Quantification of alpha4beta2* nicotinic receptors in the rat brain with microPET and 2-[18F]F-A-85380

The radioligand 2-[(18)F]F-A-85380 has been used for PET studies of the alpha4beta2* subtype of nicotinic acetylcholine receptors (nAChRs) in the living brain of humans and nonhuman primates. In order to extend the capacity of microPET to quantify neuroreceptors in rat brain, we carried out studies of 2-[(18)F]F-A-85380 to measure the apparent binding potential BP* in individual rats, which were studied repeatedly over several months. Using a bolus-plus-infusion paradigm, 2-[(18)F]F-A-85380 (specific activity 20-1300 GBq/micromol) was administered intravenously over 8 to 9 h with K(bol) values of 350 to 440 min and a mean infusion rate of 0.03+/-0.01 nmol/kg/h. Studies included a 2-h nicotine infusion initiated 2 h before the end of scanning to displace specifically bound radioactivity. Steady state binding in brain was obtained within 5 h as defined by the occurrence of constant radioactivity concentrations in brain regions and constant, free arterial plasma levels of nonmetabolized radioligand. BP* averages (+/-SEM) for thalamus, forebrain, and cerebellum were 5.9+/-0.7, 2.6+/-0.4, and 1.0+/-0.1, respectively, which are consistent with the alpha4beta2* nAChR distribution in rat brain measured in vitro. Studies of receptor occupancy determined the ED(50) to be 0.29 nmol/kg/h. The demonstration that alpha4beta2* nAChRs are quantifiable in the rat brain using PET measurements, coupled with the ability to conduct longitudinal studies over several months in the same rats, suggests potential applications to studies of chronic nicotine use, its treatment, and abnormal functioning of alpha4beta2* receptors in a rat model.     

Effects of A-134974, a novel adenosine kinase inhibitor, on carrageenan-induced inflammatory hyperalgesia and locomotor activity in rats: evaluation of the sites of action

The present study investigated 1) antihyperalgesic actions of a novel and selective adenosine kinase (AK) inhibitor, A-134974 (IC(50) = 60 pM), in the carrageenan model of thermal hyperalgesia; 2) effects of A-134974 on locomotor activity; and 3) relative contributions of supraspinal, spinal, and peripheral sites to the actions of A-134974. Systemic A-134974 (i.p.) dose dependently reduced hyperalgesia (ED(50) = 1 micromol/kg) and at higher doses, reduced locomotor activity (ED(50) = 16 micromol/kg). Administration of A-134974 intrathecally (i.t.) was more potent (ED(50) = 6 nmol) at producing antihyperalgesia than delivering the compound by intracerebralventricular (ED(50) = 100 nmol, i.c.v.) or intraplantar (ED(50) >300 nmol) routes. In contrast, i.c.v. administration of A-134974 was more effective in reducing locomotor activity than i.t. administration (ED(50) values were 1 and >100 nmol, respectively). Increasing the pretreatment time for i.t.-delivered A-134974 caused a greater reduction in locomotor activity (ED(50) = 10 nmol). This was due to diffusion of A-134974 (i.t.) to supraspinal sites. The antihyperalgesic effects of systemic A-134974 were antagonized by the adenosine receptor antagonist theophylline (THEO, 30-500 nmol) administered i.t., but not i.c.v. In the locomotor assay, i.t.-injected THEO did not antagonize hypomobility caused by systemic or i.t. administration of A-134974. However, i.c.v. infusion of THEO did block the hypomotive actions of i.c.v.-, i.t.-, and i.p.-administered A-134974. These data demonstrate that the novel AK inhibitor A-134974 potently reduces thermal hyperalgesia primarily through interactions with spinal sites, whereas its ability to depress locomotor activity is predominantly mediated by supraspinal sites.     

Peripheral and central sites of action for A-85380 in the spinal nerve ligation model of neuropathic pain

Neuronal nicotinic receptor (NNR) agonists such as ABT 594 have been shown to be effective in a wide range of preclinical models of acute and neuropathic pain. The present study, using the NNR agonist A-85380, sought to determine if NNR agonists are acting via similar or differing mechanisms to induce anti-nociception and anti-allodynia. A systemic administration of the quaternary NNR antagonist chlorisondamine (0.4 micromol/kg, intraperitoneal (i.p.)) did not alter A-85380-induced (0.75 micromol/kg, i.p.) anti-nociception in the rat paw withdrawal model of acute thermal pain. In contrast, previous studies have demonstrated that blockade of central NNRs by prior administration of chlorisondamine (10 microg i.c.v.) prevents A-85380 induced anti-nociception indicating a predominantly central site of action of NNR agonists in relieving acute pain. In the rat spinal nerve ligation model of neuropathic pain, A-85380 induced a dose-dependent anti-allodynia (0.5-1.0 micromol/kg) that was blocked by pretreatment with mecamylamine (1 micromol/kg). Interestingly, unlike acute pain, both systemic and central administration of chlorisondamine blocked A-85380-induced anti-allodynia, an effect that was determined not to be due to a non-specific effect of chlorisondamine or to chlorisondamine crossing the blood-brain barrier. The peripheral site of action was shown not to be the primary receptive field, since A-85380 had equally potent anti-allodynic effects when it was injected into either the affected or unaffected paw. In contrast, infusion of A-85380 directly onto the L5 dorsal root ganglion on the affected side resulted in a dose-dependent and marked anti-allodynia (10-20 microg) at doses that had no effect when injected systemically. This effect was blocked by pretreatment with chlorisondamine. Together these data further support the idea that different mechanisms underlie different pain states and suggest that the effects of NNR agonists in neuropathic pain may be due in part to peripheral actions of the compounds.     

