William Scott Caldwell

EPR evidence for nitric oxide production from guanidino nitrogens of L-arginine in animal tissues in vivo.

Administration of Fe(2+)-citrate complex (50 mg/kg of FeSO4 or FeCl2 plus 250 mg/kg of sodium citrate) subcutaneously in the thigh or Escherichia coli lipopolysaccharide (LPS, 1 mg/kg) intraperitoneally, (i.p.) to mice induced NO formation in the livers in vivo at the rate of 0.2-0.3 micrograms/g wet tissue per 0.5 h. The NO synthesized was specifically trapped with Fe(2+)-diethyldithiocarbamate complex (FeDETC2), formed from endogenous iron and diethyldithiocarbamate (DETC) administered i.p. 0.5 h before decapitation of the animals. NO bound with this trap resulted in the formation of a paramagnetic mononitrosyl iron complex with DETC (NO-FeDETC2), characterized by an EPR signal at g perpendicular = 2.035, g parallel = 2.02 with triplet hyperfine structure (HFS) at g perpendicular. This allowed quantification of the amount of NO formed in the livers. An inhibitor of enzymatic NO synthesis from L-arginine, NG-nitro-L-arginine (NNLA, 50 mg/kg) attenuated the NO synthesis in vivo. L-Arginine (500 mg/kg) reversed this effect. Injection of L-[guanidineimino-15N2]arginine combined with Fe(2+)-citrate or LPS led to the formation of the EPR signal of NO-FeDETC2 characterized by a doublet HFS at g perpendicular, demonstrating that the NO originates from the guanidino nitrogens of L-arginine in vivo.

A further study of FTC yield and nicotine absorption in smokers

Abstract The relationship between nicotine yield as determined by the FTC method and nicotine absorption was examined in 72 smokers in a more rigorous repetition of a previous study of 33 smokers. For this study, 113 smokers evenly distributed across four FTC “tar” yield ranges were recruited; only 72 demonstrated reasonable compliance with the study criteria with regard to sample collections and cigarette brand style consistency. Subjects recorded the number of cigarettes smoked daily and collected a 24-h urine sample and a saliva sample on 3 consecutive days. Nicotine absorption was determined by monitoring urinary excretion of nicotine and its metabolites. In addition, saliva samples were monitored for cotinine using radioimmunoassay (RIA). The correlation of the relationship for nicotine absorbed per cigarette was positive and significant (r = 0.31, P = 0.008) but weaker than in the previous study. Only smokers in the highest yield range showed any statistical difference from smokers in the lower ranges. Our results suggest that FTC nicotine yield is weakly related to nicotine absorption and that smoker-controlled factors exert a great influence on the amount of nicotine absorbed by smokers. Compensation is substantial but incomplete for the minority (by market share) of smokers at the low end of the yield scale. It is uncertain how well any alternative set of machine parameters would predict nicotine absorption for the majority of smokers, even if it were more predictive for the small number of smokers at the lower yield part of the range.

Comparison of measured and FTC.predicted nicotine uptake in smokers

Cigarette smokers have a wide variety of “tar” and nicotine yields to choose from in the current market, ranging from 0.5 mg “tar” and less than 0.05 mg nicotine to 27 mg “tar” and 1.8 mg nicotine by the Federal Trade Commission (FTC) method. To understand better the relationship between FTC nicotine yields and actual nicotine uptake in smokers, we have studied nicotine uptake in 33 smokers of self-selected products representing four “tar” groupings: 1 mg “tar” (1 MG), ultra-low “tar” (ULT), full-flavor low “tar” (FFLT), and full flavor (FF) cigarettes. These cigarette categories had mean FTC nicotine yields of 0.14, 0.49, 0.67, and 1.13 mg/cigarette, respectively. The subjects smoked their usual brand of cigarette ad libitum and provided a 24-h urine sample for total nicotine uptake analysis over a period during which the number of cigarettes smoked was recorded. Nicotine uptake was determined by monitoring urinary nicotine and its metabolites, including the glucuronide conjugates. Daily nicotine uptake was 9.1±7.3 mg (range 1–21 mg) for 1 MG, 19.2±10.0 mg (range 4–42 mg) for ULT, 21.8±9.4 mg (range 13–38 mg) for FFLT, and 37.1±14.4 mg (range 21–60 mg) for FF smokers. On a per cigarette basis, yields were 0.23±0.11, 0.56±0.23, 0.60±0.18, and 1.19±0.43 mg nicotine, respectively. Although individual variability was fairly large (CVs of 0.39–0.80), means for the different groups showed that lower FTC yield smokers not only absorb less nicotine per 24-h period, but also per cigarette smoked. These data suggest that nicotine uptake is a function of individual smoking behavior within product design limits. We conclude from these data that, while FTC yield cannot precisely predict nicotine uptake for an individual smoker, it is useful in predicting and comparing actual nicotine uptake by smokers who select cigarettes with a particular FTC yield.

