Characterization of a CNS penetrant, selective M1 muscarinic receptor agonist, 77-LH-28-1.

BACKGROUND AND PURPOSE: M1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimer's disease and schizophrenia. However, the discovery of subtype-selective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh-binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine), which bind to an allosteric site and selectively activate the M(1) mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs. EXPERIMENTAL APPROACH: In this study, we have compared the pharmacological profile of AC-42 and a close structural analogue, 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) at human recombinant, and rat native, mAChRs by calcium mobilization, inositol phosphate accumulation and both in vitro and in vivo electrophysiology. KEY RESULTS: Calcium mobilization and inositol phosphate accumulation assays revealed that both AC-42 and 77-LH-28-1 display high selectivity to activate the M1 mAChR over other mAChR subtypes. Furthermore, 77-LH-28-1, but not AC-42, acted as an agonist at rat hippocampal M1 receptors, as demonstrated by its ability to increase cell firing and initiate gamma frequency network oscillations. Finally, 77-LH-28-1 stimulated cell firing in the rat hippocampus in vivo following subcutaneous administration. CONCLUSIONS AND IMPLICATIONS: These data suggest that 77-LH-28-1 is a potent, selective, bioavailable and brain-penetrant agonist at the M1 mAChR and therefore that it represents a better tool than AC-42, with which to study the pharmacology of the M1 mAChR.

beta 1-selective adrenoceptor antagonists. 2. 4-ether-linked phenoxypropanolamines.

A series of 4-substituted phenoxypropanolamines was prepared and examined for beta-adrenoceptor activity. Some of the compounds, especially the [4-[2-[[2-(4-fluorophenyl)ethyl] oxy]ethoxy]phenoxy]propanolamines (14, 15, and 24), showed potent beta 1-blockade with virtually no beta 2-blockade at doses over a 1000 times greater. The compounds also possessed partial agonist activity. Structure-activity relationships are discussed, and conclusions are drawn about the binding sites on beta-adrenoceptors.

Beta 1-selective adrenoceptor antagonists. 3. 4-Azolyl-linked phenoxypropanolamines.

A series of 4-substituted phenoxypropanolamines has been prepared and examined for beta-adrenoceptor activity. The 4-substituents, di- and triazole ring systems connected to the phenoxy ring by different length chains, were chosen as a means of introducing cardioselectivity. This has been achieved, especially in the 1-[4-[(4-chloropyrazol-1-yl)methoxy] phenoxy]-3-(isopropylamino)-2-propanol (11), the 4-[(2H-1,2,3-triazol-2-yl)methoxy] analogue (21), and the 4-[2-(2H-1,2,3-triazol-2-yl)ethoxy] analogue (22), which show potent beta 1-blockade with selectivity ratios in excess of 100:1. Structure-activity relationships are discussed, and the optimum position of the heteroatom in the 4-substituent is defined.

A comparison of indomethacin with ibuprofen on gastrointestinal mucosal integrity in conventional and germ-free rats.

The effects of indomethacin and ibuprofen on gastrointestinal mucosal integrity were studied in conventional and germ-free rats. Only ibuprofen induced significant gastric erosion formation in both conventional and germ-free animals, demonstrating that the presence of micro-organisms is not required in this form of damage. Both indomethacin and ibuprofen caused significant intestinal damage and blood loss in germ-free animals. However, in the conventional counterparts, damage due to indomethacin was enhanced whereas that induced by ibuprofen was not. The results from the present work would suggest that non-steroidal anti-inflammatory drugs such as indomethacin, which are secreted largely in the bile, unlike ibuprofen, may act in concert with bacteria and the constituents of bile to induce, in part, intestinal damage and blood loss.

The in vitro effects of 6-methoxy-2-naphthylacetic acid, the active metabolite of nabumetone, on rat gastric mucosal eicosanoid synthesis and metabolism.