Pindolol suppresses serotonergic neuronal activity and does not block the inhibition of serotonergic neurons produced by 8-hydroxy-2-(di-n-propylamino)tetralin in awake cats.

Clinical studies have shown that pindolol can enhance the effects of antidepressant drugs, presumably by acting as an antagonist at somatodendritic 5-hydroxytryptamine (5-HT)(1A) autoreceptors, which regulate the firing rate of central serotonergic neurons. The current study characterized the action of pindolol on the single-unit activity of serotonergic neurons in the dorsal raphe nucleus of freely moving cats. (+/-)-Pindolol produced a dose-dependent inhibition of neuronal activity after i.v. (ED(50) = 0.25 mg/kg) and s.c. (ED(50) = 1.23 mg/kg) administration. The active enantiomer (-)-pindolol (1 mg/kg i.v.) also suppressed neuronal activity (maximal decrease, 88%). Upon p.o. administration, (+/-)-pindolol (10 mg/kg) produced a marked, long-acting suppression of neuronal activity similar to that observed after s.c. administration. In all cases, the reduction in firing rate produced by pindolol was completely reversed by low doses of N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]-N-(2-pyridinyl)cycloh exanecarboxamide (WAY-100635) (0.1 mg/kg i.v. or 0.2 mg/kg s.c.), a selective 5-HT(1A) antagonist. Systemic administration of (-)-tertatolol (1-5 mg/kg i.v.), another beta-adrenoceptor blocker/putative 5-HT(1A) antagonist, had no significant effect on neuronal activity. The ability of i.v. (+/-)-pindolol (0.1-1 mg/kg) to reverse the suppression of serotonergic neuronal activity produced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (10 microg/kg i.v.), a selective 5-HT(1A) agonist, also was examined. (+/-)-Pindolol had no appreciable effect on the action of 8-OH-DPAT. In contrast, the 5-HT(1A) antagonist drugs WAY-100635 (0.1 mg/kg i.v. ), 4-fluoro-N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl benzamide (0.1 mg/kg, i.v.), N-tert-butyl-3-(4-(2-methoxyphenyl)piperazin-1-yl)-2-phenylprop anamid e [(S)-WAY-100135] (0.5 mg/kg i.v.), and (-)-tertatolol (1-5 mg/kg i. v.) reversed the effect of 8-OH-DPAT to varying degrees. Overall, these results indicate that pindolol acts as an agonist rather than an antagonist at 5-HT(1A) autoreceptors in awake animals.     

Intestinal prokinesia by two esters of 4-amino-5-chloro-2- methoxybenzoic acid: involvement of 5-hydroxytryptamine-4 receptors and dissociation from cardiac effects in vivo

In five fasting, conscious dogs, we compared the prokinetic action of two selective 5-hydroxytryptamine-4 (5-HT4) receptor agonists with low affinity for 5-HT3 receptors ML10302 (2-piperidinoethyl 4-amino-5-chloro-2-methoxybenzoate) and SR59768 (2-[(3S)-3-hydroxypiperidino]ethyl 4-amino-5-chloro-2-methoxybenzoate) in the duodenum and jejunum, using cisapride as a reference compound. Heart rate and rate-corrected QT (QTc) also were monitored to assess whether or not the cardiac effects of cisapride are shared by other 5-HT4 receptor agonists. Both ML10302 and SR59768 dose-dependently stimulated spike activity in the duodenum with similar potencies (dose range, 3-300 nmol/kg i.v.; ED50 values: 24 and 23 nmol/kg i.v., respectively), mimicking the effect of cisapride (30-3000 nmol/kg i.v.). The maximal effect was achieved with the dose of 100 nmol/kg i.v. for both compounds. Similar findings were obtained in the jejunum. Atropine and GR125487 (1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl-methyl 5-fluoro-2-methoxy-1H-indole-3-carboxylate, selective 5-HT4 receptor antagonist), at doses having no effect per se, antagonized intestinal prokinesia by maximal doses of ML10302 and SR59768. Neither ML10302 nor SR59768 had any effect on heart rate or QTc at any of the doses tested, whereas cisapride, at the highest dose (3000 nmol/kg), induced tachycardia and lengthened the QTC (p <.01). In conclusion, ML10302 and SR59768 share with cisapride a similar prokinetic action in the canine duodenum and jejunum in vivo. This effect is mediated by pathways involving activation of 5-HT4 and muscarinic receptors. Unlike cisapride, which induces tachycardia and prolongs the QTc by a mechanism probably unrelated to 5-HT4 receptor activation, ML10302 and SR59768 are devoid of cardiac effects in this model.     

