Nanodelivery of Parthenolide Using Functionalized Nanographene Enhances its Anticancer Activity.

Advances in anticancer chemotherapy have been hindered by the lack of biocompatibility of new prospective drugs. One significant challenge concerns water insolubility, which compromises the bioavailability of the drugs leading to increased dosage and higher systemic toxicity. To overcome these problems, nanodelivery has been established as a promising approach for increasing the efficacy and lowering the required dosage of chemotherapeutics. The naturally derived compound, parthenolide (PTL), is known for its anti-inflammatory and anticancer activity, but its poor water solubility limits its clinical value. In the present study, we have used carboxyl-functionalized nanographene (fGn) delivery to overcome the extreme hydrophobicity of this drug. A water-soluble PTL analog, dimethylamino parthenolide (DMAPT), was also examined for comparison with the anticancer efficacy of our PTL-fGn complex. Delivery by fGn was found to increase the anticancer/apoptotic effects of PTL (but not DMAPT) when delivered to the human pancreatic cancer cell line, Panc-1. The IC50 value for PTL decreased from 39 µM to 9.5 µM when delivered as a mixture with fGn. The IC50 of DMAPT did not decrease when delivered as DMAPT-fGn and was significantly higher than that for PTL-fGn. There were significant increases in ROS formation and in mitochondrial membrane disruption in Panc-1 cells after PTL-fGn treatment as compared to PTL treatment, alone. Increases in toxicity were also seen with apoptosis detection assays using flow cytometry, ethidium bromide/acridine orange/DAPI staining, and TUNEL. Thus, fGn delivery was successfully used to overcome the poor water solubility of PTL, providing a strategy for improving the effectiveness of this anticancer agent.

NF-κB-dependent and -independent epigenetic modulation using the novel anti-cancer agent DMAPT.

The transcription factor nuclear factor-kappaB (NF-κB) is constitutively active in several cancers and is a target of therapeutic development. We recently developed dimethylaminoparthenolide (DMAPT), a clinical grade water-soluble analog of parthenolide, as a potent inhibitor of NF-κB and demonstrated in vitro and in vivo anti-tumor activities in multiple cancers. In this study, we show DMAPT is an epigenetic modulator functioning in an NF-κB-dependent and -independent manner. DMAPT-mediated NF-κB inhibition resulted in elevated histone H3K36 trimethylation (H3K36me3), which could be recapitulated through genetic ablation of the p65 subunit of NF-κB or inhibitor-of-kappaB alpha super-repressor overexpression. DMAPT treatment and p65 ablation increased the levels of H3K36 trimethylases NSD1 (KMT3B) and SETD2 (KMT3A), suggesting that NF-κB directly represses their expression and that lower H3K36me3 is an epigenetic marker of constitutive NF-κB activity. Overexpression of a constitutively active p65 subunit of NF-κB reduced NSD1 and H3K36me3 levels. NSD1 is essential for DMAPT-induced expression of pro-apoptotic BIM, indicating a functional link between epigenetic modification and gene expression. Interestingly, we observed enhanced H4K20 trimethylation and induction of H4K20 trimethylase KMT5C in DMAPT-treated cells independent of NF-κB inhibition. These results add KMT5C to the list NF-κB-independent epigenetic targets of parthenolide, which include previously described histone deacetylase 1 (HDAC-1) and DNA methyltransferase 1. As NSD1 and SETD2 are known tumor suppressors and loss of H4K20 trimethylation is an early event in cancer progression, which contributes to genomic instability, we propose DMAPT as a potent pharmacologic agent that can reverse NF-κB-dependent and -independent cancer-specific epigenetic abnormalities.

Scientific overview: 2013 BBC plenary symposium on tobacco addiction.

