A protocadherin gene cluster regulatory variant is associated with nicotine withdrawal and the urge to smoke.

Nicotine withdrawal symptoms contribute to relapse in smokers, thereby prolonging the harm caused by smoking. To investigate the molecular basis for this phenomenon, we conducted a genome-wide association study of DSM-IV nicotine withdrawal in a sample of African American (AA) and European American (EA) smokers. A combined AA and EA meta-analysis (n=8021) identified three highly correlated single nucleotide polymorphisms (SNPs) in the protocadherin (PCDH)-α, -ß and -γ gene cluster on chromosome 5 that were associated with nicotine withdrawal (P<5 × 10-8). We then studied one of the SNPs, rs31746, in an independent sample of smokers who participated in an intravenous nicotine infusion study that followed overnight smoking abstinence. After nicotine infusion, abstinent smokers with the withdrawal risk allele experienced greater alleviation of their urges to smoke, as assessed by the Brief Questionnaire on Smoking Urges (BQSU). Prior work has shown that the PCDH-α, -ß and -γ genes are expressed in neurons in a highly organized manner. We found that rs31746 mapped to a long-range neuron-specific enhancer element shown previously to regulate PCDH-α, -ß and -γ gene expression. Using Braincloud mRNA expression data, we identified a robust and specific association between rs31746 and PCDH-ß8 mRNA expression in frontal cortex tissue (P<1 × 10-5). We conclude that PCDH-α, -ß and -γ gene cluster regulatory variation influences the severity of nicotine withdrawal. Further studies on the PCDH-α, -ß and -γ genes and their role in nicotine withdrawal may inform the development of novel smoking cessation treatments and reduce the harm caused by tobacco smoking.

FKBP5 variation is associated with the acute and chronic effects of nicotine.

Stress and hormones released in response to stress influence the effects of nicotine and the severity of nicotine withdrawal. Here, we systematically examine the contribution of a stress response gene, FKBP5, to the acute and chronic behavioral effects of nicotine in smokers. Subjects were European- and African-American (EA and AA) heavy smokers who participated in an intravenous (IV) nicotine administration study (total n=169). FKBP5 rs3800373 genotype was analyzed for association to several outcomes, including nicotine withdrawal and the acute subjective, heart rate (HR), blood pressure and plasma cortisol responses to IV nicotine. Nicotine withdrawal was also examined in relation to rs3800373 allele frequencies in an independent cohort of EA and AA current smokers (n=3821). For a subset of laboratory subjects FKBP5 mRNA (n=48) expression was explored for an association to the same outcomes. The rs3800373 minor allele was associated with less severe nicotine withdrawal in laboratory subjects and the independent cohort of smokers. The rs3800373 minor allele was also associated with lower subjective ratings of negative drug effects in response to IV nicotine. Low FKBP5 mRNA expression was associated lower cortisol levels, lower subjective ratings of negative drug effects and a blunted HR response to nicotine. Stress hormone regulation via FKBP5 warrants further investigation as a potential contributor to the effects of nicotine withdrawal, which occurs commonly, and has an important role in the maintenance of smoking behavior and relapse following a quit attempt.

COMT Val158Met modulates subjective responses to intravenous nicotine and cognitive performance in abstinent smokers.

The catechol-O-methyltransferase (COMT) Val158Met polymorphism may be a risk factor for nicotine addiction. This study examined the influence of the COMT Val158Met polymorphism on subjective, physiological and cognitive effects of intravenous (IV) nicotine use in African Americans (AAs; n=56) and European Americans (EAs; n=68) smokers. Overnight abstinent smokers received saline followed by 0.5 and 1.0 mg per 70 kg doses of nicotine, administered 30 min apart. Smokers with valine (Val)/Val genotype, compared with methionine (Met) carriers, had greater negative subjective effects from IV nicotine and had more severe withdrawal severity following overnight abstinence from smoking. Women with Val/Val genotype reported greater difficulty concentrating and irritability than men with Val/Val or Met carrier genotypes. The Val/Val genotype was associated with better performance on the math task and in AA smokers it was associated with greater systolic blood pressure. These results support the rationale of pharmacologically inhibiting COMT to aid with smoking cessation among Val/Val genotype smokers.