Antagonism of leukotriene C4, leukotriene D4 and leukotriene E4 vasoconstrictor responses in the conscious rat with the peptidoleukotriene receptor antagonist SK&F 104353: evidence for leukotriene D4 receptor heterogeneity

The purpose of these experiments was to investigate the effects of the selective peptidoleukotriene receptor antagonist, SK&F 104353, on leukotriene (LT)C4, LTD4 and LTE4 vasopressor responses in conscious, normotensive rats. Steady-state plasma concentrations of SK&F 104353 at infusion rates of 0.2 mg/kg + 1 mg/kg/hr, 1 mg/kg + 3 mg/kg/hr or 2 mg/kg + 10 mg/kg/hr were 0.5, 1.6 and 9.4 micrograms/ml, respectively, indicating that the plasma concentrations of SK&F 104353 were related directly to the infusion rate. LTC4, LTD4 and LTE4 (0.17-170 nmol/kg i.v.) produced dose-dependent increases in mean blood pressure. The ED20 dose (i.e., dose required to increase blood pressure 20 mm Hg) of LTC4, LTD4 or LTE4 was 2.7 +/- 0.4, 2.2 +/- 0.3 and 109 +/- 17 nmol/kg, respectively. SK&F 104353 produced dose-dependent, parallel shifts to the right in the LTC4 dose-response curve. Administration of SK&F 104353 at doses of 0.2 mg/kg + 1 mg/kg/hr, 1 mg/kg + 3 mg/kg/hr or 2 mg/kg + 10 mg/kg/hr produced dose ratios (i.e., ratio of ED20 in presence of SK&F 104353 to that of the vehicle group) of 6, 12 and 26, respectively. Against LTD4 responses, SK&F 104353 at doses of 0.1 mg/kg + 0.3 mg/kg/hr or 0.2 mg/kg + 1 mg/kg/hr produced dose ratios of 3 and 9, respectively. At a dose of 1 mg/kg + 3 mg/kg/hr, there was no further increase in the dose ratio, whereas a dose of 2 mg/kg + 10 mg/kg/hr resulted in a dose ratio of greater than 100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-inflammatory and antinociceptive properties of BP 2-94, a histamine H(3)-receptor agonist prodrug

BP 2-94 is an azomethine prodrug of (R)-alpha-methylhistamine [(R)-alpha-MeHA], a potent and selective histamine H(3)-receptor agonist. When administered orally to mice BP 2-94 was distributed to various peripheral tissues where it released the active drug. BP 2-94 displayed anti-inflammatory and antinociceptive properties in mice. It dose-dependently inhibited carrageenan-induced paw edema with an ED(50) value of 0.17 +/- 0.05 micromol/kg (p.o.) and a maximal effect of 47%. It also reduced Freund's complete adjuvant-induced paw edema in preventive as well as in curative fashion. Repeated oral administrations of BP 2-94 reduced the pre-established Freund's complete adjuvant-induced edema with an ED(50) value of 5 +/- 2 micromol/kg (p.o.) and a maximal effect of 47%. The antiedema effects of BP 2-94 and indomethacin were additive. BP 2-94 was also efficient in reducing cyclophosphamide-induced cystitis in mice: it decreased leukocyte infiltration by 62% and plasma protein extravasation by 73% in urinary bladder. In addition, BP 2-94 displayed antinociceptive activity in the capsaicin-induced licking test via H(3)-receptor stimulation. Its antinociceptive effect was dose dependent, occurring with an ED(50) value of 0.4 +/- 0.1 micromol/kg (p.o.) and a maximal reduction of licking duration by 69%. No tolerance to the antinociceptive effect was observed after repeated administration of BP 2-94 for 3 days. These observations with BP 2-94 suggest that H(3)-receptor agonists might represent a novel class of anti-inflammatory and antinociceptive agents.     

