Nicotinic Receptor Alpha-5 Subunit Gene Polymorphism is Associated with Heavy Smoking Under a Range of Nicotine Dosing Conditions.

INTRODUCTION: This study aimed to assess the role of the rs16969968 variant of nicotinic receptor alpha-5 subunit in regulating smoking behavior and nicotine intake in response to nicotine manipulations among dependent smokers in a naturalistic environment. METHODS: Sixty-nine adults (19 females) smoking 10 or more cigarettes per day were asked to complete four 2-week study phases during which they smoked exclusively one of two types of Spectrum nicotine research cigarettes (FTC nicotine yield 0.8 and 1.6 mg, respectively), their usual brand of cigarettes, or their usual brand of cigarettes while wearing a 21-mg nicotine patch. Measurements included rs16969968 genotype, number of cigarettes per day, smoking topography, and plasma cotinine. RESULTS: Compared to controls (G/G carriers), A allele carriers reported smoking 4 to 5 more cigarettes per day across all conditions (all ps < .05). Mean total smoke volume per day and cotinine were greater in A allele carriers than in controls (ps = 0.05, 0.046, respectively). No significant genotype differences were found in smoking compensation indices for the switch from Medium to High nicotine yield cigarettes. Nicotine patch-induced reductions in cigarettes smoked per day and total smoke volume per day showed significant interactions between genotype and pre-patch levels, heavier smokers showing greater effects of genotype (p = .052 and p =.006, respectively). CONCLUSIONS: Results suggest that the rs16969968 variants regulate heaviness of smoking primarily by their impact on daily numbers of cigarettes smoked, but no genotype differences were found in smoking compensation after switching from Medium to High nicotine cigarettes. IMPLICATIONS: The differences in daily cigarette consumption between rs16969968 risk-allele carriers and controls are shown to be consistent regardless of manipulations of cigarette nicotine content and transdermal nicotine supplementation and markedly greater among dependent smokers than those observed in the general smoker populations. G/G allele carriers, relative to A allele carriers, appeared to be more sensitive to the nicotine patch manipulation, reducing their smoking to a greater extent. These findings support continued efforts in the development of personalized intervention strategies to reduce the rs16969968-conveyed genetic propensity for heavy smoking.

Effect of Electronic Cigarette Liquid pH on Retention of 11C-Nicotine in a Respiratory Tract Model.

INTRODUCTION: Based on our preliminary 11C-nicotine positron emission tomography (PET) imaging studies in humans, we speculated that greater deposition of nicotine in the respiratory tract from electronic cigarettes compared to combustible cigarettes could result from the alkaline pH of typical aerosol-producing electronic cigarette liquids (e-liquids). To address this hypothesis, we assessed the effect of e-liquid pH on the retention of nicotine in vitro using 11C-nicotine, PET, and a human respiratory tract model of nicotine deposition. AIMS AND METHODS: A single 2-second 35-mL puff was delivered to a human respiratory tract cast from a 2.8-Ohm cartomizer at 4.1 volts. Immediately after the puff, a 2-second 700-mL air wash-in volume was administered. E-liquids (glycerol and propylene glycol 50/50 vol/vol) containing 24 mg/mL nicotine were mixed with 11C-nicotine. Deposition (retention) of nicotine was assessed using a GE Discovery MI DR PET/CT scanner. Eight e-liquids with different pH values (range 5.3-9.6) were investigated. All experiments were performed at room temperature and at a relative humidity of 70%-80%. RESULTS: Retention of nicotine in the respiratory tract cast was pH dependent and the pH-sensitive component of the retention was well described by a sigmoid curve. In total, 50% of the maximal pH-dependent effect was observed at pH 8.0, which is close to the pKa2 of nicotine. CONCLUSIONS: The retention of nicotine in the respiratory tract conducting airways is dependent on the e-liquid pH. Lowering the e-liquid pH reduces retention of nicotine. Nonetheless, reduction of the pH below 7 has little effect, consistent with the pKa2 of protonated nicotine. IMPLICATIONS: Similar to combustible cigarettes, the retention of nicotine in the human respiratory tract from consumption of electronic cigarettes may have some health consequences and affect nicotine dependence. Here we demonstrated that the retention of nicotine in the respiratory tract is dependent on the e-liquid pH, and lowering pH reduces retention of nicotine in conducting airways of the respiratory tract. Therefore, e-cigarettes with low pH values would result in reduced respiratory tract nicotine exposure and faster delivery of nicotine to the central nervous system (CNS). The latter can be associated with e-cigarette abuse liability and the effectiveness of e-cigarettes as substitutes for combustible cigarettes.

