- Email: [email protected]
Gene transfer of engineered cocaine hydrolase: Lifetime expression with no immune response
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Abstracts / Drug and Alcohol Dependence 140 (2014) e2–e85
negative urines, reduction in opioid craving, improvement in mental health functional quality of life, and no new safety concerns. Financial support: Funded by Alkermes, Inc. Drs. Gastfriend, Memisoglu and Silverman are employees of Alkermes, Inc. Dr. Earley is a paid consultant to Alkermes. Injectable extended-release naltrexone (Vivitrol® ) was developed with support from National Institute on Drug Abuse Grant R43DA013531 and National Institute on Alcohol Abuse and Alcoholism Grant N43AA001002. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.203 Behavioral effects of synthetic cannabinoids marketed as “spice” Michael B. Gatch, M. Forster Pharmacology & Neuroscience, UNT Health Science Center, Fort Worth, TX, United States Aims: A number of cannabinoid compounds are being sold as “legal” alternatives to marijuana in the form of incense. As these marginally legal compounds become controlled, suppliers move to other, unregulated compounds. The purpose of these experiments was to determine whether some common synthetic cannabinoids have discriminative stimulus effects similar to 9tetrahydrocannabinol, which is thought to be the main active component in marijuana. Methods: The compounds JWH-203, JWH-250, and AM2201 were tested for locomotor stimulant effects in mice and subsequently for substitution in rats trained to discriminate 9tetrahydrocannabinol (3 mg/kg, i.p.). Results: JWH-203, JWH-250, and AM2201 each decreased locomotor activity for up to 90 min. JWH-203, JWH-250, and AM2201 each fully substituted for the discriminative stimulus effects of 9tetrahydrocannabinol at doses that did not alter rate of responding. Conclusions: JWH-203, JWH-250, and AM2201 each decreased locomotor activity for up to 90 min. JWH-203, JWH-250, and AM2201 each fully substituted for the discriminative stimulus effects of 9-tetrahydrocannabinol at doses that did not alter rate of responding. Financial support: Supported by NIH N01DA-7-8872. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.204 Trauma exposure and PTSD is associated with attenuation in fronto-limbic functional connectivity among cocaine users Michael J. Gawrysiak 1,2 , Jesse Suh 1,2 , Y. Li 2 , K. Jagannathan 2 , R. Fabianski 2 , Anna Rose Childress 2 1 Philadelphia VA Medical Center, VISN 4 MIRECC, Philadelphia, PA, United States 2 University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, United States
Aims: Individuals with substance use disorders exhibit poorer treatment outcome, greater functional impairment and higher risk for relapse when presenting with comorbid trauma exposure or posttraumatic stress disorder (PTSD). These two co-morbidities have, in several clinical populations, been linked to poor functional connectivity (FC) between the amygdala and medial prefrontal cortex (AMYG-mPFC) during tasks that probe emotional regulation. We hypothesized that this “biomarker” of emotional dysregulation might be detected even in the resting state, for cocaine-addicted individuals with co-morbid trauma and PTSD.
