989; Den Boer et al., 1991; De Vries et al., 1996) and is a key element of the vascular hypothesis (Humphrey and Feniuk, 1991; Ferrari and Saxena, 1993). In anesthetized pigs, F 11356 elicited carotid vasoconstriction in a highly selective manner with respect to systemic arterial pressure or coronary vascular resistance. F 11356 was more than 10 times more potent than naratriptan, rizatriptan, sumatriptan, or zolmitriptan in reducing carotid blood flow and consistently produced greater maximum responses. Dose dependence of carotid vasoconstriction elicited by sumatriptan and naratriptan had a bell-shaped aspect suggestive of low agonist efficacy; and activation of other mechanisms at higher doses to explain the downturn phase of the dose-response curve can be excluded because sumatriptan-induced carotid vasoconstriction is mediated exclusively by 5-HT_1B/1D receptors (De Vries et al., 1996).

In Vivo Receptor Selectivity. F 11356 failed to produce behavioral effects in rats, even at a relatively high dose (40 mg/kg), indicating that receptor selectivity of F 11356 for 5-HT_1B/1D receptors versus 5-HT_1A receptors is maintained in vivo.

Further Cardiovascular Studies. Data obtained in vitro with F 11356 in canine coronary artery rings revealed that the compound produced equivalent, low-amplitude maximal responses to sumatriptan, naratriptan, and DHE and was more potent than sumatriptan but less potent than naratriptan and DHE. In view of the high-efficacy properties of F 11356 compared with the other compounds investigated, greater maximal responses might have been expected but were not observed, which is in accord with the absence of changes in coronary vascular resistance in anesthetized pigs and the high degree of craniovascular selectivity exhibited by F 11356 in vivo described earlier. We have no obvious explanation as to why F 11356 behaves in a similar fashion to the tryptamine derivatives studied and DHE in producing weak coronary vasoconstriction. One possible reason is that 5-HT_1B receptor-effector coupling in this tissue can produce only limited contractile responses, even on maximal activation. Although relatively weak contractile responses evoked by 5-HT_1B/1D receptor agonists in isolated coronary arteries have been shown by others (Connor et al., 1997; Maassen Van Den Brink et al., 1998; Valentin et al., 1998), further studies are required to address this important issue.

Conclusion. In summary, F 11356 is a novel arylpiperazide 5-HT derivative; is a selective, high-potency agonist at 5-HT_1B/1D receptors; and distinguishes itself from tryptamine and derivatives and DHE in exerting exceptional intrinsic activity at these receptors in models relevant to the vascular and neurogenic hypotheses of migraine. By virtue of its high-efficacy properties at 5-HT_1B/1D receptors, F 11356 displays hitherto unobserved high levels of craniovascular selectivity in vivo with respect to coronary and systemic vasoconstriction. Despite high intrinsic activity at 5-HT_1B receptors, the amplitude of canine coronary vasoconstriction evoked by F 11356 was not greater than that observed with sumatriptan, naratriptan, or DHE. The level of intrinsic activity of F 11356 obtained at 5-HT_1B/1D receptors is comparable to that of the endogenous agonist 5-HT. The selective 5-HT_1B/1D receptor agonists derived from tryptamine that have been described thus far, including sumatriptan, naratriptan, rizatriptan, zolmitriptan, and eletriptan, behave as partial agonists (i.e., have low agonist efficacy at these receptors with respect to 5-HT; Connor et al., 1997; Martin et al., 1997; Pauwels et al., 1997; Willems et al., 1998), which might limit their oral clinical antimigraine effectiveness (Ferrari, 1998; Goadsby, 1998a, b). Clinical studies with a high-efficacy 5-HT_1B/1D receptor agonist, of which F 11356 is the first, are therefore justified to verify the hypothesis that the magnitude of intrinsic activity at these receptors is a key determinant of therapeutic antimigraine effectiveness. F 11356 is orally active, gains access to the central nervous system, has a long duration of action, and is well tolerated. As a putative antimigraine drug, F 11356 may exert high-efficacy agonist activity throughout the entire trigeminovascular system in which 5-HT_1B/1D receptors are located: at its peripheral sensory nerve-vessel interface and the ganglion and central (brain stem) levels. Thus, F 11356 has the potential to exert greater therapeutic antimigraine activity than other, currently available treatments.