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The role of carbonyl reductases in biotransformation of bupropion and 4-methylnitrozamino-1-(3-pyridyl)-1-butanone by human placenta
Abstracts / Drug and Alcohol Dependence 156 (2015) e102–e182
The role of carbonyl reductases in biotransformation of bupropion and 4-methylnitrozamino-1-(3-pyridyl)-1-butanone by human placenta Tatiana Nanovskaya 1 , Valentina M. Fokina 1,2 , X. Wang 1 , Cheryl Oncken 3 , Mahmoud S. Ahmed 1 , Gary Hankins 1 1
OB&GYN, UTMB, Galveston, TX, United States Pharmacology & Toxicology, UTMB, Galveston, TX, United States 3 Medicine, University of Connecticut Health Center, Farmington, TX, United States 2
Aims: Bupropion sustained release (BUP SR) is being evaluated as an aid for smoking cessation during pregnancy. The predominant metabolic pathway of BUP in placenta is reduction to erythro- (EB) and threohydrobupropion (TB), the major placental metabolite of BUP; the reaction is catalyzed primarily by 11hydroxisteroid dehydrogenase (11-HSD) and aldoketoreductases. Maternal cigarette smoking has been associated with prenatal exposure to NNK, which is the most abundant and potent carcinogen of cigarette smoke. One of the metabolic pathways of NNK is carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)1-butanol (NNAL) that initiates NNK detoxification. The goal of the current investigation is to determine placental metabolism of NNK and to identify placental enzymes responsible for its reduction. Methods: Term placentas were collected from women who smoked during pregnancy and from non-smokers. NNK metabolism was determined in vitro using placental microsomal and cytosolic subcellular fractions; the formation of NNAL was quantified by HPLC-UV. Results: The apparent Km and Vmax values for the reaction in placental subcellular fractions were determined. The formation of NNAL in placentas of heavy smokers was lower than in placentas of non-smokers: 12.1 ± 3.5 vs. 17 ± 6.3 nmol mg P−1 for cytosolic fraction (p < 0.05) and 21 ± 2.7 vs. 23.6 ± 4.6 nmol mg P−1 for microsomes. By contrast, the formation of TB in placentas of smokers, 447.9 ± 260 pmol mg P−1 , was higher than in placentas of nonsmokers (300.6 ± 102.3 pmol mg P−1 ), p < 0.05. Conclusions: These data suggest that reductive metabolisms of NNK and BUP in placenta are catalyzed by different carbonyl reductases. The identification of placental enzymes catalyzing NNK reduction is currently under investigation. Financial support: Supported by NIDA grant DA030998 to TN. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.438 Self-administration of second-generation synthetic cathinones in rats Jennifer Naylor 1 , Edward Townsend 1 , Kevin Freeman 1 , James Rowlett 2 , Bruce E. Blough 3 , Sally L. Huskinson 1 1 Psychiatry and Human Behavior, The University of Mississippi Medical Center, Jackson, MS, United States 2 University of Mississippi Medical Center, Jackson, MS, United States 3 Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, NC, United States
Aims: The commercial distribution and abuse of synthetic cathinone compounds, commonly referred to as ‘bath salts’, has increased at an alarming rate in recent years. New structural variants of these compounds, referred to as second-generation
e161
synthetic cathinones, have been reported in confiscated samples. The DEA has temporarily scheduled ten of these compounds into schedule I. The current study characterized three secondgeneration cathinones as reinforcers and compared them to the prototypical stimulant methamphetamine. Methods: Male, Sprague-Dawley rats (n = 6–8/group) were implanted with intravenous jugular catheters and trained to self-administer methamphetamine (0.05 mg/kg/inj) under a 5-response, fixed-ratio schedule of i.v. drug injection. Once self-administration was stable, synthetic cathinone compounds, alpha-pyrrolidi-nopentiophenone (alpha-PVP), 4-methyl-alpha-pyrrolidinopropiophenone (4-MePPP), and (4-methyl-N-ethylcathinone) 4-MEC were made available for self-administration in separate groups of animals. Results: All three synthetic cathinones functioned as reinforcers. There was individual-subject variability in the doses that maintained behavior above saline levels. At least one dose within the following dose ranges functioned as a reinforcer: methamphetamine (0.012–0.1 mg/kg/inj), alpha-PVP (0.003–0.1 mg/kg/inj), 4-MEC (0.4–3.2 mg/kg/inj), and 4-MePPP (0.2–3.2 mg/kg/inj). Rank order of potency the drugs’ reinforcing effects was alpha-PVP > methamphetamine > 4-MePPP > 4-MEC. Conclusions: The current findings demonstrated that three second-generation synthetic cathinones functioned as reinforcers in rats, providing some justification for permanent scheduling of these drugs by regulatory agencies. Future research in our laboratory will assess the reinforcing effectiveness of these three compounds with a behavioral economics approach. Financial support: This research was supported by the UMMC Drug Abuse Development Fund and R01 DA12970 to BB. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.439 Serotonin 5-HT2C receptors in the ventral subiculum regulate cocaine-evoked hyperactivity in rats Harshini Neelakantan 1 , Sonja J. Stutz 1 , Marcy J. Bubar 1 , Robert M. Sears 3 , Ralph J. DiLeone 3 , Kathryn A. Cunningham 1,2 1
Center for Addiction Research, University of Texas Medical Branch, Galveston, TX, United States 2 Department of Pharmacy, University of Texas Medical Branch, Galveston, TX, United States 3 Department of Psychiatry, Yale University, New Haven, CT, United States Aims: The ventral subiculum (vSUB) interconnects with limbiccorticostriatal circuit which regulates the behavioral effects of cocaine. Serotonin neurons innervate the vSUB, however, little is known about its role or the 5-HT receptors that are functionally relevant in this region. We investigated the vSUB localization of the 5-HT2C receptor (5-HT2C R) and its role in the control of cocaineevoked hyperactivity. Methods: The expression of the 5-HT2C R and co-localization with GAD 67 (marker for GABA) in the vSUB were evaluated using immunohistochemical analyses in rats (n = 3). We employed virally mediated genetic knockdown strategy to test the hypothesis that knockdown of the 5-HT2C R in the vSUB of rats (n = 5) will modulate cocaine-evoked hyperactivity. Motor activity was assessed after saline or cocaine (10–20 mg/kg) in modified open-field chambers and analyzed using repeated measures two-way ANOVA. Results: Dense immunoreactivity (IR) for 5-HT2C R was observed throughout the vSUB. Only a small proportion of 5-HT2C R-IR was co-localized with GAD 67-IR that was restricted to the distal end
The role of carbonyl reductases in biotransformation of bupropion and 4-methylnitrozamino-1-(3-pyridyl)-1-butanone by human placenta Tatiana Nanovskaya 1 , Valentina M. Fokina 1,2 , X. Wang 1 , Cheryl Oncken 3 , Mahmoud S. Ahmed 1 , Gary Hankins 1 1
OB&GYN, UTMB, Galveston, TX, United States Pharmacology & Toxicology, UTMB, Galveston, TX, United States 3 Medicine, University of Connecticut Health Center, Farmington, TX, United States 2
Aims: Bupropion sustained release (BUP SR) is being evaluated as an aid for smoking cessation during pregnancy. The predominant metabolic pathway of BUP in placenta is reduction to erythro- (EB) and threohydrobupropion (TB), the major placental metabolite of BUP; the reaction is catalyzed primarily by 11hydroxisteroid dehydrogenase (11-HSD) and aldoketoreductases. Maternal cigarette smoking has been associated with prenatal exposure to NNK, which is the most abundant and potent carcinogen of cigarette smoke. One of the metabolic pathways of NNK is carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)1-butanol (NNAL) that initiates NNK detoxification. The goal of the current investigation is to determine placental metabolism of NNK and to identify placental enzymes responsible for its reduction. Methods: Term placentas were collected from women who smoked during pregnancy and from non-smokers. NNK metabolism was determined in vitro using placental microsomal and cytosolic subcellular fractions; the formation of NNAL was quantified by HPLC-UV. Results: The apparent Km and Vmax values for the reaction in placental subcellular fractions were determined. The formation of NNAL in placentas of heavy smokers was lower than in placentas of non-smokers: 12.1 ± 3.5 vs. 17 ± 6.3 nmol mg P−1 for cytosolic fraction (p < 0.05) and 21 ± 2.7 vs. 23.6 ± 4.6 nmol mg P−1 for microsomes. By contrast, the formation of TB in placentas of smokers, 447.9 ± 260 pmol mg P−1 , was higher than in placentas of nonsmokers (300.6 ± 102.3 pmol mg P−1 ), p < 0.05. Conclusions: These data suggest that reductive metabolisms of NNK and BUP in placenta are catalyzed by different carbonyl reductases. The identification of placental enzymes catalyzing NNK reduction is currently under investigation. Financial support: Supported by NIDA grant DA030998 to TN. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.438 Self-administration of second-generation synthetic cathinones in rats Jennifer Naylor 1 , Edward Townsend 1 , Kevin Freeman 1 , James Rowlett 2 , Bruce E. Blough 3 , Sally L. Huskinson 1 1 Psychiatry and Human Behavior, The University of Mississippi Medical Center, Jackson, MS, United States 2 University of Mississippi Medical Center, Jackson, MS, United States 3 Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, NC, United States
Aims: The commercial distribution and abuse of synthetic cathinone compounds, commonly referred to as ‘bath salts’, has increased at an alarming rate in recent years. New structural variants of these compounds, referred to as second-generation
e161
synthetic cathinones, have been reported in confiscated samples. The DEA has temporarily scheduled ten of these compounds into schedule I. The current study characterized three secondgeneration cathinones as reinforcers and compared them to the prototypical stimulant methamphetamine. Methods: Male, Sprague-Dawley rats (n = 6–8/group) were implanted with intravenous jugular catheters and trained to self-administer methamphetamine (0.05 mg/kg/inj) under a 5-response, fixed-ratio schedule of i.v. drug injection. Once self-administration was stable, synthetic cathinone compounds, alpha-pyrrolidi-nopentiophenone (alpha-PVP), 4-methyl-alpha-pyrrolidinopropiophenone (4-MePPP), and (4-methyl-N-ethylcathinone) 4-MEC were made available for self-administration in separate groups of animals. Results: All three synthetic cathinones functioned as reinforcers. There was individual-subject variability in the doses that maintained behavior above saline levels. At least one dose within the following dose ranges functioned as a reinforcer: methamphetamine (0.012–0.1 mg/kg/inj), alpha-PVP (0.003–0.1 mg/kg/inj), 4-MEC (0.4–3.2 mg/kg/inj), and 4-MePPP (0.2–3.2 mg/kg/inj). Rank order of potency the drugs’ reinforcing effects was alpha-PVP > methamphetamine > 4-MePPP > 4-MEC. Conclusions: The current findings demonstrated that three second-generation synthetic cathinones functioned as reinforcers in rats, providing some justification for permanent scheduling of these drugs by regulatory agencies. Future research in our laboratory will assess the reinforcing effectiveness of these three compounds with a behavioral economics approach. Financial support: This research was supported by the UMMC Drug Abuse Development Fund and R01 DA12970 to BB. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.439 Serotonin 5-HT2C receptors in the ventral subiculum regulate cocaine-evoked hyperactivity in rats Harshini Neelakantan 1 , Sonja J. Stutz 1 , Marcy J. Bubar 1 , Robert M. Sears 3 , Ralph J. DiLeone 3 , Kathryn A. Cunningham 1,2 1
Center for Addiction Research, University of Texas Medical Branch, Galveston, TX, United States 2 Department of Pharmacy, University of Texas Medical Branch, Galveston, TX, United States 3 Department of Psychiatry, Yale University, New Haven, CT, United States Aims: The ventral subiculum (vSUB) interconnects with limbiccorticostriatal circuit which regulates the behavioral effects of cocaine. Serotonin neurons innervate the vSUB, however, little is known about its role or the 5-HT receptors that are functionally relevant in this region. We investigated the vSUB localization of the 5-HT2C receptor (5-HT2C R) and its role in the control of cocaineevoked hyperactivity. Methods: The expression of the 5-HT2C R and co-localization with GAD 67 (marker for GABA) in the vSUB were evaluated using immunohistochemical analyses in rats (n = 3). We employed virally mediated genetic knockdown strategy to test the hypothesis that knockdown of the 5-HT2C R in the vSUB of rats (n = 5) will modulate cocaine-evoked hyperactivity. Motor activity was assessed after saline or cocaine (10–20 mg/kg) in modified open-field chambers and analyzed using repeated measures two-way ANOVA. Results: Dense immunoreactivity (IR) for 5-HT2C R was observed throughout the vSUB. Only a small proportion of 5-HT2C R-IR was co-localized with GAD 67-IR that was restricted to the distal end