Serotonergic synergism: the risks and benefits of combining the selective serotonin reuptake inhibitors with other serotonergic drugs.

It has become common clinical practice to combine the selective serotonin reuptake inhibitors with other serotonergic agents for augmentation or adjunctive purposes. The empirical basis for using these combinations remains limited, but is growing. Also growing is a literature that suggests that even the most apparently benign combinations of serotonergic drugs carry at least some risk of serious pharmacokinetic or pharmacodynamic drug interactions, such as a serotonin syndrome.

Effects of cotinine on cigarette self-administration.

Previous studies have shown that cotinine, a metabolite of nicotine, antagonizes some of the effects of nicotine. One study showed that cotinine eliminates the beneficial effects of the nicotine patch in reducing cigarette withdrawal symptoms. The purpose of this study was to examine the effects of various doses of cotinine on cigarette self-administration. Subjects were randomly assigned to one of three doses of cotinine fumarate (40, 80 and 160 mg) and placebo, each for a period of 10 days, in a randomized order. Outcome variables included measures of nicotine intake and subjective responses to smoked cigarettes. Results showed no differences in the number of cigarettes smoked, carbon monoxide levels, and weights of cigarette butts across the various doses of cotinine and placebo. However, higher nicotine serum levels were observed in the 160 mg cotinine fumarate condition compared to placebo and to 40 mg cotinine fumarate. No systematic effects of cotinine on subjective responses to cigarettes were observed. Cotinine appears potentially to have a selective modulatory effect on nicotine withdrawal symptoms but not on cigarette smoking.

Glutamate, without GABA antagonists, induces synchronized discharges in intact hippocampus via NMDA receptors.

In rats under urethane anesthesia, iontophoresis of high amounts of glutamate (50-150 nA) in hippocampus caused repetitive field potentials. These synchronized discharges were best recorded in the proximal part of stratum radiatum as positive waves of 10-15 ms duration and of 0.5-5 mV amplitude. A tetrodotoxin-sensitive faster component of 2-5 ms duration was frequently superimposed on the peaks of the positive waves and was followed by a negative wave of 1-6 mV and 20-30 ms. Glutamate-evoked discharges were suppressed by iontophoresis of N-methyl-D-aspartate (NMDA) antagonists, MK-801, Mg2+ and ketamine and also by ketamine injection (i.v. 5-10 mg/kg). The population spikes evoked by fimbrial stimulation were not facilitated by glutamate and the synchronized discharges were suppressed for up to 300 ms following the stimulation, suggesting the presence of an efficient inhibition during glutamate-induced synchronized activity. Glutamate also had no effect on paired-pulse inhibition. No synchronized discharges were recorded with a second electrode separated more than 150 microns from the iontophoretic electrode, suggesting that the activity was local. These data demonstrate that high amounts of glutamate evoke synchronized discharges in hippocampus, possibly through activation of NMDA receptors. The model presented may be utilized to study the mechanisms of synchronization without disinhibition.

Maintenance of acute morphine tolerance in mice by selective blockage of kappa opioid receptors with norbinaltorphimine.

In this study we investigated the effect of the highly selective kappa opioid antagonist, norbinaltorphimine (norBNI) on the development of tolerance to a single dose of morphine. Mice were pretreated with 100 mg/kg of morphine sulfate (morphine), s.c. and 2 h later, norBNI (20 mg/kg s.c.) was administered and various times after this pretreatment, antinociceptive ED50 value of morphine was determined in the tail-flick assay. Twenty-four and 72 h after morphine injection, ED50 values of morphine were significantly increased by about 2.5-fold from those of their control mice that received saline instead of the tolerance-inducing dose of morphine. In a second set of experiments, animals were pretreated similarly with morphine and norBNI and 72 h after morphine injection, various opioid agonists were applied by the i.c.v. or i.t. route to see whether or not any cross-tolerance had developed to these agonists. The ED50 of i.c.v.-administered morphine was significantly greater than that of the non-pretreated controls. A small degree of cross-tolerance was observed with U-50,488H but not with DPDPE [D-Pen2,D-Pen5]enkephalin (DPDPE) at the supraspinal site. At the spinal site, tolerance to morphine was not observed. These results suggest that antagonism at kappa opioid sites after morphine administration, modulates positively the development of opioid tolerance.

delta-Opioid receptor binding in mouse brain: evidence for heterogeneous binding sites.

