Nicotinic acetylcholine receptors (AChR) belong to a family of proteins that form ligand-gated transmembrane ion channels. They are involved in the fast transmission of signals between cells and the control of intercellular communication in the nervous system. A variety of therapeutic agents and abused drugs, including cocaine, inhibit the AChR and monoamine transporters and interfere with nervous system function. Here we describe a mechanism-based approach to prevent this inhibition. We had previously developed presteady-state kinetic (transient kinetic) techniques, with microsecond-to-millisecond time resolutions, for investigations of reactions on cell surfaces that allow one to determine the effects of inhibitors not only on the channel-opening probability but also on the opening and closing rates of the AChR channel. The transient kinetic measurements led to two predictions. T he nicotinic acetylcholine receptor (AChR) is the prototypical member of a family of structurally related membrane proteins, the ligand-gated ion channels (1). These proteins regulate intercellular communication between the approximately 10 12 cells of the mammalian nervous system, a process considered essential for brain function (2). Many therapeutic agents and abused drugs affect their function (3). For instance, the AChR is inhibited by the anticonvulsant MK-801 [(ϩ)Ϫdizocilpine] (4-6) and by several abused drugs, including cocaine (7-9). Cocaine affects more than three million people annually in the United States alone, at an estimated cost to society of more than 100 billion dollars.Understanding the mechanism of the AChR and its inhibition is a longstanding and challenging problem (10) with major implications for medicine and drug addiction (11-12). Two decades ago, single-channel current-recording (13) measurements led to the proposal of a simple and generally accepted mechanism in which inhibitors enter the open channel and block it (14-17) (the channel-blocking mechanism, Mechanism A in Fig. 1). Although several variations of this open-channelblocking mechanism have been proposed, including the conversion of an inhibitor-bound closed-channel conformation to a blocked open-channel form (18-21) (Mechanism B, Fig. 1), the open-channel-blocking mechanism, based mainly on singlechannel current or other steady-state kinetic measurements (14-21), has met the test of time during the last 20 years. In the techniques used for those measurements, the channel-activating ligand is in quasi equilibrium (steady state) with the receptor. The question we asked was: Can additional information about the receptor-mediated reactions be obtained by using presteadystate kinetic techniques? Recently, presteady-state kinetic techniques that are suitable for measuring receptor-mediated reactions on cell surfaces in the millisecond-to-microsecond time region were developed (22-31). The time resolution of the laser-pulse photolysis technique (23-26) is sufficient to investigate the reaction before the channel has opened. One can, therefore, obtain info...

Recent evidence suggests that the P2X 7 receptor may play a role in the pathophysiology of preclinical models of pain and inflammation. Therefore, pharmacological agents that target this receptor may potentially have clinical utility as antiinflammatory and analgesic therapy. We investigated and characterized the previously reported P2X 7 antagonist N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, hydrochloride salt (AACBA; GSK314181A). In vitro, AACBA was a relatively potent inhibitor of both human P2X 7 -mediated calcium flux and quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triemethylammonio)propyl]-diiodide (YO-PRO-1) uptake assays, with IC 50 values of approximately 18 and 85 nM, respectively. Compared with the human receptor, AACBA was less potent at the rat P2X 7 receptor, with IC 50 values of 29 and 980 nM in the calcium flux and YO-PRO-1 assays, respectively. In acute in vivo models of pain and inflammation, AACBA dose-dependently reduced lipopolysaccharideinduced plasma interleukin-6 release and prevented or reversed carrageenan-induced paw edema and mechanical hypersensitivity. In chronic in vivo models of pain and inflammation, AACBA produced a prophylactic, but not therapeutic-like, prevention of the clinical signs and histopathological damage of collageninduced arthritis. Finally, AACBA could not reverse L 5 spinal nerve ligation-induced tactile allodynia when given therapeutically. Consistent with previous literature, these results suggest that P2X 7 receptors do play a role in animal models of pain and inflammation. Further study of P2X 7 antagonists both in preclinical and clinical studies will help elucidate the role of the P2X 7 receptor in pain and inflammatory mechanisms and may help identify potential clinical benefits of such molecules.