Nabumetone is a neutral non-steroidal anti-inflammatory drug with a low propensity to cause gastrointestinal (GI) damage. Previous studies, in vivo, have shown that the drug has weak effects on gastric mucosal cyclooxygenase activity, which may help to explain its favourable GI profile. The present study set out to determine whether the observed effects of nabumetone on cyclooxygenase, in vivo, parallel those of its active metabolite, 6-methoxy-2-naphthylacetic acid (6MNA), in vitro. We have also studied nabumetone and 6MNA on two other systems, namely 15-prostaglandin dehydrogenase (15-PGDH) and 5-lipoxy-genase (5-LO), which when inhibited may confer mucosal protection. The results showed that 6MNA had variable effects on cyclooxygenase activity, depending on the concentration and was less potent and less effective than indomethacin. Cyclooxygenase activity was not inhibited by the reversible inhibitor, aminopyrine, but at low concentrations stimulation was observed. Sulphasalazine inhibited 15-PGDH in a concentration-dependent manner whereas 6MNA inhibited it only at high concentrations. Nabumetone was devoid of activity. Basal 5-LO activity was attenuated by phenidone and unaltered by 6MNA but increased by nabumetone at the highest concentration. In the presence of arachidonic acid, to raise 5-LO activity, nabumetone, 6MNA, BW755C and phenidone apparently inhibited this activity. However, it was possible that both nabumetone and 6MNA inhibited a prostanoid-mediated stimulatory effect on 5-LO rather than effecting enzyme inhibition per se. Nabumetone's favourable GI profile may, therefore, relate to 6MNA having weak effects on mucosal cyclooxygenase and is unlikely to involve inhibition of prostanoid metabolism or 5-LO.

Comparison of the effects of nabumetone with indomethacin on rat gastric mucosal 6-keto-PGF1 alpha production and on bile salt-induced changes in gastric mucosal function.

The effect of nabumetone on rat gastric mucosal cyclooxygenase activity ex-vivo and in-vitro has been compared with that of indomethacin. Nabumetone was less potent and less active in inhibiting the production of gastric mucosal 6-keto-PGF1 alpha compared with indomethacin either ex-vivo or in-vitro. Anti-inflammatory doses of nabumetone failed to enhance bile salt-induced gastric erosion or mucosal permeability to dextran whereas indomethacin significantly enhanced gastric erosion and increased dextran permeability. These results suggest that nabumetone fails to promote gastric damage or increase permeability because of minimal effects on gastric mucosal cyclooxygenase.

Electrophysiological responses of crayfish oocytes to biogenic amines.

Intracellular recordings were made from immature, growing oocytes of the crayfish Pacifastacus leniusciulus. Oocytes had a relatively negative resting potential of -74.7+/-2.2 mV (n=26; range -53 to -90) and a mean input resistance of 0.86+/-0.19 MOmega (n=22; range 0.17-3.3). Octopamine induced a long-lasting response involving biphasic changes in input resistance, together with bi- or multiphasic changes in membrane potential. The resistance-decreasing phase involved (in different oocytes) membrane hyperpolarization, depolarization or both. The resistance-increasing phase was usually a depolarization. The hyperpolarizing form of the resistance-decreasing response, and the depolarizing resistance-increasing response reversed in polarity at membrane potentials of (respectively) -90 and -92 mV, suggesting increases and decreases in K(+) conductance underly the biphasic changes in input resistance. The threshold concentration for the response was remarkably low (>10(-12) M) and showed little or no dose-dependence over the concentration range 10(-12)-10(-6) M. Similar responses were evoked by dopamine and serotonin (at 10(-9) M), although a higher proportion of oocytes responded to octopamine and/or dopamine than to serotonin.

A rat gastric mucosal preparation for studying agents which affect the in vitro production of prostanoids.

