Recent progress in the development of subtype selective nicotinic acetylcholine receptor ligands.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels, which are found at the neuromuscular junction and in the central and peripheral nervous systems. The channels can be assembled from fourteen known subunits. The exact combination and function of all the channels are still not determined but in the CNS certain combinations have been identified which appear to modulate the release of specific neurotransmitters. Non-specific nAChR agonists like nicotine and epibatidine, have been shown to have interesting pharmacology but their clinical value is limited by their undesirable side effects. Selective ligands for different receptor subtypes have been reported and these compounds are probably the best tools for determining the function of the subtypes. The expectation is that some receptor subtype selective nAChR ligands will be clinically useful for the treatment of a broad range of CNS disorders. The development of stable cell lines functionally expressing specific combinations of subunits has greatly improved our understanding of ligand specificity. There have also been advances in the modelling of the ligand binding site, thanks to the discovery of a homologous snail ACh binding protein the X-ray structure of which was determined in 2001. These techniques should lead to rapid advances in the development of truly subtype selective ligands. In this review we describe recent progress in the area and describe the first 1000 fold selective low molecular weight ligands from the AstraZeneca group. We also comment on the first subtype specific channel modulators.

ARL 17477, a selective nitric oxide synthase inhibitor, with neuroprotective effects in animal models of global and focal cerebral ischaemia.

In the present studies, we have evaluated the effects of N-[4-(2-¿[(3-Chlorophenyl)methyl]amino¿ethyl)phenyl]-2-thiophenecarbo ximidamide dihydrochloride (ARL 17477) on recombinant human neuronal NOS (nNOS) and endothelial NOS (eNOS). We then carried out pharmacokinetic studies and measured cortical nitric oxide synthase (NOS) inhibition to determine that the compound crossed the blood brain barrier. Finally, the compound was evaluated in a model of global ischaemia in the gerbil and two models of transient focal ischaemia in the rat. The IC(50) values for ARL 17477 on human recombinant human nNOS and eNOS were 1 and 17 microM, respectively. ARL 17477 (50 mg/kg i.p.) produced a significant reduction in the ischaemia-induced hippocampal damage following global ischaemia when administered immediately post-occlusion, but failed to protect when administration was delayed until 30 min post-occlusion. In the endothelin-1 model of focal ischaemia, ARL 17477 (1 mg/kg i.v.) significantly attenuated the infarct volume when administered at either 0, 1 or 2 h post-endothelin-1 (P<0.05). In the intraluminal suture model, ARL 17477 at both 1 and 3 mg/kg i.v. failed to reduce the infarct volume measured at 1, 3 or 7 days post-occlusion. These results demonstrate that ARL 17477 protects against global ischaemia in gerbils and provides some reduction in infarct volume following transient middle cerebral artery occlusion in rats, indicating that nNOS inhibition may be a useful treatment of ischaemic conditions.

Evaluation of glycine site antagonists of the NMDA receptor in global cerebral ischaemia.

In the present studies we have investigated the effects of a range of glycine site antagonists of the N-methyl-d-aspartate (NMDA) receptor in the gerbil model of global cerebral ischaemia. The compounds tested were (+)-3-amino-1-hydroxy-2-pyrrolidone (HA 966, 15 mg/kg), 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinolinone) (L-701,324, 40 mg/kg), 7-chloro-3-(cyclopropylcarbonyl)-4-hydroxy-2(1H)-quinolinone) (L-701, 252, 50 mg/kg), (3-(3-hydroxyphenyl)prop-2-ynyl 7-chloro-4 hydroxy-2(1H)-quinolone-3-carboxylate) (L-701,273, 50 mg/kg), 5-nitro-6,7-dichloro-2,3-quinoxalinedione (ACEA 1021, 25 mg/kg) and [(E)-3[(phenylcarbamoyl) ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt (GV 150526A, 40 mg/kg). All compounds were administered via the i.p. route 30 min before and again at 2 h 30 min after 5 min bilateral carotid artery occlusion (BCAO) in the gerbil. For comparison we also evaluated a non-competitive NMDA antagonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine (MK-801, 2 mg/kg) and an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) antagonist, (3S,4aR, 6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)]decahydroisoquinoline-3-car boxylic acid (LY293558, 20 mg/kg). In the present studies L-701,252, L-701, 324 and L-701,273 provided a small degree of neuroprotection. ACEA 1021, GV 150526A and HA 966 failed to provide any neuroprotection, while MK-801 provided significant (20%) protection. In contrast LY293558 provided good (55%) neuroprotection. These results indicate that glycine site antagonists and competitive NMDA antagonists provide a small degree of neuroprotection in global cerebral ischaemia. In contrast, AMPA receptor antagonists provide more robust neuroprotection in global cerebral ischaemia.

Antiproliferative naphthopyrans: biological activity, mechanistic studies and therapeutic potential.