Comparison of brain nicotine accumulation from traditional combustible cigarettes and electronic cigarettes with different formulations.

Rapid brain accumulation is critical for the acute reinforcing effects of nicotine. Although nicotine formulation (free-base vs. protonated or salt) in electronic cigarette (E-cig) liquid affects user satisfaction, its impact on brain nicotine accumulation (BNA) from E-cig use has not been evaluated in comparison with traditional combustible cigarettes (C-cigs) using a within-subjects design. BNA was directly assessed with 29 adult dual users (13 females) of E-cigs and C-cigs, using [11C]nicotine and positron emission tomography (PET). Participants underwent two 15-min upper body (from chest to head) scanning sessions during which they inhaled a single puff of [11C]nicotine-labeled vapor from E-cigs with free-base nicotine or C-cig smoke in a randomized order. Seventeen of them also went through another session during which they inhaled from E-cigs with nicotine salt. A full-body scan was also conducted at each session to measure total absorbed dose of [11C]nicotine. Mean maximum nicotine concentration (Cmax) in brain following inhalation of free-base nicotine E-cig vapor was 19% and 15% lower relative to C-cig smoke and nicotine salt E-cig vapor (ps = 0.014 and 0.043, respectively). The Cmax values did not differ significantly between the C-cig and nicotine salt E-cig. Mean values of time to the maximum concentration (Tmax) were not significantly different between the two types of E-cig, but they were 64% and 40% longer than that for C-cig smoking (ps = 0.0005 and 0.004, respectively). Mean Cmax with C-cigs and free-base nicotine E-cigs were greater in females relative to males and correlated with T1/2 of lung nicotine clearance and participants' pack-years. These results suggest that while E-cigs with free-base nicotine formulation can deliver nicotine rapidly to the brain, those with nicotine salt formulation are capable of even more efficient brain nicotine delivery closely resembling combustible cigarettes. Therefore, nicotine formulation or pH in E-liquid should be considered in evaluation of E-cigs in terms of abuse liability and potential in substituting for combustible cigarettes.

Up-regulation of nicotinic acetylcholine receptors in menthol cigarette smokers.

One-third of smokers primarily use menthol cigarettes and usage of these cigarettes leads to elevated serum nicotine levels and more difficulty quitting in standard treatment programmes. Previous brain imaging studies demonstrate that smoking (without regard to cigarette type) leads to up-regulation of ß(2)*-containing nicotinic acetylcholine receptors (nAChRs). We sought to determine if menthol cigarette usage results in greater nAChR up-regulation than non-menthol cigarette usage. Altogether, 114 participants (22 menthol cigarette smokers, 41 non-menthol cigarette smokers and 51 non-smokers) underwent positron emission tomography scanning using the α(4)ß(2)* nAChR radioligand 2-[(18)F]fluoro-A-85380 (2-FA). In comparing menthol to non-menthol cigarette smokers, an overall test of 2-FA total volume of distribution values revealed a significant between-group difference, resulting from menthol smokers having 9-28% higher α(4)ß(2)* nAChR densities than non-menthol smokers across regions. In comparing the entire group of smokers to non-smokers, an overall test revealed a significant between-group difference, resulting from smokers having higher α(4)ß(2)* nAChR levels in all regions studied (36-42%) other than thalamus (3%). Study results demonstrate that menthol smokers have greater up-regulation of nAChRs than non-menthol smokers. This difference is presumably related to higher nicotine exposure in menthol smokers, although other mechanisms for menthol influencing receptor density are possible. These results provide additional information about the severity of menthol cigarette use and may help explain why these smokers have more trouble quitting in standard treatment programmes.