Methods: 36 stabilized detoxified cocaine patients were divided into three groups based on trauma status and PTSD diagnoses (NoTrauma, n = 15; TRAUMA-NoPTSD, n = 10; PTSD, n = 11), using related questions from the Addiction Severity Index and the MINI psychiatric interview. Arterial spin labeled perfusion fMRI measured resting rCBF. Perfusion data were pre-processed with SPM8, using FC analyses with AMYG as the seed region. Results: The NoTrauma group evidenced robust positive FC between AMYG-mPFC (p < .001(uncorr.), t = 11.90), a pattern that progressively diminished for the TRAUMA group (p < .001(uncorr.), t = 6.03) and was absent for the PTSD group. Conclusions: As hypothesized, cocaine patients with TRAUMA and PTSD exhibit abnormal FC in regions responsible for regulation of affect and motivation—even in the resting state. Diminished AMYG-mPFC FC in the resting state may reflect an underlying dysfunction that manifests more fully during tasks that make demands on this connectivity (e.g., regulation of appetitive craving and/or aversive motivation). Our ongoing studies will test whether this biomarker of compromised fronto-limbic connectivity can predict clinical outcomes, including relapse. Financial support: NIDA R33 DA026114 and R01 DA025906; P60 DA05186; P50 DA12756; VA VISN 4 MIRECC; Commonwealth of Pennsylvania CURE Addiction Center of Excellence. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.205 Gene transfer of engineered cocaine hydrolase: Lifetime expression with no immune response L. Geng, Y. Gao, Stephen Brimijoin Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, United States Aims: Recent animal studies show viral gene transfer of a cocaine hydrolase (CocH) based on mutated human butyrylcholinesterase lowers cocaine reward and impairs cocaine-primed reinstatement of drug seeking, but antibody response to foreign protein limits enzyme levels. Before CocH gene transfer is tested in cocaine users, to determine if mutation for fast cocaine hydrolysis stimulates immune reaction, we examined expression level and antibody responses in mice transduced with equivalent mutated mouse cholinesterase (mCocH). Methods: mCocH cDNA with five mutations (A199S/S227A/S287G/A328W/Y332G) was incorporated into plasmid for HEK 293 cell culture and viral vectors for mouse injection. Enzyme was purified from culture supernates on procainamide-Sepharose. Molar levels were established by active site titration. Mouse tail-vein blood samples were drawn for radiometric assay of cocaine hydrolase and determination of anti-CocH IgG and IgM by solid phase precipitation. Results: Substrate kinetics of mCocH showed 10-fold increased efficiency in cocaine hydrolysis (vs 1300-fold with same mutations in human CocH). Mice given 7 × 1010 viral particles (VP) of AAV vector showed a 10-fold rise in plasma CocH activity for 18 months or more, while 3 × 1011 VP generated a 30-fold rise. HDAd vector (2 × 1012 VP) was more effective, with a 300,000-fold rise of CocH activity and 500-fold increase in enzyme protein, for >6 mo at ∼50% of peak. Circulating anti-CocH IgG or IgM antibodies were not detected. Hence the enzyme mutations were weakly antigenic. Other observations (poster by Gao et al.) indicate that vector-delivered mCocH lacks toxicity across a range of systems and tissues. Conclusions: Our findings show that mCocH at extremely high levels causes little direct physiological effect on mice but will block
Abstracts / Drug and Alcohol Dependence 140 (2014) e2–e85
action of massive cocaine doses. This outcome justifies continued exploration of CocH gene transfer with regard to its potential for reducing risk of relapse into drug-seeking after a period of abstinence. Financial support: NIDA Avant-Garde Award DP1-DA31340 and R01-DA23979. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.206 Nicotine vapor inhalation escalates nicotine self-administration Nicholas W. Gilpin Physiology, Louisiana State University Health Sciences Center, New Orleans, LA, United States Aims: Humans escalate their cigarette smoking over time. This study addresses a major obstacle in the field of pre-clinical nicotine addiction research, which has been the inability to produce escalated nicotine self-administration in rats. Methods: In Experiment 1, male Wistar rats were trained to respond for nicotine in 2-h operant sessions, then exposed to chronic intermittent (12 h/day) nicotine vapor and repeatedly tested for nicotine self-administration at 8–12 h withdrawal. Rats were tested intermittently on days 1, 3 and 5 of the vapor exposure procedure, then tested on consecutive days 6–15 of nicotine vapor exposure. In Experiment 2, rats were exposed or not exposed to chronic intermittent nicotine vapor, then tested for spontaneous and precipitated somatic signs of nicotine withdrawal. Results: Rats exhibited transient increases in operant nicotine responding during intermittent testing, regardless of vapor condition, and this responding returned to baseline levels upon resumption of consecutive-days testing (i.e., nicotine deprivation effect). Nicotine vapor-exposed rats then escalated nicotine selfadministration relative to both their own baseline (∼200% increase) and non-dependent controls (∼3× higher). Eight hrs following removal from nicotine vapor, rats exhibited robust mecamylamineprecipitated somatic signs of withdrawal. There was a strong correlation between nicotine flow rate and air-nicotine concentration, and the air-nicotine concentrations used here resemble concentrations experienced by human smokers. Conclusions: These results suggest that chronic intermittent nicotine vapor inhalation produces somatic and motivational signs of nicotine dependence, the latter of which is evidenced by escalation of nicotine self-administration. Financial support: This work was funded by AA018400 and LSUHSC SOM Faculty Start-Up Funds. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.207 Association of cognitive functioning with treatment outcomes of substance-dependent adults with major depression Suzette Glasner-Edwards 1 , Mary-Lynn Brecht 1 , A. Bellows 1 , H. Chokron Garneau 1 , S.A. Brown 2 , R. Rawson 1 1 Psychiatry, UCLA Integrated Substance Abuse Programs, Los Angeles, CA, United States 2 Psychology, UCSD, San Diego, CA, United States
Aims: Many evidence-based behavioral interventions for addictions, such as cognitive behavioral therapy (CBT) and motivational interviewing (MI), assume adequate cognitive functioning. Those with relatively lower cognitive ability may be particularly vul-
e69
nerable to poorer treatment outcomes. Individuals with major depression suffer from cognitive symptoms; likewise, chronic substance use is associated with neurocognitive impairments. In a pilot randomized clinical trial (N = 70), we evaluated: (1) changes in cognitive functioning from baseline to treatment-end; and (2) the effects of baseline cognitive functioning on outcomes of a 12week continuing care intervention targeting depression and drug dependence. Methods: Group CBT plus MI (CBT-MI) was compared to a control condition (dual recovery anonymous group: DRA) among depressed, substance dependent adults who were completing day hospital treatment for co-occurring disorders. Results: A group-by-cognitive functioning interaction was observed, indicating that depressive symptom severity changed differentially over time as a function of overall cognitive ability and intervention condition, with CBT-MI participants with high cognitive ability reporting greater reductions in depression (Effect Size = 0.95), relative to those with low cognitive ability. Likewise, a greater proportion of those in the CBT-MI condition evidenced stable or improving levels of cognitive ability from baseline to treatment end, relative to those in DRA, for whom declines in cognitive functioning were more frequently observed (Effect Size = 0.28). Analyses of substance use outcomes in association with cognitive functioning and intervention condition are presently under way. Conclusions: Among substance users with comorbid depression, some recovery in cognitive ability may occur in response to CBT- and MI-based interventions, and depression treatment response to CBT-MI may vary as a function of pre-treatment cognitive ability. Financial support: This research was supported by NIDA (K23 DA020085). http://dx.doi.org/10.1016/j.drugalcdep.2014.02.208 Nicotine, HPA axis hormones and mood states in women Nathalie Goletiani 1 , A. Siegel 2 , J. Hudson 3,1 1
MIC, McLean Hospital, Belmont, MA, United States Internal Medicine, McLean Hospital, Belmont, MA, United States 3 Psychiatry, McLean Hospital, Belmont, MA, United States 2
