Introduction

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Current drug treatment for migraine headaches includes NSAIDs like ibuprofen, the ergot DHE and the 5-HT_1D agonist SUMA. NSAIDs inhibit prostaglandin synthesis and attenuate neurogenic inflammation in the trigeminovascular system (Buzzi et al., 1988). However, NSAIDs are not effective for many migraine patients and are associated with the risk of dyspepsia and gastrointestinal hemorrhage (Welch, 1993). DHE and SUMA are currently the most efficacious drugs available for migraine. DHE is associated with nausea, vomiting, abdominal pain, diarrhea and cerebral vasoconstriction (Welch, 1993). Side effects associated with SUMA are coronary vasospasm and chest heaviness (Welsh, 1993). Furthermore, both DHE and SUMA are significantly less active in humans after oral administration. Newer 5-HT_1D agonists may soon be available. These drugs are orally active and have been reported to be more potent than SUMA; however, the pharmacology of these second-generation "SUMA-like drugs" does not exclude potential cardiovascular effects. In light of these therapeutic limitations, an antimigraine drug that demonstrates oral efficacy without the cardiovascular liability of SUMA or DHE represents an improvement over current therapies.

Markowitz et al. (1987) proposed that migraine pain can occur as a result of trigeminal nerve stimulation. Factors and mediators that initiate activation of trigeminal C-fibers are unknown. However, the end result of trigeminal stimulation is a neurogenic inflammation within cephalic tissue. Neurogenic inflammation is mediated by the release of vasoactive peptides like substance P, calcitonin gene-related peptide and neurokinin A from sensory nerve terminals that innervate blood vessels in the dura mater (Buzzi et al., 1988; Markowitz et al., 1987). Once released, these neuropeptides activate postsynaptic receptors to cause vasodilation and enhance vascular permeability.

Recent evidence has demonstrated that neurogenic inflammation of the dura mater in rats and guinea pigs can be inhibited by a prejunctional histaminergic H₃ receptor mechanism (Matsubara et al., 1992). In an animal model of migraine that involves the stimulation of the trigeminal ganglia to elicit neurogenic inflammation, activation of presynaptic histamine H₃ receptors attenuated neurogenic vasculitis by inhibiting the release of neuropeptides from sensory nerve endings (Matsubara et al., 1992).

In addition, pharmacological studies have indicated that the CNS presynaptic histamine H₃ receptor may be an important regulator of arousal/sleep patterns (Tasaka et al., 1989; Lin et al., 1990; Sakai et al., 1991; Monti et al., 1991, 1996). It is well established that the classic antihistamines induce sedation in humans (Roth et al., 1987) by blockade of CNS histamine H₁ receptors (Levander et al., 1985). Conversely, studies in animals demonstrate that histamine H₁ receptor activation in the CNS produces an increase in arousal and a concomitant decrease in EEG sleep patterns (Wolf and Monnier, 1973). Previous studies have shown that activation of central histamine H₃ receptors produces locomotor hypoactivity in rats and mice (Sakai et al., 1991; Bristow and Bennet, 1993) and an increase in slow wave sleep activity and total sleep time in cats (Lin et al., 1990). However, the role of histamine H₃ receptors on sleep/arousal studies remains to be elucidated.

In light of the observations of the effects of histaminergic H₃ receptor activation on neurogenic inflammation within the dura mater and of actions on wakefulness, we characterized the CNS pharmacology of Sch 50971 (fig. 1), a selective, high affinity, orally active agonist of histamine H₃ receptors (Hey et al., 1998). It is concluded that the preclinical activity profile shows potential as a novel antimigraine and sedative agent.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Structure of Sch 50971.

	Materials and Methods

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General. Sch 50971 [(+)-trans-4-(4(R)-methyl-3(R)-pyrolidinyl)-1H-imidazole dihydrochloride] (fig. 1) was prepared by the Chemistry Department of Schering-Plough Research Institute. Pyrilamine hydrochloride, ipratropium bromide, triazolam, gallamine triethiodide, atropine sulfate from Sigma; thioperamide maleate, was obtained from Research Biochemicals. (R)--Methylhistamine HCl, diphenhydramine HCl, cimetidine, histamine HCl and diazepam were obtained from Schering-Plough Research Institute (Compound Distribution Center). Sumatriptan was a gift from Glaxo Research. Other compounds were obtained from standard suppliers. All drug doses were calculated as their free base.