Acute effects of nicotine on prolactin release in the rat: agonist and antagonist effects of a single injection of nicotine

The effects of nicotine on prolactin release were studied in conscious, unrestrained rats in which an indwelling jugular cannula allowed multiple samplings of blood after i.v. administration of nicotine. Intravenous administration of nicotine bitartrate dihydrate increases plasma prolactin concentrations in a dose-dependent manner with an ED50 of approximately 100 micrograms/kg (200 nmol/kg) and this effect is blocked completely by pretreatment with mecamylamine, indicating that it is mediated by a nicotinic cholinergic receptor. Intracerebral ventricular injection of 1 microgram of nicotine also increases plasma prolactin levels, but i.v. injection of this same amount of nicotine has no effect, indicating that nicotine acts within the brain to release prolactin. A single i.v. injection of nicotine resulted in desensitization of the prolactin response to a subsequent injection of nicotine given 1 to 2 hr later, thus confirming a previous report by Sharp and Beyer (J. Pharmacol. Exp. Ther. 238: 486-491, 1986). The prolactin response to nicotine was restored within 24 hr after a single injection. The acute desensitization after a single injection of nicotine appears to be specific to release of prolactin by nicotine because the prolactin response to morphine was unaffected 1 hr after injection of nicotine. A single injection of nicotine appears to desensitize the prolactin response to a subsequent injection of nicotine with an ED50 of approximately 20 micrograms/kg (40 nmol/kg), indicating that nicotine is even more potent in stimulating desensitization of nicotinic cholinergic receptors than in stimulating prolactin release. These results support the concept that nicotine acts as a time-averaged antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin (5-HT)3 receptor blocking activities of YM060, a novel 4,5,6,7-tetrahydrobenzimidazole derivative, and its enantiomer in anesthetized rats

YM060 [(R)-5-[(1-methyl-3-indolyl)carbonyl]-4,5,6,7-tetrahydro-1H-benzimida zol e hydrochloride], is structurally independent of other 5-HT3 receptor antagonists. We investigated in vivo 5-HT3 receptor blocking activity of YM060 and compared results with those of its enantiomer (S-form), ondansetron (GR38032F), granisetron (BRL43694), ICS205-930 [(3 alpha-tropanyl)-1H-indole-3-carboxylic acid ester], LY277359 [endo-5-chloro-2,3-dihydro-2,2-dimethyl-N-(8-methyl-8-azabicyclo[3.2.1] oct-3-yl)-7-benzofuran-carboxamide-(Z)-2-butenedioate (1:1)], Y25130 [(+-)-N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4- dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride] and zacopride [(R,S)4-amino-N-[1-azabicyclo (2.2.2)oct-3-yl]-5-chloro-2-methoxybenzamide(E)-2-butenedioat e]. YM060 injected i.v. dose-dependently inhibited the reduction in heart rate induced by 5-HT (30 micrograms/kg i.v.) in rats (von Bezold-Jarisch reflex) with an ED50 value of 0.036 (0.031-0.041) micrograms/kg (n = 3-5). Based on these values, YM060 was 53, 18, 23, 16, 11 and 4 times as potent as ondansetron, granisetron, ICS205-930, LY277359, Y25130 and zacopride, respectively. The S-form of YM060 also inhibited 5-HT-induced bradycardia, but with a potency approximately 250 times less than that of YM060 (R-form). YM060 dosed p.o. also inhibited 5-HT-induced bradycardia with an ED50 value of 0.59 (0.44-0.80) micrograms/kg (n = 3-5), indicating the drug to be 387, 66, 97, 6 and 16 times more potent than ondansetron, granisetron, ICS205-930, LY277359 and Y25130, respectively, but 2 times less potent than zacopride. Bioavailability of YM060 based on the p.o.-to-i.v. ED50 ratio (p.o./i.v. = 16) was lower than those of zacopride (2) and LY277359 (6), similar to that of Y25130 (22) and better than those of ondansetron (109), granisetron (60) and ICS205-930 (71).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiovascular effects of nicotine, chlorisondamine, and mecamylamine in the pigeon

Chlorisondamine and mecamylamine are nicotinic antagonists that produce both ganglionic and central blockade. Chlorisondamine, when administered as a large systemic dose, produces a persistent central block, despite being charged. The present study evaluated the cardiovascular effects of chlorisondamine. Shortly after administration, chlorisondamine (0.10, 1, and 10 mg/kg i.m.) lowered blood pressure significantly and decreased heart rate at the low dose (0.1 mg/kg i.m.) and increased heart rate at the high dose (10 mg/kg i.m.). Mecamylamine (1 and 10 mg/kg i.m.) also lowered blood pressure and heart rate. After both antagonists, heart rate returned to baseline values within 90 min and blood pressure within 24 h. Low doses of nicotine (0.01-0.03 mg/kg i.m.) lowered blood pressure but did not affect heart rate. Higher doses (0.10-3.2 mg/kg i.m.) transiently increased blood pressure and heart rate. Subsequent to antagonist administration, nicotine was administered to determine whether either drug blocked the cardiovascular effects of nicotine. Chlorisondamine (0.1, 1, and 10 mg/kg i.m.) administered 30 min before nicotine blocked the increases in blood pressure and heart rate. Only the high dose (10 mg/kg i.m.) of chlorisondamine administered 24 h before nicotine produced a blockade of nicotine's pressor effect. This block diminished within 3 days. Mecamylamine (1 mg/kg i.m.) antagonized only nicotine's tachycardic effect. Longer pretreatment with mecamylamine (10 mg/kg, 24 h before nicotine challenge) did not antagonize the cardiovascular effects of nicotine. Thus, chlorisondamine produces a longer lasting blockade of nicotine's cardiovascular effects than mecamylamine.     

