Bronchodilator activity of (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino] carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407), a potent, long-acting, and selective muscarinic M3 receptor antagonist.

The novel quaternary ammonium salt (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407) showed subnanomolar affinities for human muscarinic M1 (hM1), M2 (hM2), and M3 (hM3) receptors and dissociated very slowly from hM3 receptors (t(½) = 166 min) with a large part of the receptorial complex (54%) remaining undissociated at 32 h from radioligand washout. In contrast, [(3)H]CHF5407 dissociated quickly from hM2 receptors (t(½) = 31 min), whereas [(3)H]tiotropium dissociated slowly from both hM3 (t(½) = 163 min) and hM2 receptor (t(½) = 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC(50) = 9.0-9.6) and long-lasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED(50) value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3 receptors. In contrast, CHF5407 is markedly short-acting at M2 receptors, a behavior not shared by tiotropium.

Discovery of a novel class of substituted pyrrolooctahydroisoquinolines as potent and selective delta opioid agonists, based on an extension of the message-address concept.

This paper describes the design and synthesis of compounds belonging to a novel class of substituted pyrrolooctahydroisoquinolines which are potent and selective delta opioid agonists. Molecular modeling studies performed on known, selective delta ligands such as (+)-3 and the potent delta agonists SNC 80 led to the identification of the carboxamido moiety of the latter as a putative nonaromatic delta address. Insertion of this moiety onto the octahydroisoquinoline opioid message resulted in (+/-)-5b, a potent and selective delta ligand. The active enantiomer, (-)-5b, displayed nanomolar affinity for the delta receptor (Ki = 0.9 nM) with good mu/delta and kappa/delta binding selectivity ratios (140 and 1480, respectively). In addition, (-)-5b behaved as a full delta agonist in the mouse vas deferens bioassay having an IC50 = 25 nM and being antagonised in the presence of 30 nM naltrindole (NTI). These studies, based on the message-address concept, indicated that the nonaromatic (N,N-diethylamino)carbonyl moiety is a viable alternative to the classical benzene ring as a delta opioid address. Preliminary in vivo studies showed that (+/-)-5b produced a dose-related antinociception in the mouse abdominal constriction test after intracerebroventricular administration (ED50 = 1.6 micrograms/mouse).

Stepwise modulation of neurokinin-3 and neurokinin-2 receptor affinity and selectivity in quinoline tachykinin receptor antagonists.

A stepwise chemical modification from human neurokinin-3 receptor (hNK-3R)-selective antagonists to potent and combined hNK-3R and hNK-2R antagonists using the same 2-phenylquinoline template is described. Docking studies with 3-D models of the hNK-3 and hNK-2 receptors were used to drive the chemical design and speed up the identification of potent and combined antagonsits at both receptors. (S)-(+)-N-(1-Cyclohexylethyl)-3-[(4-morpholin-4-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 25, SB-400238: hNK-3R binding affinity, K(i) = 0.8 nM; hNK-2R binding affinity, K(i) = 0.8 nM) emerged as the best example in this approach. Further studies led to the identification of (S)-(+)-N-(1,2,2-trimethylpropyl)-3-[(4-piperidin-1-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 28, SB-414240: hNK-3R binding affinity, K(i) = 193 nM; hNK-2R binding affinity, K(i) = 1.0 nM) as the first hNK-2R-selective antagonist belonging to the 2-phenylquinoline chemical class. Since some members of this chemical series showed a significant binding affinity for the human mu-opioid receptor (hMOR), docking studies were also conducted on a 3-D model of the hMOR, resulting in the identification of a viable chemical strategy to avoid any significant micro-opioid component. Compounds 25 and 28 are therefore suitable pharmacological tools in the tachykinin area to elucidate further the pathophysiological role of NK-3 and NK-2 receptors and the therapeutic potential of selective NK-2 (28) or combined NK-3 and NK-2 (25) receptor antagonists.

Synthesis and NMR characterization of a novel class of thienomorphinans.

[formula: see text] Synthesis of four novel thieno derivatives 4-7 featuring the codeine skeletal backbone is reported. Characterization by 1H and 13C NMR is also discussed, along with binding profile for opioid receptors.