Kinetics of brain nicotine accumulation in dependent and nondependent smokers assessed with PET and cigarettes containing 11C-nicotine.

Tobacco smoking is a chronic, relapsing disorder that constitutes one of the primary preventable causes of death in developed countries. Two of the popular hypotheses to explain the development and maintenance of strong nicotine dependence in cigarette smokers posit (i) a rapid brain nicotine accumulation during cigarette smoking and/or (ii) puff-associated spikes in brain nicotine concentration. To address these hypotheses, we investigated the dynamics of nicotine accumulation in the smoker's brain during actual cigarette smoking using PET with 3-s temporal resolution and (11)C-nicotine loaded into cigarettes. The results of the study, performed in 13 dependent smokers (DS) and 10 nondependent smokers (NDS), suggest that puff-associated spikes in the brain nicotine concentration do not occur during habitual cigarette smoking. Despite the presence of a puff-associated oscillation in the rate of nicotine accumulation, brain nicotine concentration gradually increases during cigarette smoking. The results further suggest that DS have a slower process of brain nicotine accumulation than NDS because they have slower nicotine washout from the lungs and that DS have a tendency to compensate for their slower rate of brain nicotine accumulation compared with NDS by inhaling a larger volume of smoke. For these reasons, smokers' dependence on cigarette smoking, or the resistance of NDS to becoming dependent, cannot be explained solely by a faster brain nicotine accumulation.

Comparison of brain nicotine uptake from electronic cigarettes and combustible cigarettes.

Brain accumulation rate and magnitude are critical for the acute reinforcing effects of nicotine. Despite electronic cigarettes' (E-cigs) appeal as substitutes for traditional combustible cigarettes (C-cigs), brain nicotine accumulation (BNA) from E-cigs has not been compared with that from C-cigs using a within-subjects design. BNA was directly assessed with 16 adult dual users (10 females) of E-cigs (e-liquid pH 9.4) and C-cigs, using 11C-nicotine and positron emission tomography (PET). Participants went through two 15-min head scanning sessions during which they inhaled a single puff of E-cig vapor or C-cig smoke containing 11C-nicotine in a randomized order. A full-body scan was also conducted at each session to measure total absorbed dose of 11C-nicotine. Mean maximum concentration (Cmax) and area under curve of BNA were 22.1% and 22.7% lower, respectively, following E-cig compared with C-cig inhalation. Meanwhile, T1/2 was 2.7 times longer following inhalation of E-cig vapor relative to C-cig smoke (all ps < 0.005). Whole-body imaging indicated greater nicotine retention in the respiratory tract from vapor versus smoke inhalation (p < 0.0001). Following vapor inhalation, nicotine retention in the respiratory tract was correlated with Cmax values of BNA (rs = -0.59, p < 0.02). Our results confirm that E-cigs with alkaline pH e-liquid can deliver nicotine rapidly to the brain, albeit less efficiently than C-cigs partly due to greater airway retention of nicotine. Since brain nicotine uptake mediates reinforcement, these results help elucidate actions of E-cigs in terms of abuse liability and effectiveness in substituting for combustible cigarettes.

Rapid Brain Nicotine Uptake from Electronic Cigarettes.

This study sought to determine brain nicotine kinetics from use of the increasingly popular electronic cigarette (E-cig). Methods: In 17 E-cig users (9 men and 8 women), brain uptake of nicotine after inhalation from E-cigs was directly assessed using 11C-nicotine PET. The brain nicotine kinetics were compared with those from smoking combustible cigarettes (C-cigs). Results: A single puff of E-cig vapor caused the nicotine concentration in the brain to rise quickly (mean time to reach 50% of maximum brain nicotine concentration, 27 s), with a peak amplitude 25% higher in women than men, resembling previous observations with C-cigs. Nonetheless, the accumulation from E-cigs (24%) was less than that from C-cigs (32%) in both men and women. Conclusion: E-cigs can deliver nicotine to the brain with a rapidity similar to that of C-cigs. Therefore, to the extent that rapid brain uptake promotes smoking reward, E-cigs might maintain a degree of nicotine dependence and also serve as a noncombustible substitute for cigarettes.