This article will firstly briefly review the newer generation of immunosuppressant drugs, focusing mainly on tacrolimus (FK-506), sirolimus (rapamycin), mycophenolate mofetil (RS-61443) and leflunomide (HWA 486) and then describe work carried out at the Lilly Research Centre on analogues of leflunomide and subsequent diversion into a structurally distinct series of compounds, the naphthopyrans. A clear structure activity relationship exists within this series and selected data from a Concanavalin A stimulated T-cell proliferation assay are presented to illustrate this. Although the compounds proved to possess little in vivo activity in our rheumatoid arthritis program, examination of the compounds in in vitro and in vivo models within the diabetic complications group showed the compounds behaved as would be anticipated for inhibitors of protein kinase C, although this direct mode of action was clearly not correct. Mechanistic investigations revealed that the favoured compound 290181 blocks phorbol 12,13-dibutyrate-induced binding of transcription factor proteins to the PEA3/TRE sequence of the promoter region of the urokinase plasminogen activator gene. The compounds also showed antiproliferative effects on vascular smooth muscle cells, an in vitro activity that translated into in vivo efficacy in a rat model of restenosis. Mechanistic studies here demonstrated that 290181 blocks proliferation in the G2/M phase of the cell cycle by binding directly to a novel site on tubulin. Finally the compounds were shown to inhibit the release of neutral proteases from interleukin-1 stimulated articular chondrocytes, this activity having implications in the degenerative aspects of osteoarthritis.

Effects of Ca2+ and Na+ channel inhibitors in vitro and in global cerebral ischaemia in vivo.

In the present study we have examined the effects of the small organic molecules: NNC 09-0026 ((-)-trans-1-butyl-4-(4-dimethylaminophenyl)-3-[(4-trifluoromethyl-ph eno xy) methyl] piperidine dihydrochloride); SB 201823-A (4-[2-(3,4-dichlorophenoxy)ethyl]-1-pentyl piperidine hydrochloride); NS 649 (2-amino-1-(2,5-dimethoxyphenyl)-5-trifluoromethyl benzimidazole); CNS 1237 (N-acenaphthyl-N'-4-methoxynaphth-1-yl guanidine) and riluzole on human omega-conotoxin sensitive N-type voltage-dependent Ca2+ channel currents (ICa) expressed in HEK293 cells, on Na+ channel currents (INa) in acutely isolated cerebellar Purkinje neurones in vitro and in the gerbil model of global cerebral ischaemia in vivo. Estimated IC50 values for steady-state inhibition of ICa were as follows; NNC 09-0026, 1.1 microM; CNS 1237, 4.2 microM; SB 201823-A, 11.2 microM; NS 649, 45.7 microM and riluzole, 233 microM. Estimated IC50 values for steady-state inhibition of Na+ channel currents were as follows: NNC 09-0026, 9.8 microM; CNS 1237, 2.5 microM; SB 201823-A, 4.6 microM; NS 649, 36.7 microM and riluzole, 9.4 microM. In the gerbil model of global cerebral ischaemia the number of viable cells (mean +/- S.E.M.) per 1 mm of the CA1 was 215 +/- 7 (sham operated), 10 +/- 2 (ischaemic control), 44 +/- 15 (NNC 09-0026 30 mg/kg i.p.), 49 +/- 19 (CNS 1237 30 mg/kg i.p.), 11 +/- 2 (SB 201823-A 10 mg/kg i.p.), 17 +/- 4 (NS 649 50 mg/kg i.p.) and 48 +/- 18 (riluzole 10 mg/kg i.p.). Thus NNC 09-0026, CNS 1237 and riluzole provided significant neuroprotection when administered prior to occlusion while SB 201823-A and NS 649 failed to protect. These results indicate that the Ca2+ channel antagonists studied not only inhibited human N-type voltage-dependent Ca2+ channels but were also effective blockers of rat Na+ channels. Both NNC 09-0026 and CNS 1237 showed good activity at both Ca2+ and Na+ channels and this may contribute to the observed neuroprotection.

Identification of a novel chemical series that blocks interleukin-1-stimulated metalloprotease activity in chondrocytes.

Cartilage destruction is one of the essential features of osteoarthritis and other degenerative disease conditions of articular disease, and it may be caused by metalloproteases induced by cytokines such as interleukin-1. To search for novel chemical entities that will block the production of metalloproteases, we have utilized an in vitro system in which macrophage-conditioned medium (a source of interleukin-1) was used to stimulate rabbit articular chondrocytes in culture. Upon treatment with macrophage-conditioned medium or recombinant interleukin-1, chondrocytes synthesize and secrete collagenase, stromelysin and other proteases into the surrounding medium and fail to organize an appropriate extracellular matrix. Using this in vitro system, we have determined that a series of naphthopyran derivatives were able to block the production of neutral metalloproteases. Structural modifications of the lead compound have revealed specific requirements for activity. This class of compounds represents one of very few that are known to block the synthesis, rather than the activity, of matrix-degrading metalloproteases and thus may be beneficial in preventing the cartilage destruction associated with several degenerative diseases of the articular joint.