The interaction of the two isomers of the opioid benzodiazepine tifluadom with mu-, delta-, and kappa-binding sites and their analgesic and intestinal effects in rats.

The (+) and (-) isomers of tifluadom were assessed in rats for their opioid activities. In vitro (+)-tifluadom was almost equipotent at mu- and kappa- sites and about 10 times less potent at delta-sites: (-)-tifluadom had the same binding spectrum but was 10-20 times less potent. In vivo (+)-tifluadom delayed the hot-plate reaction time; this effect was antagonized by naloxone, but not by Ro 15-1788. (-)-Tifluadom up to 20 mg/kg had no antinociceptive effect. In the intestinal transit test analgesic doses of (+)-tifluadom did not delay the intestinal transit of a charcoal meal in rats and had weak antagonist activity against morphine-induced inhibition of intestinal transit, whereas (-)-tifluadom had neither agonist nor antagonist effect. It thus appears that (+)-and (-)-tifluadom are not selective in vitro and in vivo for one type of opioid binding site/receptor.

Differential postnatal development of mu-, delta- and kappa-opioid binding sites in rat brain.

With selective labelling techniques and analysis of saturation curves it is shown that in rat brain the concentrations of mu-, delta- and kappa-binding sites increase differentially during postnatal development. There are no changes in the binding affinities. The concentration (pmol/g brain) of kappa-sites are first to reach adult levels, namely between 7 and 14 days after birth. Adult levels of mu-sites are attained between 14 and 21 days after birth. The most striking finding is that development of delta-sites, which are not detectable 3 days after birth by the method used, lags markedly behind that of mu- and kappa-sites. The profile of development in rat brain is compared to that found previously in mouse brain.

Dopamine denervation of the nucleus accumbens induces a selective increase in the number of delta-opioid binding sites.

Selective lesions of dopamine (DA) neurons of the nucleus accumbens were made in rats by local injections of 6-hydroxydopamine (6-OHDA). Seven days after 6-OHDA, the binding affinities and capacities at mu-, delta- and kappa-opioid binding sites were determined in the nucleus accumbens by selective labelling techniques. 6-OHDA pretreatment caused a significant increase in the number of delta-opioid binding sites (+30%) while the number of mu-, kappa- and total sites was not modified. The affinity at mu-, delta- and kappa-sites was unchanged after 6-OHDA pretreatment.

Regional variations in binding capacities at mu-, delta- and kappa-opioid sites in membrane suspensions from rabbit brain.

The highest maximum binding capacities at the mu-sites of rabbit brain are in the striatum, with intermediate levels in the diencephalon, mesencephalon, cerebellum and cortex and low levels in the pons-medulla and hippocampus. For the delta-site the highest maximum binding capacity is also in the striatum; there are almost equally low levels in the other brain regions. At the kappa-sites the maximum binding capacities are highest in the diencephalon; there are intermediate levels in the cortex and striatum, and low levels in the mesencephalon, cerebellum, hippocampus and pons-medulla. The KD values lack reproducibility; there are no regional variations at the kappa-site as estimated with [3H](-)-bremazocine, but the possibility cannot be excluded that there are regional variations in the KD values for [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin at the mu-site or for [3H][D-Ala2,D-Leu5]enkephalin at the delta-site. It may be important to use saturation analysis in future investigations of the distributions of the binding sites.

Agonist and antagonist properties of some benzomorphans on nociceptive reaction and intestinal transit in rats.

The agonist and antagonist properties of bremazocine, ethylketazocine, Mr 2034 and Mr 2092, benzomorphans with the same pharmacological actions in vitro, were studied in vivo in rats. Overnight fasted animals were tested for nociceptive reaction on a 55 degrees C hot-plate and for the intestinal transit of a charcoal meal fed 5 min earlier. The antagonist activity was assessed by the drugs' ability to prevent morphine-induced constipation. Bremazocine was an agonist on the nociceptive reaction and a pure antagonist on intestinal propulsion; ethylketazocine and Mr 2034 were pure agonists in both tests and Mr 2092 was a mixed agonist-antagonist in the intestinal transit test. Thus the in vivo agonist-antagonist properties of opiates cannot always be predicted from their in vitro characteristics.