Electrical trigeminal stimulation and assessment of plasma protein extravasation within the dura mater. The assessment of Sch 50971 on plasma protein extravasation in the dura mater produced by electrical trigeminal stimulation was determine using the methods of Markowitz et al. (1987). Male Hartley guinea pigs (400-600 g; Charles River Laboratories, Wilmington, MA) were anesthetized with urethane (1.5 g/kg). The left jugular vein was cannulated for i.v. administration of drugs. Guinea pigs were mechanically ventilated (volume, 4 ml; rate, 45 breaths/min) via a tracheal cannula. Animals were placed in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA), and a bipolar stainless steel electrode (Rhodes Medical Instruments, Woodland Hills, CA) was lowered -10.0 mm from the dura mater into the trigeminal ganglia (4.0 mm lateral, -10.0 mm posterior relative to bregma). (R)--Methylhistamine (0.3 mg/kg), sumatriptan (0.3 mg/kg) and Sch 50971 (0.3, 3.0 and 30 mg/kg) administered i.v. were given 10 min before trigeminal electrical stimulation (1.2 mA, 5 Hz, 5 msec duration for 5 min). Sch 50971 (3, 10, 30 mg/kg) given p.o. was given 30 min before stimulation. Evans blue (50 mg/kg i.v.), a fluorescent dye that complexes with plasma proteins functioned as an index of plasma protein extravasation, was given 5 min before trigeminal stimulation. After electrical trigeminal stimulation, guinea pigs were perfused with 200 ml of saline via the left cardiac ventricle. The dura mater was removed from the left and right hemispheres. Dissected tissue were incubated in 1 ml formamide at 37°C for 18 hr. To quantify the amount of dye in each sample colorimetric measurements were performed using a SLT Lab instruments SLT-340 ATTC plate reader (Grodig, Salzburg). Extravasated Evans Blue was quantified by interpolation on a standard curve of dye concentration in the range of 0.3 to 30 µg/ml. The data are expressed as the ratio of the concentration of Evans blue on the stimulated/unstimulated side of the dura mater after unilateral electrical stimulation of the trigeminal ganglia.

Potentiation of pentobarbital-induced loss of righting reflex in guinea pigs. Sch 50971 and a series of standard sedating drugs were studied to determine their effects on pentobarbital-induced narcosis in guinea pigs, defined as loss of the righting reflex. In this model, sedation is defined as a potentiation of the loss of the righting reflex to a standard dose of sodium pentobarbital. Fasted male Hartley guinea pigs (400-600 g, Charles River) were orally pretreated with Sch 50971 (1-100 mg/kg), triazolam (0.3-3 mg/kg), diphenhydramine (3-30 mg/kg), diazepam (1-10 mg/kg), (R)--methylhistamine (3-100 mg/kg) or saline 30 min before receiving 18 mg/kg of sodium pentobarbital (i.p.). The onset of narcosis was defined as the time (in min) between i.p. pentobarbital dosing and the loss of the righting reflex. The duration of narcosis (sleep) was defined as the period (min) between the loss and recovery of the righting reflex. Return of the righting reflex was noted when the animal righted itself 3 times in a 1-min period. Potentiation of the loss of the righting reflex was determined by subtracting the total sleep time of the vehicle group from the experimental group.

EEG activity, spontaneous locomotor activity and sleep assessment studies in conscious guinea pigs. Adult male Hartley guinea pigs (400-500 g, Charles River) were anesthetized with a combination of xylazine (10 mg/kg s.c.) and ketamine (60 mg/kg s.c.). The animals were placed in a stereotoxic apparatus, and the surface of the skull was surgically exposed. Two gold-plated surface electrodes were placed in direct contact with the dura, above the parietal cortex, in the general somatosensory area of the brain. The exact placement used was posterior 2 to 3 mm, lateral ±2 mm (on both sides of the skull) with respect to bregma. The animals were allowed to recover for 1 week after surgery.

Statistical analysis. Statistically significant (P < .05) differences between treatments were determined by Student's t test (for either paired or unpaired observations) or by one-way analysis of variance (ANOVA) using a Dunnett's t test for multiple comparisons. For plasma protein extravasation experiments significance (P < .05) was determined by Kruskal-Wallis in conjunction with Wilcoxon's rank sign test.

Animal care and use. These studies were performed in accordance to the NIH Guide to the Care and Use of Laboratory Animals and the Animal Welfare Act in an AAALAC-accredited program.