Stimulation of serotonin2C receptors blocks the hyperactivation of midbrain dopamine neurons induced by nicotine administration

In vivo electrophysiological techniques were used to study the effect of nicotine on the basal activity of dopamine (DA)-containing neurons in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) of chloral hydrate-anesthetized rats. Acute i.v. injections of nicotine (25-400 microg/kg) caused a dose-dependent increase of the firing rate and the bursting activity of DA neurons both in the SNc and the VTA. Repeated daily injection of nicotine (1 mg/kg i.p.) for 10 consecutive days did not cause any significant change in the basal activity of DA neurons in the SNc and the VTA. Acute challenge with nicotine (25-400 microg/kg i.v.) in animals treated repeatedly with this drug caused a dose-related excitation of DA neurons in both areas. To test the hypothesis that stimulation of 5-hydroxytryptamine (5-HT, serotonin)(2C) receptors could affect nicotine-induced stimulation of DA neuronal activity, the selective 5-HT(2C) receptor agonist RO 60-0175 was used. Pretreatment with 100 microg/kg i.v. (S)-2-(chloro-5-fluoro-indo-l-yl)-l-methylethylamine 1:1 C(4)H(4)O(4) (RO 60-0175) prevented the enhancement in DA neuronal firing rate elicited by acute nicotine (25-400 microg/kg i.v.) in the SNc of both drug naive and chronically treated rats but was devoid of any significant effect in the VTA. Moreover, the dose of 300 microg/kg i.v. RO 60-0175 significantly reduced the stimulatory effect of VTA DA neurons induced by acute challenge with nicotine (25-400 microg/kg i.v.) both in drug naive and chronically treated rats. It is concluded that selective activation of 5-HT(2C) receptors can block the stimulatory action of nicotine on midbrain DA neuronal activity.     

Central and peripheral injections of the 5-HT2 agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, modify cardiovascular function through different mechanisms.

The mechanisms underlying the cardiovascular effects of central and peripheral administration of the 5-HT2 (serotonin) receptor agonist (+-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) were studied in conscious rats. Intravenous (10-1000 nmol/kg) and i.c.v. (3-300 nmol) administration of DOI produced dose-related elevations of arterial pressure without altering heart rate except after injection of the highest doses. Pretreatment with xylamidine tosylate, a 5-HT2 receptor antagonist that does not cross the blood-brain barrier, blocked the pressor response to i.v., but not i.c.v., administration of equivalent doses of DOI. Pretreatment with the vasopressin receptor antagonist d(CH2)5Tyr(Me)AVP significantly reduced the pressor response to i.c.v., but not i.v., administration of DOI. Prior ganglionic blockade with chlorisondamine amplified the pressor response to both i.v. and i.c.v. administration of DOI. Pretreatment with a combination of chlorisondamine, xylamidine and d(CH2)5Tyr(Me)AVP abolished the pressor response to i.c.v. administration of DOI. Thus, the pressor response to i.v. administration of DOI was mediated at sites outside the blood-brain barrier, most likely at vascular 5-HT2 receptors, and was not secondary to vasopressin release. Inappropriate heart rate changes attended the pressor responses to i.v. administration of DOI, suggesting an action at extravascular sites. The pressor response to i.c.v. administration of DOI resulted from a combination of vasopressin release, modulation of autonomic nervous outflow and some leakage into the periphery.     

Profound spinal tolerance after repeated exposure to a highly selective mu-opioid peptide agonist: role of delta-opioid receptors

Recent studies suggest that delta-opioid receptors play a role in the development of opioid tolerance and led us to hypothesize that highly selective mu-opioid agonists may produce less tolerance. H-2',6'-dimethyltyrosine-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA) has extraordinary selectivity for mu-receptors (K(i)(delta)/K(i)(mu) > 14,000). Daily administration of [Dmt(1)]DALDA (5 times ED(50); s.c.) for 7 days increased ED(50) 3.6-fold from 0.16 to 0.58 micromol/kg. A higher dose of [Dmt(1)]DALDA (10 times ED(50), every 12 h) for 2.5 days resulted in a 11.7 times increase in the ED(50) (1.9 micromol/kg). Complete cross-tolerance to morphine was observed, with a 3.4- and 15.1-fold shift in the morphine ED(50), respectively. We also compared the extent of spinal versus supraspinal tolerance after repeated s.c. [Dmt(1)]DALDA administration. Five doses of [Dmt(1)]DALDA (10 times ED(50), every 12 h) resulted in a 3.4 times shift in the i.c.v. ED(50) (15.4 versus 4.6 pmol/mouse) but a 44 times shift in the i.t. ED(50) (52.9 versus 1.2 pmol/mouse). Tolerance to [Dmt(1)]DALDA was associated with 30 to 35% reduction in [(3)H][Dmt(1)]DALDA binding in brain and spinal cord. Coadministration of [Dmt(1)]DALDA with delta-antagonist naltriben (NTB) reduced spinal tolerance by 50%. Even after spinal tolerance had been established, addition of a delta-antagonist (NTB or H-Tyr-TicPsi[CH(2)NH]Phe-Phe-OH) significantly enhanced the potency of i.t. [Dmt(1)]DALDA 2- to 4-fold. These results suggest that agonist activation of delta-receptors is not necessary for the development of opioid tolerance; however, delta-receptors play a modulatory role in the maintenance of the tolerant state.     