Brain nicotinic acetylcholine receptor occupancy: effect of smoking a denicotinized cigarette.

Our group recently reported that smoking a regular cigarette (1.2-1.4 mg nicotine) resulted in 88% occupancy of brain alpha4beta2* nicotinic acetylcholine receptors (nAChRs). However, this study did not determine whether nicotine inhalation or the many other pharmacological and behavioural factors that occur during smoking resulted in this receptor occupancy. If nicotine is solely responsible for alpha4beta2* nAChR occupancy from smoking, then (as estimated from our previous data) smoking a denicotinized (0.05 mg nicotine) or a low-nicotine (0.6 mg nicotine) cigarette (commonly used for research and clinical purposes) would result in substantial 23% and 78% alpha4beta2* nAChR occupancies, respectively, and a plasma nicotine concentration of 0.87 ng/ml would result in 50% alpha4beta2* nAChR occupancy (EC50). Twenty-four positron emission tomography sessions were performed on tobacco-dependent smokers, using 2-[F-18]fluoro-A-85380 (2-FA), a radiotracer that binds to alpha4beta2* nAChRs. 2-FA displacement was determined from before to 3.1 hours after either: no smoking, smoking a denicotinized cigarette, or smoking a low-nicotine cigarette. Analysis of this PET data revealed that smoking a denicotinized and a low-nicotine cigarette resulted in 26% and 79% alpha4beta2* nAChR occupancies, respectively, across three regions of interest. The EC50 determined from this dataset was 0.75 ng/ml. Given the consistency of findings between our previous study with regular cigarettes and the present study, nicotine inhalation during smoking appears to be solely responsible for alpha4beta2* nAChR occupancy, with other factors (if present at all) having either short-lived or very minor effects. Furthermore, smoking a denicotinized cigarette resulted in substantial nAChR occupancy.

NIDA522131, a new radioligand for imaging extrathalamic nicotinic acetylcholine receptors: in vitro and in vivo evaluation.

A novel radioligand, 6-chloro-3-((2-(S)-azetidinyl)methoxy)-5-(2-fluoropyridin-4-yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The K(d) and T(1/2) of dissociation of NIDA522131 binding measured at 37 degrees C in vitro were 4.9 +/- 0.4 pmol/L and 81 +/- 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the alpha(4)beta(2)* nAChRs distribution. Comparison between [(18)F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [(18)F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non-displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3-4 fold higher for [(18)F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at alpha(4)beta(2)* versus alpha(3)beta(4)* nAChRs. These results suggest that [(18)F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans.

Estimation of D2-like receptor occupancy by dopamine in the putamen of hemiparkinsonian Monkeys.

To advance understanding of the neurochemical changes in Parkinson's disease (PD), we compared D2-like dopamine receptor occupancy by dopamine in the control and lesioned putamen of four pig-tailed macaques treated unilaterally with MPTP. PET and in vitro binding techniques were used to measure binding potential (BP(*)) and density of D2-like dopamine receptors (B(max)), respectively. As would be expected in PD, relatively higher values of BP(*) and B(max) and less amphetamine-induced decrease in [(11)C]raclopride binding were observed in the lesioned compared with the contralateral putamen in each animal. The percent differences between lesioned and contralateral sides were similar whether the measurements were of [(11)C]raclopride BP(*) or B(max) values, measured in vivo and in vitro, respectively. As [(11)C]raclopride BP(*) is a measure of the density of D2-like dopamine receptors available for radioligand binding (i.e., not occupied by dopamine), these findings suggest that the fractional occupancy of receptors by endogenous dopamine in the lesioned putamen is nearly equal to that in the contralateral putamen. Therefore, the absolute number of receptors occupied by dopamine, which is a product of receptor density and fractional occupancy by dopamine, is greater in the lesioned than in the contralateral putamen. One possible explanation for the lack of differences in fractional occupancy of D2 receptors by dopamine (despite a loss in available dopamine) is a lesion-induced increase in a portion of low-affinity D2 receptors to a state of high affinity for dopamine.