In this study we investigated the characteristics of binding sites with which delta opioid receptor agonists interact in homogenates of mouse brain using Krebs-HEPES medium. [3H][D- Ser2,Leu5,Thr6]enkephalin (DSLET), [3H][D-Ala2,D-Leu5]enkephalin (DADLE) and [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) were used to label delta opioid binding sites. The analyses of the saturation binding data of these ligands (Scatchard plots) gave best fits to single rather than multiple site models. The binding capacity (Bmax) labelled by [3H]DSLET was found to be significantly greater than those of [3H]DADLE and [3H]DPDPE in brains of mice. Naltriben (the benzofuran analogue of naltrindole) was equally effective in competing for [3H]DSLET, [3H]DPDPE and [3H]DADLE binding sites. On the other hand, DADLE was significantly more potent in competing for [3H]DADLE and [3H]DPDPE binding sites than for [3H]DSLET binding sites. Also, DPDPE was more potent in competing for the binding sites of [3H]DADLE and [3H]DPDPE than for those of [3H]DSLET. DSLET was found to be equipotent in competing for [3H]DSLET, [3H]DPDPE and [3H]DADLE binding sites. These results suggest a heterogeneity of delta opioid receptors which may be explained possibly by the existence of delta opioid receptor subtypes.

Depressive symptoms modulate the subjective and physiological response to cocaine in humans.

The goal of this study was to examine the relationship between the presence of subclinical depressive symptoms and physiological and subjective responses to smoked cocaine in humans. Cocaine users without major depression, who participated in various inpatient studies, received a single 0.4 mg/kg of smoked cocaine. When the relationship between the Beck Depression Inventory (BDI) scores and various subjective and physiological responses to cocaine was examined, similar trends were found. Low BDI scores of 0-7 were associated with a smaller physiological and subjective cocaine response. In contrast, BDI ranges of 8-13 were associated with enhanced cocaine response which plateaued or declined in the higher (> 14) BDI group. These group differences were not explained by sex or body weight differences among groups. The implication of these results is that the presence of depressive symptoms may affect cocaine use behavior partly by being associated with an enhanced response to cocaine.

Carvedilol affects the physiological and behavioral response to smoked cocaine in humans.

The noradrenergic system is implicated in mediating some of the physiological effects of cocaine. The purpose of this study was to investigate whether treatment with an adrenergic blocker, carvedilol, which would be expected to attenuate the physiological effects of cocaine, would also attenuate the subjective and behavioral response to cocaine in humans. Twelve crack cocaine users participated in this double-blind, placebo-controlled outpatient study. Acute treatment with 50 mg of oral carvedilol attenuated the smoked cocaine-induced increases in heart rate, systolic and diastolic blood pressure. The number of cocaine self-administrations was lower under 25 mg carvedilol treatment condition compared with 50 mg carvedilol or placebo treatment conditions. The subjective responses to smoked cocaine deliveries were not affected by carvedilol treatment. These results suggest that acute treatment with carvedilol attenuates the physiological effects of smoked cocaine. The effects of carvedilol on cocaine self-administration need to be studied further.

Predictors of cardiovascular response to smoked cocaine in humans.

The purpose of this study was to examine the predictors of heart rate and blood pressure changes following cocaine administration. Sixty-two smoked cocaine users received a single 0.4 mg/kg dose of smoked cocaine. Male sex, African American race, higher body weight and current marijuana use predicted a greater cardiovascular response to cocaine. In contrast, higher baseline blood pressure, heart rate, amount and frequency of current cocaine use and presence of current cocaine snorting predicted a diminished cardiovascular response to cocaine. Whether these predictors of the cardiovascular response to smoked cocaine in the laboratory also predict cardiovascular complications from long-term cocaine use needs to be studied further.