Bremazocine induces antinociception, but prevents opioid-induced constipation and catatonia in rats and precipitates withdrawal in morphine-dependent rats.

Some in vivo agonist and antagonist properties of the putative k-compound bremazocine were characterized in rats. Bremazocine, at doses from 0.015-32 mg/kg i.p., delayed nociceptive reaction on a 55 degrees C hot-plate with a dose-response curve not readily fitting a single straight line; this effect was antagonized by high doses of naloxone. In the same rats bremazocine did not delay the intestinal transit of a charcoal meal fed 5 min earlier and prevented morphine-induced constipation. This antagonism appeared to be opioid-specific and competitive, with apparent pA2 value 8.56. Catatonia induced by etorphine (0.004 mg/kg s.c.) and constipation induced by etorphine (0.004 mg/kg s.c.) and D-Ala2-D-Leu5-enkephalin (0.1 mg/kg i.p.) were completely antagonized by bremazocine (0.03-8 mg/kg i.p.). Antinociception induced by morphine (10 mg/kg i.v.) and etorphine (0.004 mg/kg s.c.) was only partly prevented. Naloxone (1 mg/kg) and bremazocine (0.015-1 mg/kg i.p.) precipitated a withdrawal syndrome, evaluated as jumping frequency, in rats rendered dependent to morphine. These data suggest the involvement of more than one opioid receptor population in bremazocine action in vivo.

Antagonism by N-methyl levallorphan-methane sulphonate (SR 58002 C) of morphine-elicited acute and chronic central and peripheral effects.

The peripheral activity of the quaternary narcotic antagonist N-methyl levallorphan-methane sulphonate (SR 58002 C) at opioid sites located in the periphery and in the central nervous system (CNS), was studied by different approaches in rats after subcutaneous injection (s.c.). Pretreatment with SR 58002 C 2,8 or 32 mg/kg s.c. 10, 50 or 110 min before buprenorphine consistently reduced buprenorphine in vivo binding only in the small intestinal longitudinal muscle with attached myenteric plexus (MP), whereas naloxone (1 mg/kg s.c.) 10 min before buprenorphine lowered buprenorphine binding in MP and brain (without cerebellum). Plasma levels were not altered by SR 58002 C or naloxone. The same doses of SR 58002 C injected 10, 50 or 110 min before morphine selectively antagonized the inhibition of transit of a charcoal meal along the small intestine (mainly a peripheral effect) induced by the agonist, but did not antagonize morphine-elicited analgesia in the hot-plate test (central effect). Naloxone (1 mg/kg s.c.) injected 10 min before morphine antagonized both agonist effects simultaneously. In morphine-dependent rats SR 58002 C (0.25, 1, 4 and 32 mg/kg s.c.) induced diarrhea, dose-dependently, in most animals within the first 30 min, while jumping, measured in the same rats, occurred in some animals, not dose-dependently, from 60 min on. Naloxone (1 mg/kg s.c.) induced both effects in most rats. These findings suggest that, although SR 58002 C probably penetrates the blood-brain barrier in some morphine-dependent rats, it discriminates peripheral and CNS opioid effects.

Interaction of U-69,593 with mu-, alpha- and kappa-opioid binding sites and its analgesic and intestinal effects in rats.

The kappa-opioid compound U-69,593 was studied in rats in vitro in binding assays to assess its selectivity at the single types of opioid sites and in vivo to assess its analgesic activity and effect on intestinal propulsion. In vitro the U-69,593 inhibition curve of [3H]-(-)-bremazocine binding suppressed at mu- and alpha-sites was biphasic and the inhibition constant (Kl) at the high-affinity site (10-18 nM) was two orders of magnitude smaller than the Kl at the low-affinity site. The Kl at mu- and alpha-sites were respectively 3.3 and 8.5 microM. Thus [3H]-(-)-bremazocine, suppressed at mu- and alpha-sites, may still bind more than one site, which U-69,593 might distinguish. In vivo U-69,593 i.p. prolonged the reaction time of rats on a 55 degrees C hot-plate and the dose of naloxone required to antagonize this effect was 40 times the dose that antagonized morphine-induced antinociception, suggesting the involvement of the kappa-receptor. In the intestinal transit test U-69,593 at doses between 0.5 and 15 mg/kg i.p. only slightly slowed intestinal transit of a charcoal meal in rats with no dose-relation; it partly but significantly antagonized morphine-induced constipation. These results suggest that the kappa-type of opioid receptor, with which U-69,593 interacts may induce analgesia, but has no appreciable role in the mechanisms of opioid-induced inhibition of intestinal transit in rats.