(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 (UFP-102), a highly potent and selective agonist of the nociceptin/orphanin FQ receptor

A novel ligand for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-102), has been generated by combining in the N/OFQ-NH(2) sequence two chemical modifications, [Arg(14),Lys(15)] and [(pF)Phe(4)], that have been previously demonstrated to increase potency. In vitro, UFP-102 bound with high affinity to the human NOP receptor, showed at least 200-fold selectivity over classical opioid receptors, and mimicked N/OFQ effects in CHO(hNOP) cells, isolated tissues from various species, and mouse cortical synaptosomes releasing 5-hydroxytryptamine. UFP-102 showed similar maximal effects but higher potency (2- to 48-fold) relative to N/OFQ. The effects of UFP-102 were sensitive to NOP-selective antagonists J-113397 [(+/-)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] (pA(2) = 7.75-8.12) and UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2))(pA(2) = 6.91-7.33) but not to naloxone, and no longer observed in tissues taken from NOP receptor knockout mice (NOP(-/-)). In vivo, UFP-102 (0.01-0.3 nmol i.c.v.) mimicked the pronociceptive action of N/OFQ (0.1-10 nmol i.c.v.) in the mouse tail withdrawal assay, displaying higher potency and longer lasting effects. The action of UFP-102 was not apparent in NOP(-/-) mice. Similar results were obtained measuring locomotor activity in mice. In conscious rats, UFP-102 (0.05 nmol i.c.v.) produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate. These effects were comparable with those evoked by N/OFQ at 5 nmol. Collectively, these findings demonstrate that UFP-102 behaves as a highly potent and selective NOP receptor agonist that produces long-lasting effects in vivo.     

MEN16132, a novel potent and selective nonpeptide kinin B2 receptor antagonist: in vivo activity on bradykinin-induced bronchoconstriction and nasal mucosa microvascular leakage in anesthetized guinea pigs

We have tested the activity of 4-(S)-amino-5-(4-[4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl] piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride (MEN16132), a novel nonpeptide kinin B(2) receptor antagonist, on bradykinin (BK)-induced inflammatory responses, bronchoconstriction, and hypotension in guinea pigs. After i.v. (1-10 nmol/kg i.v.), intratracheal (i.t.) (10-100 nmol/kg i.t.), or aerosol (0.01-0.1 mM/5 min) administration, MEN16132 inhibited in a dose-dependent manner the bronchoconstriction induced by BK (10 nmol/kg i.v.). MEN16132 was more potent and possessed a longer duration of action as compared with the peptide B(2) receptor antagonist icatibant (HOE140; H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH trifluoroacetate). After i.v. administration, its inhibitory effect on bronchoconstriction lasted more than 8 h at 30 nmol/kg. When administered by i.v. or i.t. routes, the dose completely inhibiting bronchoconstriction also partially reduced the hypotensive response to BK, whereas after aerosol administration, the inhibitory effect was limited to respiratory level. Intranasal (i.n.) administration of MEN16132 (0.01-0.3 nmol/nostril) reduced, in a dose-dependent and long-lasting manner, the nasal mucosa plasma protein extravasation induced by BK (100 nmol/nostril), and it exerted a complete inhibition at about 30-fold lower dose than icatibant. At 1 nmol/nostril, MEN16132 activity was significant for at least 6 h with no systemic effect measured as inhibition of BK-induced hypotension, and at 10 nmol/nostril, the inhibitory effect lasted for more than 15 h with only a weak effect on hypotension. These findings indicate that in vivo MEN16132 is a potent kinin B(2) receptor antagonist with long duration of action, both after i.v. and local administration. A complete and prolonged inhibition of BK-induced bronchoconstriction or nasal inflammation can be achieved with MEN16132 topical administration (aerosol or i.n.) at doses devoid of systemic effects.     

N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-nitrophenyl) cyclohexanecarboxamide: a novel pre- and postsynaptic 5-hydroxytryptamine(1A) receptor antagonist active on the lower urinary tract.