Greater nicotinic acetylcholine receptor density in smokers than in nonsmokers: a PET study with 2-18F-FA-85380.

UNLABELLED: Assays of human postmortem brain tissue have revealed that smokers have greater densities of high-affinity nicotinic acetylcholine receptors (nAChRs) in several brain regions than do nonsmokers or exsmokers. Quantitative PET imaging of nAChRs in humans has recently been reported using the alpha4beta2* subtype-specific radioligand 2-(18)F-FA-85380 (2FA). METHODS: We used PET and 2FA to measure total volumes of distribution corrected for the free fraction of 2FA in plasma (V(T)/f(P)) in 10 nonsmokers and 6 heavy smokers (>14 cigarettes/d; abstinent for >36 h). Dynamic PET scans were performed over 8 h, commencing immediately after a bolus injection of 2FA. Anatomic sampling was performed on PET images that were coregistered to MR images acquired from each volunteer. Data were analyzed by Logan plots and by 1- and 2-tissue-compartment models using unbound, unmetabolized arterial 2FA concentration as the input function. RESULTS: All modeling methods yielded similar results. V(T)/f(P) was significantly higher in smokers than in nonsmokers in all brain regions tested, except the thalamus. We used measures of V(T)/f(P) and estimates of nondisplaceable volume of distribution and found 25%-200% higher values in smokers than in nonsmokers for the volume of distribution for the specific binding compartment in the frontal cortex, midbrain, putamen, pons, cerebellum, and corpus callosum. These findings were consistent with voxel-based analysis using statistical parametric mapping. CONCLUSION: Our findings suggest that PET with 2FA can be used to study the role of nicotine-induced upregulation of nAChRs in active smokers and during smoking cessation.

Validation of an extracerebral reference region approach for the quantification of brain nicotinic acetylcholine receptors in squirrel monkeys with PET and 2-18F-fluoro-A-85380.

UNLABELLED: The aim of the present study was to explore the applicability of an extracerebral reference region for the quantification of cerebral receptors with PET. METHODS: Male squirrel monkeys underwent quantitative PET studies of cerebral nicotinic acetylcholine receptors (nAChRs) with 2-(18)F-fluoro-A-85380 (2-FA). Data from dynamic PET scans were analyzed with various compartment- and non-compartment-based models, including a simplified reference tissue model (SRTM). Nondisplaceable volume-of-distribution (VDnd) values were determined in regions of interest after the blockade of 2-FA-specific binding by nicotine infusion. Binding potential values, estimated with the cerebellum and muscle as reference regions, were compared and the reproducibility of measurements was determined. RESULTS: One- and 2-tissue-compartment modeling and linear graphic analysis provided similar total volume-of-distribution (VD(T)) values for each studied region. VD(T) values were high in the thalamus, intermediate in the cortex and midbrain, and low in the cerebellum and muscle, consistent with the distribution pattern of nAChR containing alpha(4) and beta(2) receptor subunits (alpha(4)beta(2)*). The administration of nicotine at 2 mg/kg/d via an osmotic pump resulted in a nearly complete saturation of 2-FA-specific binding and led to very small changes in volumes of distribution in the cerebellum and muscle (-9% +/- 4% [mean +/- SEM] and 0% +/- 6%, respectively), suggesting limited specific binding of the radioligand in these areas. VD(T) measured in muscle in 15 monkeys was reasonably constant (3.0 +/- 0.2, with a coefficient of variation of 8%). VDnd in studied brain regions exceeded VD(T) in muscles by a factor of 1.3. With this factor and with muscle as a reference region, BP* values calculated for studied brain regions with the SRTM were in good agreement with those obtained with the cerebellum as a reference region. Significant correlations were observed between BP* values estimated with these 2 approaches. The reproducibilities of BP* measurements obtained with the 2 methods were comparable, with coefficients of variation of less than 11% and 13% for the thalamus and the cortex, respectively. CONCLUSION: These results suggest that the accurate quantification of nAChRs can be performed with 2-FA and a reference region outside the brain, providing a novel approach for the quantification of brain receptors when no suitable cerebral reference region is available.