Nicotine dependence plays a critical role in addiction to tobacco products, and thus contributes to a variety of devastating tobacco-related diseases (SGR 2014). Annual costs associated with smoking in the US are estimated to be between $289 and $333 billion. Effective interventions for nicotine dependence, especially in smokers, are a critical barrier to the eradication of tobacco-related diseases. This overview highlights research presented at the Plenary Symposium of Behavior, Biology and Chemistry: Translational Research in Addiction Conference (BBC), hosted by the UT Health Science Center San Antonio, on March 9-10, 2013. The Plenary Symposium focused on tobacco addiction, and covered topics ranging from basic science to national policy. As in previous years, the meeting brought together globally-renowned scientists, graduate student recruits, and young scientists from underrepresented populations in Texas and other states with the goal of fostering interest in drug addiction research in young generations.

Region-specific effects of N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide on nicotine-induced increase in extracellular dopamine in vivo.

BACKGROUND AND PURPOSE: Systemic administration of N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), an antagonist of nicotinic acetylcholine receptors (nAChRs) attenuated the nicotine-induced increase in dopamine levels in nucleus accumbens (NAcc). EXPERIMENTAL APPROACH: Using in vivo microdialysis, we investigated the effects of local perfusion of the novel nAChR antagonist bPiDDB into the NAcc or ventral tegmental area (VTA) on increased extracellular dopamine in NAcc, induced by systemic nicotine. We also examined the concentration-dependent effects of bPiDDB on the acetylcholine (ACh)-evoked response of specific recombinant neuronal nAChR subtypes expressed in Xenopus oocytes, using electrophysiological methods. KEY RESULTS: Nicotine (0.4 mg kg(-1), s.c.) increased extracellular dopamine in NAcc, which was attenuated by intra-VTA perfusion of mecamylamine (100 microM). Intra-VTA perfusion of bPiDDB (1 and 10 microM) reduced nicotine-induced increases in extracellular dopamine in NAcc. In contrast, intra-NAcc perfusion of bPiDDB (1 or 10 microM) failed to alter the nicotine-induced increase in dopamine in NAcc. Intra-VTA perfusion of bPiDDB alone did not alter basal dopamine levels, compared to control, nor the increased dopamine in NAcc following amphetamine (0.5 mg kg(-1), s.c.). Using Xenopus oocytes, bPiDDB (0.01-100 microM) inhibited the response to ACh on specific combinations of rat neuronal nAChR subunits, with highest potency at alpha3beta4beta3 and lowest potency at alpha6/3beta2beta3. CONCLUSIONS AND IMPLICATIONS: bPiDDB-Sensitive nAChRs involved in regulating nicotine-induced dopamine release are located in the VTA, rather than in the NAcc. As bPiDDB has properties different from the prototypical nAChR antagonist mecamylamine, further development may lead to novel nAChR antagonists for the treatment of tobacco dependence.

Effects of nornicotine enantiomers on intravenous S(-)-nicotine self-administration and cardiovascular function in rats.

RATIONALE: Previous neurochemical evidence indicates that R(+)-nornicotine is more potent than S(-)-nornicotine in evoking dopamine release in rat nucleus accumbens slices. OBJECTIVE: The current study tested the hypothesis that R(+)-nornicotine is also more potent than S(-)-nornicotine in selectively decreasing intravenous S(-)-nicotine self-administration in rats. RESULTS: After acute pretreatment (1-10 mg/kg for each enantiomer), R(+)-nornicotine was more potent than S(-)-nornicotine in decreasing S(-)-nicotine self-administration; in contrast, within the same dose range, the nornicotine enantiomers were equipotent in decreasing sucrose-maintained responding. This enantioselectivity does not likely reflect a difference in bioavailability, since similar levels of nornicotine were recovered from the brain 60 min after injection (5.6 mg/kg for each enantiomer). With repeated pretreatment, tolerance did not develop to the rate-decreasing effect of either nornicotine enantiomer (3 or 5.6 mg/kg) with respect to the decrease in S(-)-nicotine self-administration, although the enantioselectivity dissipated across repeated pretreatments. While both enantiomers acutely produced a similar increase in blood pressure and heart rate, tolerance developed to the blood pressure effects of R(+)-nornicotine, but not to the effects of S(-)-nornicotine, across repeated treatments. CONCLUSION: Both R(+)- and S(-)-nornicotine may have potential utility as a novel tobacco use cessation agent.