Aims: To determine the acute effects of cigarette smoking on hypothalamic–pituitary–adrenal axis hormones and mood states in healthy nicotine dependent participants as a function of the menstrual cycle. Methods: The acute effects of cigarette smoking on hypothalamic–pituitary–adrenal axis hormones and mood states were studied in 17 healthy nicotine dependent participants during the mid-follicular and mid-luteal phases of the menstrual cycle. Due to observation of a possible bimodal distribution of progesterone levels within the luteal phase group, we also performed a set of a posteriori analyses. Therefore, we divided the luteal group into a low progesterone group and a high progesterone group. Results: Due to observation of a possible bimodal distribution of progesterone levels within the luteal phase group, we also performed a set of a posteriori analyses. Therefore, we divided the luteal group into a low progesterone group and a high progesterone group. These analyses revealed that the high progesterone group, compared with the follicular group, displayed lower increases in ratings of rush and high from baseline and less decrease from baseline in craving, that were statistically significant and represented large effects. Additionally, the high progesterone luteal group dis-
Abstracts / Drug and Alcohol Dependence 140 (2014) e2–e85
negative urines, reduction in opioid craving, improvement in mental health functional quality of life, and no new safety concerns. Financial support: Funded by Alkermes, Inc. Drs. Gastfriend, Memisoglu and Silverman are employees of Alkermes, Inc. Dr. Earley is a paid consultant to Alkermes. Injectable extended-release naltrexone (Vivitrol® ) was developed with support from National Institute on Drug Abuse Grant R43DA013531 and National Institute on Alcohol Abuse and Alcoholism Grant N43AA001002. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.203 Behavioral effects of synthetic cannabinoids marketed as “spice” Michael B. Gatch, M. Forster Pharmacology & Neuroscience, UNT Health Science Center, Fort Worth, TX, United States Aims: A number of cannabinoid compounds are being sold as “legal” alternatives to marijuana in the form of incense. As these marginally legal compounds become controlled, suppliers move to other, unregulated compounds. The purpose of these experiments was to determine whether some common synthetic cannabinoids have discriminative stimulus effects similar to 9tetrahydrocannabinol, which is thought to be the main active component in marijuana. Methods: The compounds JWH-203, JWH-250, and AM2201 were tested for locomotor stimulant effects in mice and subsequently for substitution in rats trained to discriminate 9tetrahydrocannabinol (3 mg/kg, i.p.). Results: JWH-203, JWH-250, and AM2201 each decreased locomotor activity for up to 90 min. JWH-203, JWH-250, and AM2201 each fully substituted for the discriminative stimulus effects of 9tetrahydrocannabinol at doses that did not alter rate of responding. Conclusions: JWH-203, JWH-250, and AM2201 each decreased locomotor activity for up to 90 min. JWH-203, JWH-250, and AM2201 each fully substituted for the discriminative stimulus effects of 9-tetrahydrocannabinol at doses that did not alter rate of responding. Financial support: Supported by NIH N01DA-7-8872. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.204 Trauma exposure and PTSD is associated with attenuation in fronto-limbic functional connectivity among cocaine users Michael J. Gawrysiak 1,2 , Jesse Suh 1,2 , Y. Li 2 , K. Jagannathan 2 , R. Fabianski 2 , Anna Rose Childress 2 1 Philadelphia VA Medical Center, VISN 4 MIRECC, Philadelphia, PA, United States 2 University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, United States
Aims: Individuals with substance use disorders exhibit poorer treatment outcome, greater functional impairment and higher risk for relapse when presenting with comorbid trauma exposure or posttraumatic stress disorder (PTSD). These two co-morbidities have, in several clinical populations, been linked to poor functional connectivity (FC) between the amygdala and medial prefrontal cortex (AMYG-mPFC) during tasks that probe emotional regulation. We hypothesized that this “biomarker” of emotional dysregulation might be detected even in the resting state, for cocaine-addicted individuals with co-morbid trauma and PTSD.