Synthesis of 2-(Pyridin-3-yl)-1-azabicyclo[3.2.2]nonane, 2-(Pyridin-3-yl)-1-azabicyclo[2.2.2]octane, and 2-(Pyridin-3-yl)-1-azabicyclo[3.2.1]octane, a Class of Potent Nicotinic Acetylcholine Receptor-Ligands

In an attempt to generate nicotinic acetylcholine receptor (nAChR) ligands selective for the alpha4beta2 and alpha7 subtype receptors we designed and synthesized constrained versions of anabasine, a naturally occurring nAChR ligand. 2-(Pyridin-3-yl)-1-azabicyclo[2.2.2]octane, 2-(pyridin-3-yl)-1-azabicyclo[3.2.2]nonane, and several of their derivatives have been synthesized in both an enantioselective and a racemic manner utilizing the same basic synthetic approach. For the racemic synthesis, alkylation of N-(diphenylmethylene)-1-(pyridin-3-yl)methanamine with the appropriate bromoalkyltetrahydropyran gave intermediates which were readily elaborated into 2-(pyridin-3-yl)-1-azabicyclo[2.2.2]octane and 2-(pyridin-3-yl)-1-azabicyclo[3.2.2]nonane via a ring opening/aminocyclization sequence. An alternate synthesis of 2-(pyridin-3-yl)-1-azabicyclo[3.2.2]nonane via the alkylation of N-(1-(pyridin-3-ylethylidene)propan-2-amine has also been achieved. The enantioselective syntheses followed the same general scheme, but utilized imines derived from (+)- and (-)-2-hydroxy-3-pinanone. Chiral HPLC shows that the desired compounds were synthesized in >99.5% ee. X-ray crystallography was subsequently used to unambiguously characterize these stereochemically pure nAChR ligands. All compounds synthesized exhibited high affinity for the alpha4beta2 nAChR subtype ( K i < or = 0.5-15 nM), a subset bound with high affinity for the alpha7 receptor subtype ( K i < or = 110 nM), selectivity over the alpha3beta4 (ganglion) receptor subtype was seen within the 2-(pyridin-3-yl)-1-azabicyclo[2.2.2]octane series and for the muscle (alpha1betagammadelta) subtype in the 2-(pyridin-3-yl)-1-azabicyclo[3.2.2]nonane series.

Docking studies of benzylidene anabaseine interactions with α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding proteins (AChBPs): application to the design of related α7 selective ligands.