A convenient and reproducible method for measuring the production of prostanoids from either the rat gastric mucosa or whole corpus tissue is described. Use of selective radio-immunoassay was employed to measure production of 6-keto- PGF1 alpha, the stable hydrolysis product of PGI2, and PGE2. The mucosal tissue generated approximately 10 times more 6-keto-PGF1 alpha than PGE2. Gastric muscle tissue, however, produced 2-3 times more 6-keto-PGF1 alpha than the mucosa. Exogenous arachidonic acid was found to elevate the amount of both prostanoids although the rate of formation of 6-keto-PGF1 alpha appeared to be less than that of PGE2. A possible explanation for this was that PGE2 production, in part, was non-enzymic. Mepacrine, a phospholipase inhibitor, was found to inhibit both the control amounts of 6-keto-PGF1 alpha and the increased production of this prostanoid due to exogenous phospholipase activity. The generation of prostanoids was also inhibited by indomethacin in a concentration-related manner which suggested that the method is suitable for screening non-steroidal anti-inflammatory agents in vitro or ex vivo. Preliminary results with the above method have been reported elsewhere.

Neural control of muscle relaxation in echinoderms.

Smooth muscle relaxation in vertebrates is regulated by a variety of neuronal signalling molecules, including neuropeptides and nitric oxide (NO). The physiology of muscle relaxation in echinoderms is of particular interest because these animals are evolutionarily more closely related to the vertebrates than to the majority of invertebrate phyla. However, whilst in vertebrates there is a clear structural and functional distinction between visceral smooth muscle and skeletal striated muscle, this does not apply to echinoderms, in which the majority of muscles, whether associated with the body wall skeleton and its appendages or with visceral organs, are made up of non-striated fibres. The mechanisms by which the nervous system controls muscle relaxation in echinoderms were, until recently, unknown. Using the cardiac stomach of the starfish Asterias rubens as a model, it has been established that the NO-cGMP signalling pathway mediates relaxation. NO also causes relaxation of sea urchin tube feet, and NO may therefore function as a 'universal' muscle relaxant in echinoderms. The first neuropeptides to be identified in echinoderms were two related peptides isolated from Asterias rubens known as SALMFamide-1 (S1) and SALMFamide-2 (S2). Both S1 and S2 cause relaxation of the starfish cardiac stomach, but with S2 being approximately ten times more potent than S1. SALMFamide neuropeptides have also been isolated from sea cucumbers, in which they cause relaxation of both gut and body wall muscle. Therefore, like NO, SALMFamides may also function as 'universal' muscle relaxants in echinoderms. The mechanisms by which SALMFamides cause relaxation of echinoderm muscle are not known, but several candidate signal transduction pathways are discussed here. The SALMFamides do not, however, appear to act by promoting release of NO, and muscle relaxation in echinoderms is therefore probably regulated by at least two neuronal signalling systems acting in parallel. Recently, other neuropeptides that influence muscle tone have been isolated from the sea cucumber Stichopus japonicus using body wall muscle as a bioassay, but at present SALMFamide peptides are the only ones that have been found to have a direct relaxing action on echinoderm muscle. One of the Stichopus japonicus peptides (holothurin 1), however, causes a reduction in the magnitude of electrically evoked muscle contraction in Stichopus japonicus and also causes 'softening' of the body wall dermis, a 'mutable connective tissue'. It seems most likely that this effect of holothurin 1 on body wall dermis is mediated by constituent muscle cells, and the concept of 'mutable connective tissue' in echinoderms may therefore need to be re-evaluated to incorporate the involvement of muscle, as proposed recently for the spine ligament in sea urchins.

Comparison of the anti-inflammatory activity and gastrointestinal irritancy of nabumetone, ibuprofen, and diclofenac in rats following chronic administration.

Nabumetone, a novel nonsteroidal anti-inflammatory drug, has aroused considerable interest due to mounting evidence suggesting a diminished potential for causing gastrointestinal mucosal irritancy. In the present study, rats were administered equally effective oral suspensions of either nabumetone (79mg/kg), ibuprofen (88mg/kg), diclofenac (11.5mg/kg), or a control suspension (n = 8 or 9 per group) daily for one month and subsequently assessed for various indices of anti-inflammatory activity, gastrointestinal irritancy, and prostaglandin inhibitory activity. Daily doses were five times the ID25 for carrageenan-induced paw inflammation as obtained in previous studies. At the end of the administration period, nabumetone maintained effective antiinflammatory activity, but was devoid of gastrointestinal irritancy. In contrast, ibuprofen and diclofenac were associated with marked and significant gastrointestinal mucosal damage. The findings of the present study support published preclinical and clinical studies establishing nabumetone as an effective anti-inflammatory agent with a favourable gastrointestinal safety profile.