D-optimal design applied to binding saturation curves of an enkephalin analog in rat brain.

The D-optimal design, a minimal sample design that minimizes the volume of the joint confidence region for the parameters, was used to evaluate binding parameters in a saturation curve with a view to reducing the number of experimental points without loosing accuracy in binding parameter estimates. Binding saturation experiments were performed in rat brain crude membrane preparations with the opioid mu-selective ligand [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO), using a sequential procedure. The first experiment consisted of a wide-range saturation curve, which confirmed that [3H]-DAGO binds only one class of specific sites and non-specific sites, and gave information on the experimental range and a first estimate of binding affinity (Ka), capacity (Bmax) and non-specific constant (k). On this basis the D-optimal design was computed and sequential experiments were performed each covering a wide-range traditional saturation curve, the D-optimal design and a splitting of the D-optimal design with the addition of 2 points (+/- 15% of the central point). No appreciable differences were obtained with these designs in parameter estimates and their accuracy. Thus sequential experiments based on D-optimal design seem a valid method for accurate determination of binding parameters, using far fewer points with no loss in parameter estimation accuracy.

Morphine inhibits gastrointestinal transit in the rat primarily by impairing propulsive activity of the small intestine.

The effect of a single dose of morphine on small intestinal transit was studied in fasted rats by feeding a charcoal test meal either by stomach tube to normal animals or through a duodenal cannula to chronically implanted rats. The percentage of small intestine traversed by charcoal in 5 min was reduced by morphine (0.7 mg/kg s.c.) given 5 min before the meal to a similar extent in normal and implanted rats. In contrast administration of atropine (0.8 mg/kg s.c.) 30 min before the meal reduced charcoal transit only in normal rats fed the test meal by stomach tube. Inhibition of gastrointestinal transit in the rat by systemically administered morphine is mainly due to impaired small intestinal propulsion and does not depend to any significant extent on a direct action on the stomach delaying gastric emptying.

The opioid kappa-selective compound U-50,488H does not inhibit intestinal propulsion in rats.

The selective kappa-compound U-50,488H, at doses producing strong central pharmacological effects (0.5-32 mg kg-1 i.p.) did not delay the intestinal transit of a charcoal meal and had little or no antagonist action against morphine-induced constipation. It appears that kappa-opioid receptors are probably not involved in the mechanisms responsible for opioid-induced constipation.

Central and peripheral inhibition of gastrointestinal transit in rats: narcotics differ substantially by acting at either or both levels.

The roles of local intestinal and centrally mediated opiate-specific mechanisms underlying gastrointestinal transit inhibiton by five typical narcotic analgesics were assessed by the rat charcoal meal test. The doses (mg kg-1 s.c.) reducing the progression of charcoal to 50% of drug-free controls in 5 min (ID50) were approximately 1 for morphine and methadone, 0.5 and 40 for diamorphine and pethidine (all given 25 min before charcoal) and 0.001 for etorphine (10 min before charcoal). The delay in test meal travel caused by these ID50 doses was completely prevented by i.p. naloxone. Intracerebroventricular (i.c.v.) naloxone fully antagonized pethidine and etorphine but had no effect on morphine. Morphine, but either pethidine nor etorphine, was antagonized by i.p. N-methyl naloxone (a peripheral antagonist). Diamorphine and methadone were partially antagonized by i.c.v. naloxone or i.p. N-methyl naloxone.

The peripheral narcotic antagonist N-allyl levallorphan-bromide (CM 32191) selectively prevents morphine antipropulsive action and buprenorphine in-vivo binding in the rat intestine.