AChBPs isolated from Lymnaea stagnalis (Ls), Aplysia californica (Ac) and Bulinus truncatus (Bt) have been extensively used as structural prototypes to understand the molecular mechanisms that underlie ligand-interactions with nAChRs [1]. Here, we describe docking studies on interactions of benzylidene anabaseine analogs with AChBPs and α7 nAChR. Results reveal that docking of these compounds using Glide software accurately reproduces experimentally-observed binding modes of DMXBA and of its active metabolite, in the binding pocket of Ac. In addition to the well-known nicotinic pharmacophore (positive charge, hydrogen-bond acceptor, and hydrophobic aromatic groups), a hydrogen-bond donor feature contributes to binding of these compounds to Ac, Bt, and the α7 nAChR. This is consistent with benzylidene anabaseine analogs with OH and NH(2) functional groups showing the highest binding affinity of these congeners, and the position of the ligand shown in previous X-ray crystallographic studies of ligand-Ac complexes. In the predicted ligand-Ls complex, by contrast, the ligand OH group acts as hydrogen-bond acceptor. We have applied our structural findings to optimizing the design of novel spirodiazepine and spiroimidazoline quinuclidine series. Binding and functional studies revealed that these hydrogen-bond donor containing compounds exhibit improved affinity and selectivity for the α7 nAChR subtype and demonstrate partial agonism. The gain in affinity is also due to conformational restriction, tighter hydrophobic enclosures, and stronger cation-π interactions. The use of AChBPs structure as a surrogate to predict binding affinity to α7 nAChR has also been investigated. On the whole, we found that molecular docking into Ls binding site generally scores better than when a α7 homology model, Bt or Ac crystal structure is used.

Discovery of novel α7 nicotinic acetylcholine receptor ligands via pharmacophoric and docking studies of benzylidene anabaseine analogs

Based on pharmacophore elucidation and docking studies on interactions of benzylidene anabaseine analogs with AChBPs and α7 nAChR, novel spirodiazepine and spiroimidazoline quinuclidine series have been designed. Binding studies revealed that some of hydrogen-bond donor containing compounds exhibit improved affinity and selectivity for the α7 nAChR subtype in comparison with most potent metabolite of GTS-21, 3-(4-hydroxy-2-methoxybenzylidene)-anabaseine. Hydrophobicity and rigidity of the ligand also contribute into its binding affinity. We also describe alternative pharmacophoric features equidistant from the carbonyl oxygen atom of the conserved Trp-148 of the principal face, which may be exploited to further design diverse focused libraries targeting the α7 nAChR.

First total synthesis of (+/-)-3-hydroxy-11-norcytisine: structure confirmation and biological characterization.

The first total synthesis of the natural product 3-hydroxy-11-norcytisine (1), structurally related to cytisine (2), a benchmark ligand at neuronal nicotinic acetylcholine receptors (NNRs), has been achieved. The synthesis permits the unambiguous confirmation of the structure originally proposed for 1 and has enabled initial biological characterization of 1 and its related compounds against NNRs.

Development of Nicotinic Agonists for the Treatment of Alzheimer’s Disease

Anatomical, physiological, and pharmacological evidence has implicated the cholinergic systems of the brain in memory function (Becker, 1991). Not surprisingly, these systems are severely compromised in Alzheimer’s disease (AD), including deficiencies in the acetylcholine (ACh) synthesizing enzyme choline acetyltransferase, in the high affinity choline uptake system, and in the synthesis and release of ACh (Becker, 1991). These deficiencies correlate with the loss of memory function associated with the disease. As a result, many therapeutic strategies have focused on boosting ACh levels. Much attention has also been given to muscarinic cholinergie receptors as potential targets for AD therapeutics. More recent findings have established that nicotinic cholinergic receptors are severely altered in AD brains, being decreased by 50% or more (Nordberg et al., 1989). Animal studies have shown that activation of nicotinic receptors by nicotine can evoke ACh release (Rowell and Winkler, 1984), improve learning and memory (Hodges et al., 1992), prevent degeneration of cortical neurons (Sjak-Shie et al., 1990) and increase the number of CNS receptors (Marks et al., 1987). Two groups have experimented with nicotine as a treatment for AD. Newhouse et al. (1988) administered nicotine to AD patients and observed a decrease in intrusion errors during cognitive testing. Likewise, Jones et al. (1992) administered nicotine to AD patients and reported improvements in attention and information processing. These findings suggest that nicotinic agonists may have therapeutic potential for the treatment of AD.