N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-nitrophenyl) cyclohexanecarboxamide (Rec 15/3079) was synthesized with the aim of obtaining a novel compound with 5-hydroxytryptamine (5-HT)(1A) antagonistic properties and activity in controlling bladder function at the level of the central nervous system. Rec 15/3079 showed a selective high affinity for the 5-HT(1A) receptor (K(i) = 0.2 nM). At the human recombinant 5-HT(1A) receptor, Rec 15/3079 acted as a competitive, neutral antagonist in that it did not modify basal [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to HeLa cell membranes but shifted the activation isotherm to 5-HT to the right, in a parallel manner, with a pK(b) value of 10.5. Accordingly, Rec 15/3079 (i.v.) potently antagonized 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT)-induced hypothermia in mice (ID(50) = 20 microg/kg) and 8-OH-DPAT-induced forepaw treading in rats (ID(50) = 36 microg/kg). In vitro Rec 15/3079 was poorly active in antagonizing carbachol-induced bladder (pD'(2) = 5.03) and norepinephrine-induced urethral (apparent pK(b) = 6) contractions. However, in anesthetized rats, Rec 15/3079 (10-100 microg/kg i.v.) blocked isovolumic bladder contractions with no effect on their amplitude. In conscious rats and guinea pigs with bladders filled with saline, Rec 15/3079 (300-1000 microg/kg i.v.) increased bladder volume capacity (BVC) without affecting bladder contractility. In conscious rats with bladders filled with dilute acetic acid, Rec 15/3079 (300 microg/kg i.v.) reversed the decrease of BVC induced by the acid. To evaluate apparent selective effect on lower urinary tract reflexes, Rec 15/3079 was tested in experimental models for sedative, analgesic, anxiolytic, and antidepressant activity. Rec 15/3079 showed only a slight decrease in the duration of immobility in the behavioral despair test (antidepressant activity) at 1 mg/kg i.v. No anxiolytic activity was observed at 10 mg/kg i.v. No effect was observed in the hot plate test, but Rec 15/3079 increased tail-flick latencies after 3 to 10 mg/kg i.v. In conclusion, these studies demonstrate that Rec 15/3079 is endowed with favorable effects on bladder function, and it is devoid of unwanted side effects at the level of central nervous system at doses at least 10-fold higher than those active on the bladder.     

Effects of systemically administered dynorphin A(1-17) in rhesus monkeys.

The effects of i.v. dynorphin A(1-17) and its main nonopioid biotransformation fragment, dynorphin A(2-17), were compared in rhesus monkeys with those of the selective kappa-opioid agonist, U69, 593, in assays of operant behavior, thermal antinociception, and neuroendocrine function (prolactin release). Dynorphin A(1-17) (0. 1-3.2 mg/kg i.v.) and U69,593 (0.001-0.032 mg/kg s.c.) decreased rates of schedule-controlled (fixed ratio 20) food-reinforced responding, whereas dynorphin A(2-17) (1-3.2 mg/kg i.v.) was ineffective. Pretreatment studies with the opioid antagonist quadazocine (0.32 mg/kg s.c.) revealed that the operant effects of dynorphin A(1-17) were not mediated by kappa- or micro-opioid receptors. A different profile was observed in the warm water tail withdrawal assay of thermal antinociception, where both dynorphin A(1-17) and A(2-17) (0.032-3.2 mg/kg i.v., n = 4) were modestly effective in 50 degrees C water, and both were ineffective in 55 degrees C water. By comparison, U69,593 (0.032-0.18 mg/kg s.c.) was maximally effective in 50 degrees C water and partially effective in 55 degrees C. kappa-opioid agonists increase serum levels of prolactin in animals and humans. Dynorphin A(1-17) (ED(50) = 0.0011 mg/kg i.v.), similar to U69,593 (ED(50) = 0.0030 mg/kg i.v.), was very potent in increasing serum prolactin levels in follicular phase female rhesus monkeys, whereas dynorphin A(2-17) (0.32 mg/kg i.v.) was ineffective. The effects of dynorphin A(1-17) and U69,593 on serum prolactin were both antagonized by quadazocine (0.32 mg/kg s.c.) in a surmountable manner, consistent with opioid receptor mediation. The present studies show that serum prolactin levels are a sensitive quantitative endpoint to study the systemic effects of the endogenous opioid peptide, dynorphin A(1-17), in primates.     