Effect of a novel nicotinic receptor antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide, on nicotine self-administration and hyperactivity in rats.

RATIONALE AND OBJECTIVE: Recent work has shown that the novel compound N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB) may selectively block nicotinic acetylcholine receptors involved in regulating dopamine release. The current experiments examined the acute effect of bPiDDB on nicotine self-administration, sucrose-maintained responding, and nicotine-induced changes in acute and sensitized locomotor activity. METHODS: Rats were first trained to respond for either nicotine (i.v.) or sucrose pellets using a standard two-lever operant conditioning procedure using a fixed ratio 5 schedule of reinforcement and were then pretreated with bPiDDB (0, 0.3, 1, or 3 mg kg(-1)) 15 min prior to the session. In separate experiments, rats were assessed for nicotine-induced changes in locomotor activity following pretreatment with bPiDDB (1 or 3 mg kg(-1)) or mecamylamine (1 mg kg(-1)); pretreatments were assessed with both acute and repeated nicotine (0.4 mg kg(-1)) treatment. RESULTS: Results showed that bPiDDB dose-dependently decreased nicotine self-administration, but not sucrose-maintained responding. In the locomotor experiments, bPiDDB attenuated the hyperactivity produced by acute and repeated nicotine; however, this effect was not robust compared to mecamylamine. In contrast to mecamylamine, bPiDDB did not block the initial hypoactivity produced by acute nicotine. CONCLUSION: Since bPiDDB decreased nicotine self-administration specifically, this novel nicotinic receptor antagonist may constitute a lead for the development of a clinically useful treatment for tobacco dependence.

Once weekly administration of nicotine produces long-lasting locomotor sensitization in rats via a nicotinic receptor-mediated mechanism.

RATIONALE: Chronic nicotine administration results in dynamic changes in neuronal function, expressed as behavioral sensitization in animals and addiction in smokers. OBJECTIVES: The present study was undertaken to determine whether once-weekly nicotine injection produces sensitization to the locomotor-activating properties of nicotine as a result of nicotinic receptor activation. METHODS: Once weekly for 6 weeks, rats were administered (s.c.) two saline injections or saline and nicotine (0.35 mg/kg), and locomotor activity was monitored. Rats remained in the home cage for 21 days, and subsequently were injected with the appropriate treatment to determine whether sensitization persisted. Rats were also injected with saline or mecamylamine (1.2 mg/kg) followed by saline or nicotine once weekly for 6 weeks to determine the effect of mecamylamine and whether it inhibited nicotine-induced hyperactivity. A separate group was injected with saline and nicotine once weekly for 4 weeks; on week 5, mecamylamine and nicotine were administered to determine whether mecamylamine inhibited the expression of sensitization. Separate groups were injected with mecamylamine and nicotine once weekly for 5 weeks or 6 weeks; on week 6 or week 9, respectively, saline and nicotine were injected to determine whether mecamylamine inhibited the initiation of sensitization. RESULTS: Sensitization to the locomotor-activating properties of nicotine developed following four nicotine injections across a 28-day period and persisted following 21 days of no drug treatment. Mecamylamine did not alter activity but attenuated both the initiation and expression of sensitization. CONCLUSIONS: Nicotinic receptor activation following once-weekly nicotine administration produces long-lasting behavioral sensitization, suggesting that even infrequent nicotine exposure initiates neuroadaptive processes associated with nicotine addiction.

Neuronal nicotinic acetylcholine receptor binding affinities of boron-containing nicotine analogues.