Effects of phenytoin on cocaine self-administration in humans.

The goal of this pilot study was to determine the effects of phenytoin on cocaine self-administration in a human laboratory model. Subjects were randomized to either phenytoin (n = 6) or placebo (n = 7). Those assigned to phenytoin treatment received a single oral loading dose of 20 mg/kg. The phenytoin and placebo treatment groups did not differ in the number of tokens valued at $5, exchanged for cocaine. Similarly, the cardiovascular and subjective response to cocaine administration did not show a statistically significant treatment effect. In this laboratory model, phenytoin did not alter either the self-administration or effects of cocaine.

Cross-tolerance studies in the spinal cord of beta-FNA-treated mice provides further evidence for delta opioid receptor subtypes.

In this study we investigated the development of cross-tolerance among intrathecally (i.t.)- administered mu and delta opioid receptor selective peptides in beta-funaltrexamine (beta-FNA)-treated mice. Tolerance to the antinociceptive effect of i.t. administered DPDPE was accomplished by administration of 16 nmol/mouse of DPDPE, i.t. 3 hr before testing in beta-FNA-treated mice (10 mumol/kg, s.c., 24 hr before the experiment). Cross-tolerance developed to the antinociceptive effect of i.t. administered DADLE but not to those of DSLET or DAMGO. DSLET (0.1 nmol/mouse i.t.) administration in beta-FNA-treated mice resulted in tolerance development to its antinociceptive effect. The same pretreatment resulted in a marginally significant increase in the antinociceptive ED50 value of DPDPE. There was no cross-tolerance to the antinociceptive effect of i.t. administered DADLE or DAMGO. These results provide further evidence for the existence of delta opioid receptor subtypes where DADLE and DPDPE interact with one site and DSLET with a different one.

7-Benzylidenenaltrexone (BNTX): a selective delta 1 opioid receptor antagonist in the mouse spinal cord.

Recent reports provided evidence that at least two delta opioid receptors may mediate antinociception in mice. In this study, we studied further the involvement of delta opioid receptor subtypes in mediating antinociception at spinal sites in mice using subtype selective agonists and antagonists. The antinociceptive ED50 values (95% C.I.) of i.t. administered DPDPE [(D-Pen2, D-Pen5)enkephalin] (delta 1 receptor agonist) and DELT II [(D-Ala2)deltorphin II] (delta 2 receptor agonist) were 6.3 (5.2-7.6) and 6.4 (5.4-7.7) nmol/mouse, respectively. Administration of BNTX, s.c. increased the antinociceptive ED50 value of DPDPE 5.9-fold whereas that of DELT II was not changed significantly. On the other hand administration of naltriben (NTB, the benzofuran derivative of naltrindole), s.c. increased the antinociceptive ED50 value of DELT II 12.5-fold but did not alter that of DPDPE. Similarly administration of BNTX, i.t. increased the antinociceptive ED50 value of DPDPE 4-fold without altering significantly that of DELT II. NTB given i.t. enlarged the antinociceptive ED50 of DELT II 11-fold without affecting significantly that of DPDPE. BNTX, s.c. did not alter the antinociceptive ED50 values of the mu-agonists, DAMGO [(D-Ala2,MePhe4,Gly-ol5) enkephalin] and morphine or that of the kappa-agonist, U50,488H [trans(+/-)-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl-cyclohexyl]benzeneacetamide] These results demonstrate that BNTX is highly selective for delta 1 opioid receptors at spinal sites. Also, the present data provide for the involvement of both delta 1 and delta 2 opioid receptors in mediating antinociception at spinal sites in mice.

Progesterone treatment during the early follicular phase of the menstrual cycle: effects on smoking behavior in women.