Methods: 36 stabilized detoxified cocaine patients were divided into three groups based on trauma status and PTSD diagnoses (NoTrauma, n = 15; TRAUMA-NoPTSD, n = 10; PTSD, n = 11), using related questions from the Addiction Severity Index and the MINI psychiatric interview. Arterial spin labeled perfusion fMRI measured resting rCBF. Perfusion data were pre-processed with SPM8, using FC analyses with AMYG as the seed region. Results: The NoTrauma group evidenced robust positive FC between AMYG-mPFC (p < .001(uncorr.), t = 11.90), a pattern that progressively diminished for the TRAUMA group (p < .001(uncorr.), t = 6.03) and was absent for the PTSD group. Conclusions: As hypothesized, cocaine patients with TRAUMA and PTSD exhibit abnormal FC in regions responsible for regulation of affect and motivation—even in the resting state. Diminished AMYG-mPFC FC in the resting state may reflect an underlying dysfunction that manifests more fully during tasks that make demands on this connectivity (e.g., regulation of appetitive craving and/or aversive motivation). Our ongoing studies will test whether this biomarker of compromised fronto-limbic connectivity can predict clinical outcomes, including relapse. Financial support: NIDA R33 DA026114 and R01 DA025906; P60 DA05186; P50 DA12756; VA VISN 4 MIRECC; Commonwealth of Pennsylvania CURE Addiction Center of Excellence. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.205 Gene transfer of engineered cocaine hydrolase: Lifetime expression with no immune response L. Geng, Y. Gao, Stephen Brimijoin Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, United States Aims: Recent animal studies show viral gene transfer of a cocaine hydrolase (CocH) based on mutated human butyrylcholinesterase lowers cocaine reward and impairs cocaine-primed reinstatement of drug seeking, but antibody response to foreign protein limits enzyme levels. Before CocH gene transfer is tested in cocaine users, to determine if mutation for fast cocaine hydrolysis stimulates immune reaction, we examined expression level and antibody responses in mice transduced with equivalent mutated mouse cholinesterase (mCocH). Methods: mCocH cDNA with five mutations (A199S/S227A/S287G/A328W/Y332G) was incorporated into plasmid for HEK 293 cell culture and viral vectors for mouse injection. Enzyme was purified from culture supernates on procainamide-Sepharose. Molar levels were established by active site titration. Mouse tail-vein blood samples were drawn for radiometric assay of cocaine hydrolase and determination of anti-CocH IgG and IgM by solid phase precipitation. Results: Substrate kinetics of mCocH showed 10-fold increased efficiency in cocaine hydrolysis (vs 1300-fold with same mutations in human CocH). Mice given 7 × 1010 viral particles (VP) of AAV vector showed a 10-fold rise in plasma CocH activity for 18 months or more, while 3 × 1011 VP generated a 30-fold rise. HDAd vector (2 × 1012 VP) was more effective, with a 300,000-fold rise of CocH activity and 500-fold increase in enzyme protein, for >6 mo at ∼50% of peak. Circulating anti-CocH IgG or IgM antibodies were not detected. Hence the enzyme mutations were weakly antigenic. Other observations (poster by Gao et al.) indicate that vector-delivered mCocH lacks toxicity across a range of systems and tissues. Conclusions: Our findings show that mCocH at extremely high levels causes little direct physiological effect on mice but will block
Abstracts / Drug and Alcohol Dependence 140 (2014) e2–e85
action of massive cocaine doses. This outcome justifies continued exploration of CocH gene transfer with regard to its potential for reducing risk of relapse into drug-seeking after a period of abstinence. Financial support: NIDA Avant-Garde Award DP1-DA31340 and R01-DA23979. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.206 Nicotine vapor inhalation escalates nicotine self-administration Nicholas W. Gilpin Physiology, Louisiana State University Health Sciences Center, New Orleans, LA, United States Aims: Humans escalate their cigarette smoking over time. This study addresses a major obstacle in the field of pre-clinical nicotine addiction research, which has been the inability to produce escalated nicotine self-administration in rats. Methods: In Experiment 1, male Wistar rats were trained to respond for nicotine in 2-h operant sessions, then exposed to chronic intermittent (12 h/day) nicotine vapor and repeatedly tested for nicotine self-administration at 8–12 h withdrawal. Rats were tested intermittently on days 1, 3 and 5 of the vapor exposure procedure, then tested on consecutive days 6–15 of nicotine vapor exposure. In Experiment 2, rats were exposed or not exposed to chronic intermittent nicotine vapor, then tested for spontaneous and precipitated somatic signs of nicotine withdrawal. Results: Rats exhibited transient increases in operant nicotine responding during intermittent testing, regardless of vapor condition, and this responding returned