Cigarette smoking saturates brain alpha 4 beta 2 nicotinic acetylcholine receptors.

CONTEXT: 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy) pyridine (2-F-A-85380, abbreviated as 2-FA) is a recently developed radioligand that allows for visualization of brain alpha 4 beta 2* nicotinic acetylcholine receptors (nAChRs) with positron emission tomography (PET) scanning in humans. OBJECTIVE: To determine the effect of cigarette smoking on alpha 4 beta 2* nAChR occupancy in tobacco-dependent smokers. DESIGN: Fourteen 2-FA PET scanning sessions were performed. During the PET scanning sessions, subjects smoked 1 of 5 amounts (none, 1 puff, 3 puffs, 1 full cigarette, or to satiety [2(1/2) to 3 cigarettes]). SETTING: Academic brain imaging center. PARTICIPANTS: Eleven tobacco-dependent smokers (paid volunteers). Main Outcome Measure Dose-dependent effect of smoking on occupancy of alpha 4 beta 2* nAChRs, as measured with 2-FA and PET in nAChR-rich brain regions. RESULTS: Smoking 0.13 (1 to 2 puffs) of a cigarette resulted in 50% occupancy of alpha 4 beta 2* nAChRs for 3.1 hours after smoking. Smoking a full cigarette (or more) resulted in more than 88% receptor occupancy and was accompanied by a reduction in cigarette craving. A venous plasma nicotine concentration of 0.87 ng/mL (roughly 1/25th of the level achieved in typical daily smokers) was associated with 50% occupancy of alpha 4 beta 2* nAChRs. CONCLUSIONS: Cigarette smoking in amounts used by typical daily smokers leads to nearly complete occupancy of alpha 4 beta 2* nAChRs, indicating that tobacco-dependent smokers maintain alpha 4 beta 2* nAChR saturation throughout the day. Because prolonged binding of nicotine to alpha 4 beta 2* nAChRs is associated with desensitization of these receptors, the extent of receptor occupancy found herein suggests that smoking may lead to withdrawal alleviation by maintaining nAChRs in the desensitized state.

A simplified method for the measurement of nonmetabolized 2-[18F]F-A-85380 in blood plasma using solid-phase extraction.

Quantification of alpha(4)beta(2)* nicotinic acetylcholine receptors using 2-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[(18)F]FA) and positron emission tomography (PET) imaging requires measurement of nonmetabolized radioligand in blood plasma, which was previously accomplished using high-performance liquid chromatography (HPLC). Here, we introduce a one-step solid-phase extraction (SPE) method for measuring the concentration of nonmetabolized 2-[(18)F]FA. This method allows many samples to be processed in a short period of time. SPE effectively separated 2-[(18)F]FA from radioactive metabolites typically observed in blood plasma after administration of radioligand in humans. Measurements of the 2-[(18)F]FA parent fraction in healthy human volunteers obtained using the SPE method were nearly identical to those obtained using HPLC (1.3+/-5% average underestimation of SPE), and reproducibility was good within and between runs (2% and 6% coefficient of variation, respectively). SPE recovery of 2-[(18)F]FA from blood plasma was not appreciably diminished (3+/-0.6%) by a larger volume of blood plasma loaded onto the cartridge, suggesting the possibility of increasing the plasma sample volume at later times in a PET study to improve measurement sensitivity. 2-[(18)F]FA was stable in blood stored on ice over 8 h and in saline at low concentrations (<2 MBq/ml) but not at high concentrations (ca. 130 MBq/ml). Using SPE, the elimination half-life and full body distribution volume of 2-[(18)F]FA in healthy human volunteers were estimated as 4.2+/-0.8 h and 220+/-70 L, respectively. These results suggest that SPE is the method of choice for the determination of the plasma 2-[(18)F]FA concentration when measurement of individual metabolites is not required.

Measurement of the alpha4beta2* nicotinic acetylcholine receptor ligand 2-[(18)F]Fluoro-A-85380 and its metabolites in human blood during PET investigation: a methodological study.