The goals of this study were (1) to examine the feasibility of administering progesterone to women during the early follicular phase when the endogenous estradiol and progesterone levels are low, and (2) to investigate the effects of oral progesterone treatment on smoking behavior in female smokers. Twelve subjects had two experimental sessions, within 3-9 days after the beginning of their menses. In each experimental session, subjects received a single 200-mg dose of progesterone or placebo, orally. Two and a half hours after the medication treatment, subjects were assessed for subjective response to two puffs of a cigarette and then started the self-administration period in which they had the option to exchange their token for two puffs of cigarette, 15 min apart. Subjects had low levels of estradiol and progesterone before the first and second sessions. Plasma progesterone levels peaked in 2 h following progesterone treatment. Progesterone treatment attenuated the craving for and subjective effects from smoking. Under progesterone treatment, there was a trend for decreased smoking behavior. These preliminary results suggest that the early follicular phase of the menstrual cycle may be a useful interval to investigate the effects of exogenous progesterone in female smokers. The effects of progesterone on nicotine dependence need to be studied further.

Intravenous cocaine increases plasma epinephrine and norepinephrine in humans.

Cocaine has been shown to activate the sympathoadrenal system in both animal and human studies. Controlled human studies have found inconclusive results regarding whether acute cocaine treatment elevates plasma epinephrine and norepinephrine concentrations. The purpose of this study was to investigate whether commonly abused doses of cocaine increase plasma epinephrine and norepinephrine concentrations in humans, in a double-blind, placebo-controlled study. Five male cocaine users were given an intravenous injection of 0.46 mg/kg dose of cocaine or placebo, on two consecutive days. Plasma epinephrine and norepinephrine concentrations were significantly increased in response to cocaine injection compared to placebo. Peak plasma epinephrine and norepinephrine concentrations were reached 3 and 12 min after cocaine injection, respectively. While changes in epinephrine levels following cocaine were correlated with systolic blood pressure and heart rate changes, changes in plasma norepinephrine were correlated with diastolic blood pressure and heart rate changes following cocaine administration. These results suggest that plasma epinephrine and norepinephrine can be used as a measure for cocaine induced sympathoadrenal system activation.

Effects of labetalol treatment on the physiological and subjective response to smoked cocaine.

Adrenergic receptors mediate some of the physiological and possibly behavioral effects of cocaine. The purpose of this study was to investigate the effect of treatment with a peripherally acting adrenergic blocking drug labetalol on the cardiovascular and subjective response to repeated deliveries of smoked cocaine. In this double-blind, placebo-controlled, crossover study, 12 cocaine users were treated with a single 100 or 200 mg dose of labetalol, or placebo in each of three experimental sessions. Starting 2 h after the medication treatment, subjects received three doses of 0.4 mg/kg smoked cocaine, 30 min apart. Labetalol treatment significantly attenuated the cocaine-induced increases in heart rate and systolic blood pressure. This effect of labetalol on the cardiovascular response did not decrease with repeated cocaine deliveries. The subjective response to smoked cocaine deliveries was not affected by labetalol treatment. These results suggest that labetalol effectively attenuates the systolic blood pressure and heart rate increases induced by repeated doses of smoked cocaine, but does not alter subjective effects.

Sex and menstrual cycle differences in the subjective effects from smoked cocaine in humans.

To investigate sex and menstrual cycle effects in response to cocaine administration, data from existing studies were analyzed. First, responses to a single delivery of 0.4 mg/kg smoked cocaine were investigated. Women reported lower ratings for measures of paranoid/suspicious and heart racing/pounding than did men. In addition, women in the luteal phase reported diminished ratings for a measure of feel high than did both women in the follicular phase of the menstrual cycle and men. Second, responses to up to 6 deliveries of 0.4 mg/kg smoked cocaine were investigated. Women, compared with men, had lower ratings on feel high, heart racing/pounding, and feel stimulated. Results suggest that there are significant sex and menstrual phase differences in the subjective effects of cocaine.

Characteristics of research volunteers for inpatient cocaine studies: focus on selection bias.

In order to investigate the selection bias of subjects for inpatient human cocaine studies, characteristics of 859 potential subjects were examined. Excluded subjects compared with accepted group were more likely to be single and male, currently use drugs other than cocaine, have a history of intravenous cocaine use, and have medical or mental health problems or physical complaints. Subjects who were accepted but did not participate, compared with participants, were likely to spend more money on cocaine. These results suggest that potential subjects who were accepted to our research studies may not accurately represent all potential subjects for several important subject characteristics.