A series of boron-containing nicotine (NIC) analogues 7-9 was synthesized and evaluated for binding to alpha4beta2 and alpha7 nicotinic receptors. Compound ACME-B inhibited [3H]methyllycaconitine binding to rat brain membranes with a similar potency compared to NIC (Ki = 2.4 and 0.77 microM, respectively), but was markedly less potent in inhibiting [3H]NIC binding when compared to NIC (Ki = 0.60 microM and 1.0 nM, respectively). Thus, tethering a two-carbon bridge between the 2-pyridyl and 3'-pyrrolidino carbons of NIC or 7 affords analogues that bind to the alpha7 receptor in a manner similar to NIC, but with a dramatic loss of affinity for the alpha4beta2 receptor.

Accumulation of nicotine and its metabolites in rat brain after intermittent or continuous peripheral administration of [2'-(14)C]nicotine.

Concentrations of nicotine, cotinine, and nornicotine in brain and blood following both intermittent and continuous administration of [2'-(14)C]nicotine to rats were determined to assess nicotine metabolite accumulation in brain following repeated nicotine administration. For intermittent studies, rats were administered s.c. 1 to 10 doses of nicotine (0.3 mg/kg, 15 or 25 microCi of [2'-(14)C]nicotine; 30-min interinjection interval). For continuous administration studies, rats were implanted s.c. with an osmotic minipump delivering nicotine (0.8 mg/kg/day, 25 or 50 microCi of [2'-(14)C]nicotine for 1-21 days). Whole brain and trunk blood was collected. The concentration of [2'-(14)C]nicotine and its metabolites was determined via high-pressure liquid radiochromatography. Brain concentrations of nicotine, cotinine, and nornicotine increased 2-, 12-, and 9-fold, respectively, following 10 injections, reaching a plateau following the fifth injection. Brain blood ratios indicate an enhanced preferential distribution of nornicotine to brain with increasing numbers of injections. Across the 21-day period of continuous infusion, blood nicotine and nornicotine concentrations remained relatively constant, whereas concentrations in brain increased approximately 4-fold. Generally, cotinine concentrations in brain and blood did not change across the infusion period. Brain/blood ratios indicate an increase in nicotine distribution into brain across days of nicotine infusion. Results demonstrate that both nicotine and its metabolites accumulate in brain following repeated nicotine administration, and indicate that brain nicotine concentration can not be extrapolated from plasma cotinine or nicotine concentrations. Thus, nornicotine accumulation following repeated nicotine administration suggests that this metabolite plays a contributory role in the neuropharmacological effects of nicotine.

Nornicotine, a nicotine metabolite and tobacco alkaloid: desensitization of nicotinic receptor-stimulated dopamine release from rat striatum.

Nornicotine, a major tobacco alkaloid and nicotine metabolite, accumulates in rat brain in pharmacologically relevant concentrations following repeated nicotine administration. Nornicotine-evoked striatal dopamine release is Ca2+-dependent, stereoselective and sensitive to nicotinic receptor antagonists, indicating nicotinic receptor-mediation. The present study determined if S-(-)-nornicotine desensitizes nicotinic receptors and if cross-desensitization to S-(-)-nicotine occurs. S-(-)-Nicotine (10 and 100 nM) diminished [3H]overflow from [3H]dopamine-preloaded rat striatal slices following subsequent superfusion with 10 microM S-(-)-nicotine (46% and 74%, respectively) or 10 microM S-(-)-nornicotine (59% and 81%, respectively). S-(-)-Nornicotine (1 and 10 microM) diminished the response to subsequent superfusion with 10 microM S-(-)-nornicotine (85% and 97%, respectively) or 10 microM S-(-)-nicotine (82% and 88%, respectively). Thus, similar to S-(-)-nicotine, S-(-)-nornicotine desensitizes nicotinic receptors. but with approximately 12-fold lower potency. Cross-desensitization suggests involvement of common nicotinic receptor subtypes. Therefore, S-(-)-nicotine metabolites, such as nornicotine, have neuropharmacologically relevant effects.

Contributory role for nornicotine in nicotine neuropharmacology: nornicotine-evoked [3H]dopamine overflow from rat nucleus accumbens slices.