2-[(18)F]fluoro-A-85380 (2-[(18)F]FA) is a new radioligand for noninvasive imaging of alpha4beta2* nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET) in human brain. In most cases, quantification of 2-[(18)F]FA receptor binding involves measurement of free nonmetabolized radioligand concentration in blood. This requires an efficient and reliable method to separate radioactive metabolites from the parent compound. In the present study, three analytical methods, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and solid phase extraction (SPE) have been tested. Reversed-phase TLC of deproteinized aqueous samples of plasma provides good estimates of 2-[(18)F]FA and its metabolites. However, because of the decreased radioactivity in plasma samples, this method can be used in humans over the first 2 h after radioligand injection only. Reliable quantification of the parent radioligand and its main metabolites was obtained using reversed-phase HPLC, followed by counting of eluted fractions in a well gamma counter. Three main and five minor metabolites of 2-[(18)F]FA were detected in human blood using this method. On average, the unchanged 2-[(18)F]FA fraction in plasma of healthy volunteers measured at 14, 60, 120, 240 and 420 min after radioligand injection was 87.3+/-2.2%, 74.4+/-3%, 68.8+/-5%, 62.3+/-8% and 61.0+/-8%, respectively. In patients with neurodegenerative disorders, the values corresponding to the three last time points were significantly lower. The fraction of nonmetabolized 2-[(18)F]FA in plasma determined using SPE did not differ significantly from that obtained by HPLC (+gamma counting) (n=73, r=.95). Since SPE is less time-consuming than HPLC and provides comparable results, we conclude that SPE appears to be the most suitable method for measurement of 2-[(18)F]FA parent fraction during PET investigations.

A programmable smoke delivery device for PET imaging with cigarettes containing 11C-nicotine.

INTRODUCTION: PET imaging with 11C-nicotine-loaded cigarettes is a valuable tool to directly assess fast nicotine kinetics and its neuropharmacological role in tobacco dependence. To eliminate variations among puffs inhaled by subjects, this work aimed to develop a programmable smoke delivery device (SDD) to produce highly reproducible and adjustable puffs of cigarette smoke for PET experiments. NEW METHOD: The SDD was built around a programmable syringe pump as a smoking machine to draw a puff of smoke from a 11C-nicotine-loaded cigarette and make it available for a subject to take the smoke into the mouth and then inhale it during PET data acquisition. Brain nicotine time activity curves and total body absorbed 11C-nicotine doses (TAD) were measured in smokers who inhaled a single puff of smoke via the SDD from a 11C-nicotine-loaded cigarette. RESULTS: Nearly identical brain nicotine kinetics were observed between participants who inhaled a puff of smoke through the SDD and those who inhaled directly from a cigarette. COMPARISON WITH EXISTING METHODS: This new device minimizes puff variations that exist with earlier smoke delivery apparatuses which could introduce confounding factors. CONCLUSIONS: The SDD is effective in delivering 11C-nicotine from the study cigarettes. Despite a 2-s increase in aging of smoke delivered through the SDD versus smoke taken directly from a cigarette, the difference in brain nicotine kinetics after 11C-nicotine delivery with and without use of the SDD is negligible. This refined device may be useful for future research on the deposition and pharmacokinetics of nicotine inhaled with tobacco smoke.

Impact of short access nicotine self-administration on expression of α4ß2* nicotinic acetylcholine receptors in non-human primates.

RATIONALE: Although nicotine exposure upregulates the α4ß2* subtype of nicotinic acetylcholine receptors (nAChRs), the upregulation of nAChRs in non-human primates voluntarily self-administering nicotine has never been demonstrated. OBJECTIVES: The objective of the study is to determine if short access to nicotine in a non-human primate model of nicotine self-administration is sufficient to induce nAChRs upregulation. METHODS: We combined a nicotine self-administration paradigm with in vivo measure of α4ß2* nAChRs using 2-[(18)F]fluoro-A-85380 (2-FA) and positron emission tomography (PET) in six squirrel monkeys. PET measurement was performed before and after intravenous nicotine self-administration (unit dose 10 µg/kg per injection). Monkeys were trained to self-administer nicotine under a fixed-ratio (FR) schedule of reinforcement. Intermittent access (1 h daily per weekday) to nicotine was allowed for 4 weeks and levels of α4ß2* nAChRs were measured 4 days later. RESULTS: This intermittent access was sufficient to induce upregulation of α4ß2* receptors in the whole brain (31 % upregulation) and in specific brain areas (+36 % in amygdala and +62 % in putamen). CONCLUSIONS: These results indicate that intermittent nicotine exposure is sufficient to produce change in nAChRs expression.