	Results

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Effects of Sch 50971 on neurally induced Evans blue extravasation in the dura mater of guinea pigs. Electrical stimulation of the left trigeminal ganglia in control guinea pigs produced an increase in the concentration of Evans blue found on the ipsilateral side of the dura mater (ratio > 2.0) compared to the contralateral side (fig. 2A). In a sham group of guinea pigs (n = 8) in which the stimulating electrode was lowered to the level of the trigeminal ganglia but not stimulated a ratio of 0.71 ± 0.18 was observed. In guinea pigs given Sch 50971 at doses of 0.3, 3, and 30 mg/kg i.v. the ratios were 2.58 ± 1.21, 1.27 ± 0.06 and 1.58 ± 0.20 respectively, compared with a control group ratio of 2.10 ± 0.28. The minimum effective i.v. dose of Sch 50971 was 3 mg/kg. (R)--methylhistamine (0.3 mg/kg i.v.) and sumatriptan (0.3 mg/kg i.v.) inhibited Evans blue extravasation produced by trigeminal stimulation (fig. 2A). Neither treatment had any effects on the concentration of dye in the contralateral side (data not shown). The effects of Sch 50971 were mediated by histamine H₃ receptors because the H₃ antagonist thioperamide (3 mg/kg i.v.) blocked the inhibitory actions of Sch 50971 on plasma extravasation (fig. 2B). Figure 2C shows the effects of orally administered Sch 50971 (3, 10 and 30 mg/kg) on plasma leakage due to electrical trigeminal stimulation. Sch 50971 (10 and 30 mg/kg p.o.) inhibited Evans blue extravasation.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of Sch 50971 on extravasation of Evans blue in the dura mater. Extravasation of Evans blue is expressed as the ratio of the concentration of Evans blue found on the stimulated/unstimulated side of the dura mater after unilateral electrial stimulation of the trigeminal ganglia. A, Effects of Sch 50971 (3 mg/kg i.v., n = 9), sumatriptan (0.3 mg/kg i.v., n = 9), and (R)--methylhistamine (0.3 mg/kg i.v., n = 8) compared to their respective control groups. B, Thioperamide (3 mg/k, i.v., n = 8) blocked the effects of Sch 50971 (3 mg/kg i.v., n = 8) on extravasation of Evans blue into the dura mater. C, Effects of oral Sch 50971 (3-30 mg/kg, n = 6-9 per group) on neurogenic extravasation of Evans blue in the dura mater. Values represent mean ± S.E.M. *P < .05 compared with controls, **P < .05 compared with Sch 50971.

Effect of Sch 50971 on the pentobarbital-induced loss of righting reflex in guinea pigs. Sch 50971 was compared with a number of agents with established sedative activity in man for effects on pentobarbital-induced loss of righting reflex in guinea pigs. Oral Sch 50971 caused a dose-dependent enhancement of the narcosis induced by pentobarbital as indicated by the potentiation of the pentobarbital-induced loss of the righting reflex (fig. 3). The ED_{40 min} is defined as the dose needed to increase the loss of righting reflex 40 min beyond the time of recovery of the righting reflex for the group given only pentobarbital. The ED_{40 min} for Sch 50971 was 7.0 mg/kg. Triazolam and diazepam also produced dose-dependent increases in pentobarbital narcosis (fig. 3). The ED_{40 min} for triazolam was 0.5 mg/kg and for diazepam was 2.3 mg/kg. The maximum responses with the benzodiazepine sedatives were greater than for Sch 50971 (data not shown). The sedating H₁ antagonist diphenhydramine potentiated the responses to pentobarbital. The ED_{40 min} for diphenhydramine was 14.1 mg/kg (fig. 3). Doses of diphenhydramine greater than 30 mg/kg produced convulsions in 4 of 11 animals. In contrast, the nonsedating antihistamine, terfenadine (100-300 mg/kg) did not potentiate the loss of the righting reflex induced by pentobarbital (fig. 3). The histamine H₃ agonist, (R)--methylhistamine, also potentiated pentobarbital-induced loss of righting reflex. The ED_{40 min} for (R)--methylhistamine was 23.4 mg/kg (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Potentiation of pentobarbital-induced loss of righting reflex with oral Sch 50971 (), sedating benzodiazepine-type drugs, triazolam (), diazepam (), and standard sedating and nonsedating antihistamines, terfenadine (), diphenhydramine (). Values represent mean ± S.E.M. (n = 6-15).