Effects of the stereoisomers of nicotine and nornicotine on schedule-controlled responding and physiological parameters of dogs

The behavioral and physiological effects of five tobacco alkaloids were assessed in two different test paradigms. One group of beagle dogs (N = 5) responded under a multiple fixed-interval (FI) 300 sec, fixed-ratio (FR) 30 response schedule of food presentation. With both l-nicotine (0.1-3.0 mumol/kg i.m.) and l-nornicotine (0.1-10.0 mumol/kg i.m.), FI response rates, when averaged across the entire session, were increased at low doses and decreased at high doses; FR response rates were only decreased. d-Nicotine (0.1-30.0 mumol/kg i.m.), and d- and dl-nornicotine (0.1-10.0 mumol/kg i.m.) produced a transient increase in FI response rates at low doses. The effects of the same five compounds on pupillary diameter, rectal temperature and heart rate were measured in a second group of beagle dogs (N = 6). Both pupillary diameter and rectal temperature were decreased initially by l-nicotine (0.6 mumol/kg i.v.), d-nicotine (6.0 mumol/kg i.v.) and d-, l- and dl-nornicotine (3.0 mumol/kg i.v.); base-line levels were recovered within 60 min. Aside from differences in potency, the time courses of action were similar for all five compounds. Increases in heart rate also were produced by all five compounds, but there were marked differences in the duration of action. l-Nicotine and d- and dl-nornicotine produced appreciable, sustained increases in heart rate, whereas d-nicotine and l-nornicotine produced moderate, brief increases. The combined results of the present experiments indicate the stereoisomers of nicotine and nornicotine are behaviorally and physiologically active, and may contribute in varying amounts to the pharmacologic actions of tobacco.     

Pharmacological properties of (2R)-N-[1-(6-aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide: a novel mucarinic antagonist with M(2)-sparing antagonistic activity

We evaluated the pharmacological profiles of (2R)-N-[1-(6- aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide(compound A), which is a novel muscarinic receptor antagonist with M(2)-sparing antagonistic activity. Compound A inhibited [(3)H]NMS binding to cloned human muscarinic m1, m2, m3, m4, and m5 receptors expressed in Chinese hamster ovary cells with K(i) values (nM) of 1.5, 540, 2.8, 15, and 7.7, respectively. In isolated rat tissues, compound A inhibited carbachol-induced responses with 540-fold selectivity for trachea (K(B) = 1.2 nM) over atria (K(B) = 650 nM). In in vivo rat assays, compound A inhibited acetylcholine-induced bronchoconstriction and bradycardia with intravenous ED(50) values of 0.022 mg/kg and >/=10 mg/kg, respectively. Furthermore, in dogs, compound A (0.1-1 mg/kg p.o.) dose dependently shifted the methacholine concentration-respiratory resistance curves. In mice, compound A (10 mg/kg i.v.) did not inhibit oxotremorine-induced tremor. The brain/plasma ratio (K(p)) of compound A (3 mg/kg i.v.) was 0.13 in rats; this K(p) was less than that of scopolamine (1.7) and darifenacin (0.24). The inhibition of compound A (3 mg/kg i.v.) on ex vivo binding in rat cerebral cortex was almost similar to that of NMS. These findings demonstrate that compound A has high selectivity for M(3) receptors over M(2) receptors, displays a potent, oral M(3) antagonistic activity without inhibition of central muscarinic receptors because of low brain penetration. It is well known that central muscarinic antagonists may have diverse CNS effects, and M(2) receptors regulate cardiac pacing and act as autoreceptors in the lung and bladder. Thus, compound A may have fewer cardiac or CNS side effects than nonselective compounds.     

Nicotine elevates rat plasma ACTH by a central mechanism

Nicotine is a potent secretagogue for the release of adrenocorticotropin (ACTH) from the anterior pituitary in vivo. However, the location of its action is unknown; knowledge of this is essential for elucidating its mechanism. Our studies show that cytisine, a peripherally acting nicotinic cholinergic agonist, given i.v. at doses equimolar or greater than nicotine, failed to elevate plasma ACTH levels, whereas nicotine (0.01 and 0.03 mg/kg b.wt.) had significant effects. Nicotine (10(-7)-10(-4) M) had no effect on the secretion of beta-endorphin by anterior pituicytes in vitro, nor did it potentiate the action of corticotropin-releasing factor (10(-9) or 10(-8) M). Intracerebroventricular injection of nicotine (1-20 micrograms) significantly elevated ACTH levels. Moreover, ACTH responses to nicotine delivered into the hypothalamic region of the third ventricle were significantly greater than those elicited by injection into the upper region. Additional studies were conducted to determine the earliest age at which nicotine stimulates ACTH. The response to i.p. nicotine (1 or 2 mg/kg b.wt.) was present but diminished during the postnatal period, whereas maximal responses comparable to mature rats were attained by day 15. To establish whether nicotine has a central effect in younger animals, nicotinic antagonists were tested. Hexamethonium (2 mg/kg b.wt.), a peripherally acting antagonist, was ineffective against nicotine (0.025 and 2.0 mg/kg b.wt.), whereas mecamylamine (2 mg/kg b.wt.), inhibitory at both peripheral and central sites, blocked the ACTH response. Thus, whether administered peripherally or centrally, nicotine activates central mechanisms mediating the release of ACTH; it appears that the target(s) for nicotine are within the hypothalamus or brainstem.