The assumed low ability of the quaternary narcotic antagonist N-allyl levallorphan-bromide (CM 32191) to cross the blood-brain barrier and its selectivity in relieving the peripherally-elicited antipropulsive action of morphine while preserving analgesia has been tested. To ascertain the extent of penetration of CM 32191 into the cns, its relative potency in preventing the in-vivo binding of high specific activity [3H]buprenorphine in the rat cns and small intestine was compared. Pretreatment was with CM 32191 at 16, 30 or 60 mg kg-1 s.c., 20, 60 or 120 min before buprenorphine, the concentrations of which in cerebrum and spinal cord were comparable with control values, but were consistently reduced in the intestine (longitudinal muscle with attached myenteric plexus). Pretreatment with naloxone (20 min, 0.5 or 1 mg kg-1 s.c.) lowered buprenorphine binding in intestine and cns. Neither narcotic antagonist produced significant changes in buprenorphine plasma concentrations. The peripheral selectivity of CM 32191, methyl naloxone and naloxone was examined by investigating in the same rats nociception in the hot plate (central opiate-sensitive mechanism) and the transit of a charcoal meal along the small intestine (local opiate-sensitive mechanism). Both effects were inhibited by morphine (5 mg kg-1 i.v.). Naloxone (10 min pretreatment, 0.5 or 1 mg kg-1 s.c.) did not selectively antagonize intestinal action of the morphine since the relief of charcoal transit inhibition was consistently associated with complete loss of analgesia.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective delta opioid receptor agonists for inflammatory and neuropathic pain.

In the last decade a number of selective and potent non-peptidic agents became available to explore the usefulness of the delta-opioid receptor in modulation of pain of different origins. As a continuing effort in this field, potent and selective delta-opioid agonists based on the pyrrolomorphinan framework have been designed, synthesised and characterised biologically in our laboratories. In animal models, a selected compound of interest, SB 235863, has proved the concept that selective delta-opioid agonists may have great potential as pain relief agents in inflammatory and neuropathic pain conditions. Importantly, such a compound was free of the unwanted side effects usually associated with narcotic analgesics such as morphine.

Acute and chronic antiparkinsonian effects of the novel nociceptin/orphanin FQ receptor antagonist NiK-21273 in comparison with SB-612111.

BACKGROUND AND PURPOSE: Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor antagonists have been proposed as a novel therapeutic approach to Parkinson's disease. Main limitations of previous studies were the use of structurally similar compounds and the evaluation of their acute effects only. We report here on the acute and long-term antiparkinsonian effects of the novel compound 2-[3-[4-(2-chloro-6-fluoro-phenyl)-piperidin-1-ylmethyl]-2-(morpholine-4-carbonyl)-indol-1-yl]-acetamide (NiK-21273) in comparison with the potent and selective NOP receptor antagonist SB-612111. EXPERIMENTAL APPROACH: Basic pharmacological properties of NiK-21273 were studied in cell lines and isolated tissues (mouse and rat vas deferens). Antiparkinsonian effects were studied in reserpinized mice and 6-hydroxydopamine hemilesioned rats under both acute and chronic administration protocols. KEY RESULTS: In vitro, NiK-21273 behaved as a potent (pA(2) 7.7) and selective NOP receptor antagonist. In vivo, it reduced hypokinesia in reserpinized mice at 0.1 and 1 but not 10 mg·kg(-1), whereas SB-612111 (0.01-1 mg·kg(-1)) provided a dose-dependent antiparkinsonian effect. NiK-21273 ameliorated motor performance in 6-hydroxydopamine hemilesioned rats at 0.5 and 5 but not 15 mg·kg(-1). SB-612111 replicated these effects in the 0.01-1 mg·kg(-1) range without loss of efficacy. Both antagonists synergized with L-DOPA at subthreshold doses. Chronic administration of NiK-21273 provided delayed improvement in baseline activity at 0.5 and 1.5 mg·kg(-1), although tolerance to the higher dose was observed. Conversely, SB-612111 (1 mg·kg(-1)) maintained its effects over time without modifying baseline activity. CONCLUSIONS AND IMPLICATIONS: NOP receptor antagonists provide motor benefit in parkinsonism models although the 'therapeutic' window and long-term effects may vary between compounds.