Effects of Sch 50971 on EEG, spontaneous locomotor activity and sleep in conscious guinea pigs. To determine the effect on CNS function and assess the sedative activity of Sch 50971, its effects on EEG, spontaneous locomotor activity and sleep time were studied concurrently in the same animals. Oral Sch 50971 (10 mg/kg) produced a slight but not statistically significant increase in relative delta EEG power during the first 3 hr after dosing (fig. 4). Oral triazolam (1 mg/kg) caused a significant decrease in relative delta power during the 6 hr of the experiment. Sch 50971 (10 mg/kg) did not significantly alter theta or alpha relative EEG power (fig. 4). In contrast, triazolam (1 mg/kg) produced significant disruption in relative EEG power by reducing theta activity (fig. 4) and increasing alpha activity. The overall effect of the Sch 50971 EEG relative power spectra profile is to increase slow-wave EEG activity (increased delta wave) an action which is consistent with a sedative effect and increase in total sleep time. This action occurred without altering theta activity (component of REM) and alpha wave activity. Sch 50971 also produced a small but significant decrease of beta wave activity (associated with wakefulness) 2 to 4 hr after treatment (fig. 4). In contrast, triazolam produced significant disruptions of the overall EEG spectral profile, with large increases in alpha (component of light sleep) and beta activity and concomitant decreases in delta and theta activity. Oral diphenhydramine (30 mg/kg) produced disruptions in the overall EEG spectral profile, producing a decrease in delta and theta wave activity (data not shown).

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Effect of oral Sch 50971 (, n = 9) and triazolam (, n = 10) on relative delta wave power (0-4 Hz), theta wave power (4-8 Hz), alpha wave power (8-14 Hz) and beta wave power (14-30 Hz) in guinea pigs over a 6-hr period. Control animals (, n = 35) received methylcellulose. Values represent mean ± S.E.M. * Statistical difference compared with control group was *P < .05.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effect of oral Sch 50971 (, n = 9) and triazolam (, n = 10) on spontaneous locomotor activity and on total distance moved in conscious free roaming guinea pigs over a 6-hr period. Control animals (, n = 35) received methylcellulose. Values represent mean ± S.E.M. *Statistical difference compared with control group was *P < .05.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of oral Sch 50971 (, n = 9) and triazolam (, n = 10) on total sleep time in guinea pigs over a 6-hr period. Control animals (, n = 35) received methylcellulose. Values represent mean ± S.E.M. *Statistical difference compared with control group was *P < .05.

	Discussion

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In the current study, we confirm the observations of Matsubara et al. (1992), who demonstrated that activation of histamine H₃ receptors inhibited plasma protein extravasation in the dura mater of rats after electrical stimulation of the trigeminal ganglia. This action has been reported to be due to prejunctional inhibition of neuropeptide release from sensory axons by activation of histamine H₃ receptors in the dura mater (Ishikawa and Sperelakis, 1987; Matsubara et al., 1992). Recently, it has been demonstrated that prejunctional histamine H₃ receptors may regulate tachykinin release in response to nonadrenergic, noncholinergic sensory nerve excitation (Ohkubo et al., 1995). We found that Sch 50971 inhibits neurogenic dura mater protein extravasation in the guinea pig. The effects of Sch 50971 are mediated by histamine H₃ receptors because pretreatment with thioperamide blocks the inhibitory actions of Sch 50971 on protein leakage into the dura mater. It is proposed that activation of prejunctional H₃ receptors with (R)--methylhistamine or Sch 50971 in the current study inhibited neuropeptide release from trigeminal nerve terminals; however, this issue was not directly addressed in the present study. Nevertheless, the present study shows that Sch 50971 given by either the i.v. or oral route of administration inhibits neurogenic inflammation in an experimental model of migraine.

Sumatriptan is a 5-HT_1D receptor agonist that is currently available for the treatment of migraine. Sumatriptan activates serotonin receptors on the heart and in the vasculature to produce moderate to severe cardiovascular side effects (Welsh, 1993). The cardiovascular effects of central and peripheral histamine H₃ receptor activation have been well characterized (Malinowska and Schlicker, 1991, 1993; Hey et al., 1992; McLeod et al., 1991, 1993, 1996; Coruzzi et al., 1995). Activation of peripheral histamine H₃ receptors with (R)--methylhistamine produces a decrease in total peripheral resistance (McLeod et al., 1993). These findings indicate that activation of prejunctional histamine H₃ receptors maybe potential antimigraine agents without the vasoconstrictor liability of 5-HT receptor agonists. Further studies are needed to address these issues, especially those determining the actions of histamine H₃ receptor activation on coronary and cerebral blood flow.