Nornicotine is a tobacco alkaloid and an active nicotine metabolite, which accumulates in brain to pharmacologically relevant concentrations following repeated nicotine administration to rats. Furthermore, nornicotine is self-administered by rats, indicating that it has reinforcing efficacy and may contribute to nicotine dependence. Since drugs of abuse activate the mesolimbic dopamine (DA) system to produce rewarding effects, the present study tested the hypothesis that nornicotine evokes DA release from nucleus accumbens in a nicotinic receptor-mediated manner. Rat nucleus accumbens slices were preloaded with [3H]DA and superfused for 60 min in the absence and presence of a range of alkaloid concentrations. Superfusate samples were collected and alkaloid-evoked [3H]overflow was determined. S(-)-Nornicotine (EC(50) value = 3.0 microM), R(+)-nornicotine (EC(50) value = 0.48 microM), and S(-)-nicotine (EC(50) value = 70 nM) evoked [3H]overflow in a concentration-dependent manner. For each nornicotine enantiomer, 0.3 microM was the lowest concentration to evoke significant [3H]overflow. Dihydro-beta-erythroidine (DHbetaE, 10 microM), a classical nicotinic receptor antagonist, inhibited the S(-)-nornicotine-evoked [3H]overflow, indicating the involvement of nicotinic receptors. Furthermore, the effect of S(-)-nornicotine was calcium-dependent, consistent with a nicotinic receptor-mediated mechanism. Whereas S(-)-nornicotine was found previously to be more potent in the striatum, R(+)-nornicotine was more potent than its enantiomer in nucleus accumbens, suggesting the involvement of different nicotinic receptor subtypes in these brain regions. Thus, the results of the current study indicate that nornicotine stimulated DA release from nucleus accumbens in a nicotinic receptor-mediated manner, further supporting the hypothesis that nornicotine contributes to tobacco dependence.

Nornicotine pretreatment decreases intravenous nicotine self-administration in rats.

RATIONALE: Nicotine has been shown to be effective as a treatment for reducing tobacco dependence. However, few studies have examined the effect of other nicotinic agonists to determine if they can also decrease nicotine self-administration. OBJECTIVE: The present study determined if nornicotine, a tobacco alkaloid and major nicotine metabolite in brain, could reduce nicotine self-administration in rats. METHODS: Each rat was prepared with an indwelling jugular catheter and trained to self-administer intravenous nicotine (0.03 mg/kg per infusion). After nicotine self-administration stabilized, rats were pretreated with either (-)-nicotine (0, 0.1, 0.3, and 1.0 mg/kg free base) or (+/-)-nornicotine (0, 1, 3, 5.6, and 10.0 mg/kg free base) and assessed for nicotine self-administration. A separate group of rats was maintained on sucrose reinforced responding and pretreated with nornicotine to determine the specificity of the pretreatment effect. In another group of rats, the time course of the pretreatment effect of either (-)-nicotine (0.56 and 1.0 mg/kg) or (+/-)-nornicotine (5.6 and 10.0 mg/kg) was examined. RESULTS: Nicotine and nornicotine each produced a dose-dependent decrease in nicotine self-administration. Furthermore, the decrease in nicotine self-administration in response to the 5.6 mg/kg nornicotine pretreatment was specific to nicotine self-administration, as this dose did not decrease sucrose reinforced responding in tolerant animals. In addition, within the dose range tested, the suppressant effect of nornicotine had a two-fold longer duration than that of nicotine (120 versus 60 min). CONCLUSION: These results suggest that nornicotine may be an effective treatment for tobacco dependence.

Lobeline inhibits nicotine-evoked [(3)H]dopamine overflow from rat striatal slices and nicotine-evoked (86)Rb(+) efflux from thalamic synaptosomes.