Conditional expression in corticothalamic efferents reveals a developmental role for nicotinic acetylcholine receptors in modulation of passive avoidance behavior.

Prenatal nicotine exposure has been linked to attention deficit hyperactivity disorder and cognitive impairment, but the sites of action for these effects of nicotine are still under investigation. High-affinity nicotinic acetylcholine receptors (nAChRs) contain the beta2 subunit and modulate passive avoidance (PA) learning in mice. Using an inducible, tetracycline-regulated transgenic system, we generated lines of mice with expression of high-affinity nicotinic receptors restored in specific neuronal populations. One line of mice shows functional beta2 subunit-containing nAChRs localized exclusively in corticothalamic efferents. Functional, presynaptic nAChRs are present in the thalamus of these mice as detected by nicotine-elicited rubidium efflux assays from synaptosomes. Knock-out mice lacking high-affinity nAChRs show elevated baseline PA learning, whereas normal baseline PA behavior is restored in mice with corticothalamic expression of these nAChRs. In contrast, nicotine can enhance PA learning in adult wild-type animals but not in corticothalamic-expressing transgenic mice. When these transgenic mice are treated with doxycycline in adulthood to switch off nAChR expression, baseline PA is maintained even after transgene expression is abolished. These data suggest that high-affinity nAChRs expressed on corticothalamic neurons during development are critical for baseline PA performance and provide a potential neuroanatomical substrate for changes induced by prenatal nicotine exposure leading to long-term behavioral and cognitive deficits.

The biology of tobacco and nicotine: bench to bedside.

Strong epidemiologic evidence links smoking and cancer. An increased understanding of the molecular biology of tobacco-related cancers could advance progress toward improving smoking cessation and patient management. Knowledge gaps between tobacco addiction, tumorigenesis, and cancer brought an interdisciplinary group of investigators together to discuss "The Biology of Nicotine and Tobacco: Bench to Bedside." Presentations on the signaling pathways and pathogenesis in tobacco-related cancers, mouse models of addiction, imaging and regulation of nicotinic receptors, the genetic basis for tobacco carcinogenesis and development of lung cancer, and molecular mechanisms of carcinogenesis were heard. Importantly, new opportunities to use molecular biology to identify and abrogate tobacco-mediated carcinogenesis and to identify high-risk individuals were recognized.

Synthesis and structure-activity relationship of a novel series of aminoalkylindoles with potential for imaging the neuronal cannabinoid receptor by positron emission tomography.

A new series of CB(1) ligands with high binding affinity (K(i) = 0.7-100 nM) and moderate lipophilicity (cLogD(7.4)) in the range of 2.1-4.5 has been synthesized. A structure-activity relationship study demonstrated that for the studied set of aminoalkylindoles, the molecular dipole of the ground state conformation within the series was inversely related to the affinity. The racemic ligand with highest affinity (0.7 nM), 3-(4-fluoronaphthoyl)-1-(N-methylpiperidin-2-ylmethyl)indole, was radiolabeled with (18)F. This radioligand specifically labeled CB(1) receptors in mouse brain and accumulated in regions of high versus low CB(1) receptor density in a ratio of 1.6. The displaceable radioactivity of one enantiomer in the brains of mice determined in a pretreatment study using the CB(1) antagonist N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716) was nearly double that of the racemate for the same determination; therefore, the active enantiomer is a candidate for PET studies in animals. A pretreatement study for the other enantiomer found no displaceable radioactivity in the same group of mice; this result suggested the enantiomer was inactive.