It is well established that the classical antihistamines induce sedation in man (Roth et al., 1987) by blockade of CNS H₁ receptors (Levander et al., 1985). Conversely, studies in animals demonstrate that H₁ receptor activation in the CNS produces an increase in arousal and a concomitant decrease in EEG sleep patterns (Wolf and Monnier, 1973; Monti et al., 1991). Activation of the CNS H₃ receptors produces locomotor hypoactivity in rats and mice (Bristow and Bennet, 1993; Sakai et al., 1991) and an increase in slow wave sleep EEG activity and total sleep time in cats (Lin et al., 1990). These actions are blocked by the histamine H₃ antagonist thioperamide. Interestingly, thioperamide given alone causes arousal effects that are blocked not only by an H₃ agonist, but also they are reversed by a sedating H₁ antagonist. These studies show that sleep/wakefulness is intimately controlled by a balance of H₃ and H₁ receptor activation in the CNS, with H₃ receptor activation promoting sleep.

Based on the evidence that CNS H₃ mechanisms may be important in the regulation of sleep/arousal, we studied the potential for Sch 50971 to exhibit sedative properties. We selected the guinea pig to perform the EEG/sleep studies due to the known sensitivity of this species to histamine and histaminergic mechanisms that closely resemble those in humans (Levi et al., 1982). The effects of Sch 50971 were compared to the effects of diazepam and triazolam, standard benzodiazepine sedatives, and diphenhydramine, a sedating H₁ antihistamine in man. Sch 50971 penetrated the CNS and potentiated the loss of righting reflex induced by pentobarbital, in a dose-related manner by activation of central histamine H₃ receptors. A central H₃ mechanism for Sch 50971 was provided by the finding that the effect of Sch 50971 was blocked by central (i.c.v.) administration of thioperamide. In contrast i.c.v. thioperamide did not block the effects in this model of the benzodiazepine sedative, diazepam. Sch 50971 was short acting (between 2 and 4 hr) and, in a subacute tolerance study, no tolerance developed to the sedative effect of Sch 50971. Although triazolam was ~14-fold more potent than Sch 50971, tolerance developed to its sedative effects.

The sleep promoting action and EEG effects of Sch 50971 and triazolam were also compared. The EEG spectral activity bands were divided into delta waves (0-4 Hz; associated with slow wave deep sleep), theta waves (4-8 Hz; associated with REM sleep), alpha waves (8-14 Hz; associated with light sleep) and beta waves (14-30 Hz). In these studies, Sch 50971 effects on EEG spectral activity were dramatically different from the effects observed with triazolam, and were more consistent with physiological sleep patterns. Sch 50971 produce a small but nonsignificant increase in relative delta wave activity during the first 4 hr without affecting relative theta wave or alpha wave activity. Sch 50971 also produced a decrease in relative beta wave activity. Consistent with these effects on EEG patterns, Monti et al. (1996) showed that the H₃ agonist BP 2.94 produced a similar profile in rats characterized by an increase in delta slow wave sleep after oral dosing. In contrast to Sch 50971, triazolam produced significant disruptions in EEG patterns characterized by increases in alpha and beta wave activity, along with concomitant decreases in delta and theta wave activity. In these studies, both Sch 50971 (10 mg/kg) and triazolam (1 mg/kg) significantly increased sleep time over the 6-hr study period. It is important to note that at doses of Sch 50971 and triazolam that produced similar decreases of locomotor activity and total distance moved, Sch 50971 increased sleep time while maintaining a physiological EEG profile. In contrast, triazolam produced significant disruption of EEG patterns, indicating that the sleep was not physiological in nature. No definitive EEG profile indicative of sedation was observed with diphenhydramine at doses of 30 mg/kg p.o.

The present findings also suggest that there is a difference between the relative rank order potency of Sch 50971 and (R)--methylhistamine in the migraine model vs. the sleep induction study. It should be noted that a potency comparison between Sch 50971 and (R)--methylhistamine in these models is complicated by the fact that in the drugs were given by two different routes (i.v. in the migraine model and p.o. in the sleep studies). Pharmacokinetic differences could also explain activity differences between Sch 50971 and (R)--methylhistamine. Specifically, the experimental migraine model has been proposed to involve a perivascular action and activity may not necessitate a drug crossing the blood brain barrier. In contrast, for sedation to occur it is most likely that CNS penetration is necessary. Thus, it is possible that Sch 50971 displays greater potency in the sleep studies because of its greater relative ability to penetrate the blood brain barrier faster.

In summary, Sch 50971 is an orally active, potent and selective agonist of histamine H₃ receptors. Sch 50971 may act to ameliorate the sequelae of migraine and related vascular headaches, where activation of histamine H₃ receptors are potentially beneficial. Furthermore, Sch 50971 decreases activity and promotes EEG activity consistent with physiological sleep. Sch 50971 is well tolerated and is devoid of the side effects liability such as tolerance and respiratory depressant activity associated with some sedating drugs.