The present study evaluated the interaction of lobeline with neuronal nicotinic acetylcholine receptors using two in vitro assays, [(3)H] overflow from [(3)H]dopamine ([(3)H]DA)-preloaded rat striatal slices and (86)Rb(+) efflux from rat thalamic synaptosomes. To assess agonist interactions, the effect of lobeline was determined and compared to S(-)-nicotine. To assess antagonist interactions, the ability of lobeline to inhibit the effect of S(-)-nicotine was determined. Both S(-)-nicotine (0.1-1 microM) and lobeline (>1.0 microM) evoked [(3)H] overflow from superfused [(3)H]DA-preloaded striatal slices. However, lobeline-evoked [(3)H] overflow is mecamylamine-insensitive, indicating that this response is not mediated by nicotinic receptors. Moreover, at concentrations (<1.0 microM) which did not evoke [(3)H] overflow, lobeline inhibited S(-)-nicotine (0.1-10 microM)-evoked [(3)H] overflow, shifting the S(-)-nicotine concentration-response curve to the right. S(-)-Nicotine (30 nM-300 microM) increased (EC(50) value=0.2 microM) (86)Rb(+) efflux from thalamic synaptosomes. In contrast, lobeline (1 nM-10 microM) did not evoke (86)Rb(+) efflux, and the lack of intrinsic activity indicates that lobeline is not an agonist at this nicotinic receptor subtype. Lobeline completely inhibited (IC(50) value=0.7 microM) (86)Rb(+) efflux evoked by 1 microM S(-)-nicotine, a concentration which maximally stimulated (86)Rb(+) efflux. Thus, the results of these in vitro experiments demonstrate that lobeline inhibits the effects of S(-)-nicotine, and suggest that lobeline acts as a nicotinic receptor antagonist.

A simple high performance liquid chromatographic method for the quantification of total cotinine, total 3'-hydroxycotinine and caffeine in the plasma of smokers.

A simple isocratic HPLC procedure has been developed for the quantification of caffeine and the nicotine metabolites cotinine, 3'-hydroxycotinine, cotinine glucuronide and 3'-hydroxycotinine glucuronide in the plasma of smokers. The glucuronide conjugates were determined indirectly via initial basic hydrolysis of the analyte sample followed by quantification of the resulting deconjugation product. Plasma was basified, extracted with dichloromethane, evaporated, the residue dissolved water and an aliquot part was analyzed by HPLC. The method utilized a Partisil-10 SCX cation-exchange column and an isocratic mobile phase of sodium phosphate buffer: methanol (92:8 v/v, 0.1 M, adjusted to pH 4.8 with triethylamine) at a flow rate of 1.5 ml/min. UV detection was at 254 nm. All solutes were separated with good resolution, and quantification was determined using an internal standard of N,N-diethylnicotinamide. The retention times were: caffeine 5.1 min, 3'-hydroxycotinine 7.2 min, N,N-diethylnicotinamide 9.5 min, and cotinine 15.5 min. Detection limits for caffeine, 3'-hydroxycotinine, cotinine, and total cotinine were 10 ng/ml; the detection limit for total 3'-hydroxycotinine was 20 ng/ml. The inter-day and intra-day variations for all analytes were between 1 and 8%. This analytical method is suitable for the determination of caffeine and nicotine metabolite levels in large numbers of clinical samples.

Determination of nicotine N-1-glucuronide, a quaternary N-glucuronide conjugate, in human biological samples.