Nitric Oxide Function in an Echinoderm.

In vertebrates, nitric oxide (NO) is synthesized from L-arginine by NO synthase (NOS) and regulates relaxation of smooth muscle by activating the cyclic-GMP (cGMP) generating enzyme soluble guanylyl cyclase (SGC). Here we show that the NO-cGMP pathway mediates relaxation of the cardiac stomach in the starfish Asterias rubens. The NO-donors hydroxylamine, S-nitrosoglutathione (SNOG) and S-nitroso-N-acetylpenicillamine (SNAP) and the NOS substrate L-arginine cause relaxation of the cardiac stomach. The relaxing effect of SNAP is blocked by the SGC inhibitor 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and the relaxing effect of L-arginine is inhibited by ODQ and the NOS inhibitor Nw-monomethyl-L-arginine (L-NMMA). ODQ and methylene blue also cause contraction, which may be due to inhibition of the relaxing action of NO produced by cells in the cardiac stomach. These results suggest that NO is synthesized in the cardiac stomach and regulates relaxation by activating SGC. NO-cGMP-mediated relaxation of the cardiac stomach may be important during feeding in starfish where the relaxed stomach is everted through an oral opening and over the digestible parts of prey. The discovery of NO-cGMP-mediated relaxation in an echinoderm demonstrates that regulation of smooth muscle tone by this signaling pathway also occurs in animals other than vertebrates.

Gastrointestinal irritancy, antiinflammatory activity, and prostanoid inhibition in the rat. Differentiation of effects between nabumetone and etodolac.

Many nonsteroidal antiinflammatory drugs have the ability to cause gastrointestinal damage in both animals and man. The aim of the present study was to compare nabumetone, a nonacidic drug, with etodolac on rat gastric mucosal damage and prostanoid synthesis, while concurrently measuring prostanoid production during edema formation in a carrageenan model of paw inflammation. The results showed that both drugs inhibited paw exudate prostaglandin E2 and edema significantly, but they did not inhibit gastric prostanoid production 4 hr after dosing. Gastric damage, however, was observed with etodolac. Additional time-course studies showed that over a 4-hr period, etodolac, unlike nabumetone, markedly inhibited gastric mucosal prostaglandin E2 production, which was associated with gastric erosion formation. Further studies demonstrated that nabumetone did not induce gastrointestinal damage or blood loss when administered to rats in a high antiinflammatory oral dose. In contrast, etodolac produced marked gastrointestinal damage and bleeding, which was evident for up to 48 hr after the dose. It is suggested that nabumetone's favorable gastrointestinal irritancy profile may relate, in part, to its nonacidic nature and to its differential effects on prostanoid production.

Anti-inflammatory efficacy and gastrointestinal irritancy: comparative 1 month repeat oral dose studies in the rat with nabumetone, ibuprofen and diclofenac.

Repeat oral dosing of nabumetone for 1 month maintains anti-inflammatory efficacy in a carrageenan model of paw oedema yet does not cause gastrointestinal damage. In contrast, ibuprofen or diclofenac, while also retaining anti-inflammatory activity, produced marked gastrointestinal irritancy as evidenced by mucosal damage and blood loss.

Nabumetone, an effective anti-inflammatory agent, lacks gastrointestinal irritancy in the rat when dosed orally for one month: comparison with tiaprofenic acid and etodolac.