Xiabin Chen et al.(1) describe pioneering work with a modified drug-metabolizing enzyme, a butyrylcholinesterase (BChE) analog, intended to prolong BChE activity in humans, hoping that the result would be accelerated cocaine metabolism. This work is exciting to those involved in pharmacokinetic approaches to drug toxicity; such constructs promise to be the first drug-metabolizing enzymes used as therapeutic agents. The analog described (1) seems to be the third of its type; this analog is apparently the longest lasting in the series (2, 3).Cocaine is metabolized three ways: (i) BChE, in blood, hydrolyzes the benzoate link producing ecgonine methyl ester (EME); (ii) carbonyl esterase, in the liver, hydrolyzes the other ester link, producing benzoyl ecgonine (BE); and (iii) oxidative enzymes in the liver remove the N-methyl group producing norcocaine (NC) (4). These initial products are further metabolized, in particular EME to ecgonine and NC to norcocaine methyl ester. Benzoic acid (BA) can be formed from both EME and NC. Chen et al. (1) assayed BA as the sole indicator of metabolite formation. EME has been considered to be inactive, especially because it lacks the properties of cocaine; NC shares at least some of those properties. Measurement of BA by Chen et al. (1), although demonstrating that cocaine metabolism occurred, failed to differentiate between initial metabolites with significant differences in properties. Other work has shown that the shorter-acting analogs of BChE preferentially convert cocaine to EME (2, 3). EME is not inactive (5). It crosses the blood-brain barrier and can reverse effects of scopolamine, a CNSactive antimuscarinic compound with related chemistry. It can improve cognition in aged rats, at the same time showing anxiolytic activity. EME can alleviate inhibition by cocaine at a novel regulatory site on nicotinic acetylcholine receptors; this site is implicated in adverse effects of cocaine (6). Furthermore, EME blocks the convulsion-inducing effect of cocaine (7), providing an example of a drug metabolite pharmacologically reversing the effect of its precursor. Thus, at least two of the modified hydrolases (2, 3) pioneer novel drug metabolism technology by creation of significant concentrations of a metabolic product with novel pharmacological mechanisms.Chen et al.(1) leave unanswered the question of whether their enzyme initially catalyzes EME formation. EME, BE, and NC could have been assayed individually. Chen et al. do not tell us whether EME concentrations were raised in their experiments, and thus whether there might be a component of pharmacological alleviation of the effects of cocaine by EME in the action of the enzyme, in contrast to the probable properties of RBP-8000 (2) and TV-1380 (3), which significantly increase the concentrations of EME in plasma. We look forward to reports of future experiments that clarify the route to benzoic acid with this enzyme, and to learning whether this approach uses EME as a product of acceleration of the metabolism of cocaine or as a p...

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An in vitro effect of (+)MK-801 (dizocilpine), an inhibitor of the glutamate/NMDA and nicotinic acetylcholine receptors, on the Aβ[1−42] and Aβ[1−40] peptides is described and compared to that of memantine. Memantine has been approved by the U.S. Food and Drug Administration for the treatment of mild-moderate Alzheimer's disease. Both compounds accelerated the formation of a β-sheet structure by Aβ[1−42], (+)MK-801 more rapidly than memantine, as observed in a thioflavin T fluorescence assay. The acceleration was followed by a decrease in the fluorescence signal that was not observed when the ligand was absent. Nuclear magnetic resonance spectra of the soluble peptides in the presence and absence of (+)MK-801 demonstrated that the monomeric form did not bind (+)MK-801 and that in the presence of (+)MK-801 the concentration of the monomeric form progressively decreased. Small angle X-ray scattering confirmed that the presence of (+)MK-801 resulted in a more rapid and characteristic transition to an insoluble form. These results suggest that (+)MK-801 and memantine accelerate the transition of Aβ [1−42] and to ThT-negative insoluble forms.