[Methyl-d3]-N-1-beta-D-glucopyranosyl-(+/-)-nicotinium inner salt ((+/-)-[methyl-d3]nicotine N-1-glucuronide) was synthesized from (+/-)-[methyl-d3]nicotine via reaction with methyl-2,3,4-tri-O-acetyl-1-bromodeoxy-alpha-D-glucopyranouronate, followed by deprotection with 1 M aqueous NaOH and purification by preparative TLC. Nicotine N-glucuronide was identified and determined directly in smokers' urine. A solid phase extraction method was used to partially isolate the material from urine. Subsequent determination was by thermospray-LC/MS using the synthetic d3-labeled nicotine N-glucuronide as internal standard. The identified urinary component had the same retention time as a synthetic standard and gave the same mass spectrum. The thermospray mass spectrum was characterized from the protonated molecular ion (m/z 339) and the protonated aglycone ion (m/z 163). Quantitative results from this direct method were compared with those from an indirect method, which calculated the nicotine glucuronide in the biological sample from the amount of nicotine released following treatment of the sample with the deconjugating enzyme, beta-glucuronidase. On average, the concentration of nicotine N-glucuronide determined by the direct method was 34% greater than that determined by the indirect method. Concentrations of nicotine N-glucuronide in urine ranged from 2.2 to 7.6 nmol/ml with a limit of detection of 1.3 nmol/ml.

Nornicotine is self-administered intravenously by rats.

RATIONALE: Nicotine is a tobacco alkaloid known to be important in the acquisition and maintenance of tobacco smoking. However, other constituents in tobacco may contribute to the dependence liability. OBJECTIVE: The present report sought to determine whether nornicotine, a tobacco alkaloid and metabolite of nicotine, has a reinforcing effect. METHODS: Rats were prepared with a jugular catheter, then were allowed to self-administer intravenously either S(-)-nicotine (0.03 mg/kg/infusion), RS(+/-)-nornicotine (0.3 mg/kg/infusion) or saline using a two-lever operant procedure. The response requirement for each infusion was incremented gradually from a fixed ratio 1 (FR1) to FR5. When responding stabilized on the FR5, other doses of nicotine (0.01 mg/kg/infusion and 0.06 mg/kg/infusion) and nornicotine (0.075, 0.15, and 0.6 mg/kg/infusion) were tested for their ability to control responding. RESULTS: Similar to nicotine, rats self-administered nornicotine significantly above saline control levels. Within the dose ranges tested, both nicotine and nornicotine yielded relatively flat dose-response functions. Extinction of responding was evident when saline was substituted for nornicotine, and responding was reinstated when nornicotine again was available. The rate of nornicotine self-administration was similar between rats tested with either 24-h or 48-h inter-session intervals. CONCLUSION: These results indicate that nornicotine contributes to the dependence liability associated with tobacco use.

Pharmacological similarities between native brain and heterologously expressed alpha4beta2 nicotinic receptors.

1 We studied the pharmacological properties of native rat brain and heterologously expressed rat alpha4beta2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti-alpha4 antibody mAb 299. 2 Immunodepletion with the anti-beta2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained beta2. 3 The association and dissociation rate constants for 30 pM +/-[3H]-epibatidine binding to alpha4beta2 receptors expressed in oocytes were 0.02+/-0.01 and 0.03+/-0.01 min-1 (+/-standard error, degrees of freedom=7 - 8) at 20 - 23 degrees C. 4 The Hill coefficients for +/-[3H]epibatidine binding to the native brain, alpha4beta2 receptors expressed in oocytes, and alpha4beta2 receptors expressed in CV-1 cells (using recombinant adenovirus) were 0.69 - 0.70 suggesting a heterogeneous receptor population. Fits of the +/-[3H]-epibatidine concentration-binding data to a two-site model gave KD s of 8 - 30 and 560 - 1,200 pM. The high-affinity sites comprised 73 - 74% of the native brain and oocyte alpha4beta2 receptor population, 85% of the CV-1 alpha4beta2 receptor population. 5 The expression of rat alpha4beta2 receptors in CV-1 cells using vaccinia viral infection-transfection resulted in a more homogeneous receptor population (Hill coefficient of 1. 0+/-0.2). Fits of the +/-[3H]-epibatidine binding data to a single-site model gave a KD of 40+/-3 pM. 6 DHbetaE (IC50=260-470 nM) and the novel nicotine analogue NDNI (IC50=7-10 microM) inhibited 30 pM+/-[3H]-epibatidine binding to the native brain and heterologously expressed alpha4beta2 receptors equally well. 7 The results show that alpha4beta2-containing nicotinic receptors in the rat brain and heterologously expressed rat alpha4beta2 receptors have similar affinities for +/-[3H]-epibatidine, DHbetaE, and NDNI.