OBJECTIVE: To determine the effects of nabumetone, compared with tiaprofenic acid and etodolac, on anti-inflammatory efficacy and gastrointestinal irritancy in the rat when dosed orally for one month at a high anti-inflammatory dose. METHODS: Carrageenan paw edema was used as a model of inflammation. Gastrointestinal mucosal integrity was assessed by concurrently measuring ulcer formation. mucosal and tissue prostanoid production and plasma haptoglobin. Haemoglobin, present in the cecal contents, was used as a measure of blood loss. RESULTS: Nabumetone, tiaprofenic acid and etodolac inhibited inflammation. Etodolac induced marked gastrointestinal damage and blood loss whereas tiaprofenic acid caused only gastric damage. Nabumetone was found not to alter mucosal integrity. CONCLUSION: Nabumetone proved to be an effective anti-inflammatory agent that was devoid of gastrointestinal irritancy.

Neutropenia does not prevent etodolac- or indomethacin-induced gastrointestinal damage in the rat.

Neutrophils have been implicated in the acute formation of gastric mucosal erosions induced by nonsteroidal antiinflammatory drugs. The aims of the present study were to determine, in rats, the role of neutrophils in the pathogenesis of etodolac- and indomethacin-induced gastrointestinal ulceration and blood loss. Both drugs caused gastrointestinal ulceration, which was associated with increased blood loss, a rise in plasma haptoglobin concentration, and a rise in the number of circulating neutrophils. A marked infiltration of neutrophils occurred only in ileal tissue. Pretreatment with a selective antineutrophil serum induced a significant neutropenia, which failed to inhibit either etodolac- or indomethacin-induced gastrointestinal ulceration and blood loss. A further study demonstrated that the antineutrophil serum did not prevent gastric erosions induced by indomethacin, but it inhibited carrageenan paw edema, which is dependent, in part, on neutrophil infiltration and activation. It is concluded that neutrophils do not contribute to gastrointestinal ulceration and blood loss induced by nonsteroidal antiinflammatory drugs. Furthermore, in contrast with previous studies, our results provide no evidence that neutrophils contribute to indomethacin-induced acute gastric erosion formation.

Anti-inflammatory and gastrointestinal effects of nabumetone or its active metabolite, 6MNA (6-methoxy-2-naphthylacetic acid): comparison with indomethacin.

6MNA, the active metabolite of the non-acidic anti-inflammatory drug nabumetone, was investigated using intravenous administration for effects on (a) carrageenan paw oedema and gastric irritancy compared to either oral nabumetone or both oral and intravenous indomethacin when given acutely and (b) gastrointestinal irritancy when given in repeat dosing studies. An oral dose of nabumetone or intravenous 6MNA produced effective anti-inflammatory activity together with significant inhibition of paw exudate PGE2. An anti-inflammatory oral dose of nabumetone or intravenous 6MNA produced minimal effects on gastric 6-keto-PGF1 alpha production, with an absence of gastric damage, in contrast to indomethacin. In repeat dose studies, 6MNA failed to induce gastrointestinal damage even at doses where general toxicity was evident. These results show that in the rat 6MNA, the active metabolite of nabumetone, is an effective anti-inflammatory drug but, even in very high intravenous doses, does not have the propensity to induce gastrointestinal damage.

Antiinflammatory and gastrointestinal effects of nabumetone or its active metabolite, 6-methoxy-2-naphthylacetic acid (6MNA). Comparative studies with indomethacin.

6MNA, the active metabolite of the nonacidic antiinflammatory drug nabumetone, was investigated using intravenous administration for effects on (1) carrageenan paw edema and gastric irritancy compared with either oral nabumetone or both oral and intravenous indomethacin when given acutely and (2) gastrointestinal irritancy when given in repeat dosing studies. Oral doses of nabumetone or intravenous 6MNA produced effective antiinflammatory activity together with significant inhibition of paw exudate PGE2. Antiinflammatory oral doses of nabumetone or intravenous 6MNA produced minimal effects on gastric 6-keto PGF1 alpha production with an absence of gastric damage, in contrast with indomethacin. In repeat dose studies, 6MNA failed to induce gastrointestinal damage even at doses where general toxicity was evident. These results show that in the rat, 6MNA is an effective antiinflammatory drug but even in very high intravenous doses does not have the propensity to induce gastrointestinal damage.