Metanicotine: A nicotinic agonist with central nervous system selectivity—in vitro and in vivo characterization

A growing body of evidence suggests that disruption of nicotinic cholinergic systems may be an important factor in the etiology of a number of different diseases, ranging from neurodegenerative diseases, such as Alzheimers and Parkinsons, to ulcerative colitis. The mechanistic basis for such diverse nicotinic effects is likely to lie in the ever growing number of potential receptor subtypes. Therefore, the development of receptor subtype‐selective probes is essential to understand the emerging complexity of nicotinic cholinergic systems and the mechanisms underlying diseases that may involve these systems. Toward this end, we have evaluated the nicotinic agonist metanicotine, (E)‐N‐methyl‐4‐(3‐pyridinyl)‐3‐butene‐1‐amine, using the following in vitro and in vivo methods: 1) receptor binding and up‐regulation, 2) neurotransmitter release and ion flux in synaptosomes/cells, 3) in vivo microdialysis in rats, 4) reversal of scopolamine‐induced amnesia in a step‐through passive‐avoidance paradigm, 5) water maze performance in mice, 6) radial‐arm maze performance in brain‐lesioned rats, 7) changes in heart rate and blood pressure, and 8) physiological depression of body temperature, locomotor activity, acoustic startle, and respiration rate. Our in vitro results indicate that metanicotine binds with high affinity to the major receptor subtype in brain (α4β2), evokes dopamine release from striatal synaptosomes and Rb+ efflux from thalamic synaptosomes, but does not activate ganglionic, muscle, or other peripheral type nicotinic receptors. These results suggest that metanicotine is selective for α4‐containing central nervous system (CNS) nicotinic receptors and has reduced selectivity for peripheral nervous system (PNS) receptor subtypes. These conclusions are further supported by in vivo studies with metanicotine showing enhanced cognitive effects and significantly lower peripheral effects. Our in vivo results indicate that metanicotine increases the release of acetylcholine, norepinephrine, dopamine, and serotonin in cortex and is equal to or better than nicotine on measures of cognitive enhancement. By comparison, metanicotine is significantly less potent than nicotine in increasing heart rate and blood pressure and in causing physiological depression. These results are consistent with in vitro data indicating metanicotines CNS receptor selectivity, and they suggest that this ligand may be a suitable tool for probing the relationships that underlie the complex central and peripheral pharmacology of nicotinic cholinergic systems. Furthermore, metanicotine may be a good lead candidate for developing nicotinic agonists as CNS therapeutics with reduced peripheral side effects. Drug Dev. Res. 38:169–176

RJR-2403: A CNS-Selective Nicotinic Agonist with Therapeutic Potential

In recent years molecular cloning studies have resulted in the discovery of multiple genes encoding a variety of structural and functional subunits for nicotinic cholinergic receptors (nAChR) (Galzi and Changeux, 1995), 11 having neuronal localization (α2–α9, β2–β4) and 5 expressed in skeletal muscle (α1, β1,γ, δ, e). This molecular diversity opens up the possibility of complex receptor subtype expression both in the autonomic (PNS) and central (CNS) nervous systems, possibly explaining the complex pharmacological effects of nicotine and other classic nicotinic cholinergic agonists that do not discriminate among the various receptor subtypes expressed in the CNS and PNS. The importance of understanding this complexity is emphasized by the growing body of evidence suggesting that compromised CNS nicotinic cholinergic neurotransmission may play a key role in a variety of CNS and PNS pathologies. In this regard, there is growing interest in the use of nicotinic agonists for the treatment of Alzheimer’s disease (AD) (Williams et al., 1994). One consistent feature of this disease is a decline in the function of cholinergic systems. The loss of neurons that release acetylcholine, a key neurotransmitter in learning and memory mechanisms, initially motivated an intense but disappointing search for replacement therapies targeting muscarinic receptors. On the other hand, epidemiological studies have reported an inverse correlation between the incidence of AD and smoking (van Duijn and Hofman, 1991), supporting the notion that nicotinic pharmacology underlies an apparent protective effect.