Residence times and half-lives of nicotine metabolites in rat brain after acute peripheral administration of [2'-(14)C]nicotine.

The residence times of nicotine and its metabolites in rat brain after acute peripheral nicotine administration were determined. We hypothesize that nicotine metabolites will reach pharmacologically significant concentrations in brain. Cotinine, nornicotine, and norcotinine were structurally identified by dual label radiochemical and gas chromatography-mass spectrometric analysis as biotransformation products of nicotine present in rat brain after s. c. injection of S(-)-nicotine. Two unidentified minor metabolites were also detected in brain. The half-lives in brain of nicotine metabolites were determined after a single s.c. injection of [2'-(14)C]-(+/-)nicotine (0.8 mg/kg) and analysis of radiolabeled metabolites by high pressure-liquid radiochromatography. The brain half-lives of nicotine, cotinine, and nornicotine were 52, 333, and 166 min, respectively. Peak brain concentrations of nicotine metabolites were 300, 70, and 7 nM for cotinine, nornicotine, and norcotinine, respectively. Even with potential accumulation of cotinine in brain after chronic nicotine administration, it is likely that the brain concentration of cotinine will be insufficient to produce neuropharmacological effects resulting from activation of nicotinic receptors to induce dopamine release. Conversely, the concentration of nornicotine in brain after acute nicotine approaches the range found to be neuropharmacologically active. It is likely that nornicotine will accumulate in brain on chronic nicotine administration based on the brain half-life of this metabolite. Importantly, nornicotine is also a major alkaloidal component of tobacco. Thus, as a consequence of tobacco use, alkaloidal and metabolically formed nornicotine may reach concentrations in brain sufficient to produce pharmacological effects.

Acute and chronic effects of nornicotine on locomotor activity in rats: altered response to nicotine.

RATIONALE: Nicotine, a tobacco alkaloid, is known to be important in the acquisition and maintenance of tobacco smoking. Nornicotine, an active nicotine metabolite, stimulates nicotinic receptors and may produce psychomotor effects similar to nicotine. OBJECTIVE: The present study determined the effects of acute and repeated administration of nornicotine on locomotor activity and compared its effects with those of nicotine. METHODS: R(+)-Nornicotine (0.3-10 mg/kg), S(-)-nornicotine (0.3-10 mg/kg), S(-)-nicotine (0.1-1 mg/kg) or saline was administered s.c. to rats acutely or repeatedly (eight injections at 48-h intervals). Activity was recorded for 50 min immediately after each injection. RESULTS: S(-)-Nicotine produced transient hypoactivity, followed by dose-related hyperactivity. Repeated S(-)-nicotine administration resulted in tolerance to the hypoactivity and sensitization to the hyperactivity. Subsequent testing following a saline injection revealed evidence of conditioned hyperactivity. Acute administration of 0.3 mg/kg or 1 mg/kg R(+)- or S(-)-nornicotine produced no effect. Transient hypoactivity was observed at 3 mg/kg and 10 mg/kg R(+)-nornicotine and at 10 mg/kg S(-)-nornicotine. However, rebound hyperactivity was not observed following acute administration of either nornicotine enantiomer, suggesting that nornicotine-induced psychomotor effects differ qualitatively from those of S(-)-nicotine. Repeated R(+)-nornicotine resulted in tolerance to the transient hypoactivity, however hyperactivity was not observed. Repeated S(-)-nornicotine resulted in tolerance to the hypoactivity and the appearance of hyperactivity. Repeated administration of either nornicotine enantiomer resulted in a dose-dependent alteration in response to a 1 mg/kg S(-)-nicotine challenge, suggesting some commonalities in the mechanism of action. CONCLUSION: Nornicotine likely contributes to the neuropharmacological effects of nicotine and tobacco use.