to baseline levels upon resumption of consecutive-days testing (i.e., nicotine deprivation effect). Nicotine vapor-exposed rats then escalated nicotine selfadministration relative to both their own baseline (∼200% increase) and non-dependent controls (∼3× higher). Eight hrs following removal from nicotine vapor, rats exhibited robust mecamylamineprecipitated somatic signs of withdrawal. There was a strong correlation between nicotine flow rate and air-nicotine concentration, and the air-nicotine concentrations used here resemble concentrations experienced by human smokers. Conclusions: These results suggest that chronic intermittent nicotine vapor inhalation produces somatic and motivational signs of nicotine dependence, the latter of which is evidenced by escalation of nicotine self-administration. Financial support: This work was funded by AA018400 and LSUHSC SOM Faculty Start-Up Funds. http://dx.doi.org/10.1016/j.drugalcdep.2014.02.207 Association of cognitive functioning with treatment outcomes of substance-dependent adults with major depression Suzette Glasner-Edwards 1 , Mary-Lynn Brecht 1 , A. Bellows 1 , H. Chokron Garneau 1 , S.A. Brown 2 , R. Rawson 1 1 Psychiatry, UCLA Integrated Substance Abuse Programs, Los Angeles, CA, United States 2 Psychology, UCSD, San Diego, CA, United States
Aims: Many evidence-based behavioral interventions for addictions, such as cognitive behavioral therapy (CBT) and motivational interviewing (MI), assume adequate cognitive functioning. Those with relatively lower cognitive ability may be particularly vul-
e69
nerable to poorer treatment outcomes. Individuals with major depression suffer from cognitive symptoms; likewise, chronic substance use is associated with neurocognitive impairments. In a pilot randomized clinical trial (N = 70), we evaluated: (1) changes in cognitive functioning from baseline to treatment-end; and (2) the effects of baseline cognitive functioning on outcomes of a 12week continuing care intervention targeting depression and drug dependence. Methods: Group CBT plus MI (CBT-MI) was compared to a control condition (dual recovery anonymous group: DRA) among depressed, substance dependent adults who were completing day hospital treatment for co-occurring disorders. Results: A group-by-cognitive functioning interaction was observed, indicating that depressive symptom severity changed differentially over time as a function of overall cognitive ability and intervention condition, with CBT-MI participants with high cognitive ability reporting greater reductions in depression (Effect Size = 0.95), relative to those with low cognitive ability. Likewise, a greater proportion of those in the CBT-MI condition evidenced stable or improving levels of cognitive ability from baseline to treatment end, relative to those in DRA, for whom declines in cognitive functioning were more frequently observed (Effect Size = 0.28). Analyses of substance use outcomes in association with cognitive functioning and intervention condition are presently under way. Conclusions: Among substance users with comorbid depression, some recovery in cognitive ability may occur in response to CBT- and MI-based interventions, and depression treatment response to CBT-MI may vary as a function of pre-treatment cognitive ability. Financial support: This research was supported by NIDA (K23 DA020085). http://dx.doi.org/10.1016/j.drugalcdep.2014.02.208 Nicotine, HPA axis hormones and mood states in women Nathalie Goletiani 1 , A. Siegel 2 , J. Hudson 3,1 1
MIC, McLean Hospital, Belmont, MA, United States Internal Medicine, McLean Hospital, Belmont, MA, United States 3 Psychiatry, McLean Hospital, Belmont, MA, United States 2
Aims: To determine the acute effects of cigarette smoking on hypothalamic–pituitary–adrenal axis hormones and mood states in healthy nicotine dependent participants as a function of the menstrual cycle. Methods: The acute effects of cigarette smoking on hypothalamic–pituitary–adrenal axis hormones and mood states were studied in 17 healthy nicotine dependent participants during the mid-follicular and mid-luteal phases of the menstrual cycle. Due to observation of a possible bimodal distribution of progesterone levels within the luteal phase group, we also performed a set of a posteriori analyses. Therefore, we divided the luteal group into a low progesterone group and a high progesterone group. Results: Due to observation of a possible bimodal distribution of progesterone levels within the luteal phase group, we also performed a set of a posteriori analyses. Therefore, we divided the luteal group into a low progesterone group and a high progesterone group. These analyses revealed that the high progesterone group, compared with the follicular group, displayed lower increases in ratings of rush and high from baseline and less decrease from baseline in craving, that were statistically significant and represented large effects. Additionally, the high progesterone luteal group dis-