Neuropsychopharmacology (2005) 30, S210-S270 © 2005 Nature Publishing Group All rights reserved 0893-133X/05 S210 www.neuropsychopharmacology.org Wednesday, December 14, 2005 2. White Matter Changes in Cocaine Dependence Poster Session III - Wednesday Jay Nierenberg*, Laurie Nash, Matthew J Hoptman, Jan Hrabe, Babak A Ardekani, Gregory Bunt, Kelvin O Lim and John Rotrosen 1. Design, Synthesis and Application of Novel Tropane-Based Nathan S. Kline Institute for Psychiatric Research, Orangeburg, Photoaffinity Labels Toward the Identification of Binding NY, USA Domains on the Dopamine Transporter Amy H Newman*, Mu-Fa Zou, Jianjing Cao, Joo H Cha, John R Sponsor: Travel Awardee, Young Investigator Memorial, 2005 Lever, Theresa Kopajtic, Jonathan L Katz, M. L Parnas and Roxanne Vaughan Background: Cocaine Dependence (CD) is a serious problem that Medicinal Chemistry Section, NIDA-IRP, NIH, Baltimore, MD, USA often is refractory to treatment. Evidence suggests that CD is associ- ated with white matter (WM) deficits that may underlie changes in Sponsor: Jonathan Javitch mood, cognition and behavior. We studied 46 patients with CD and 17 controls using MRI at 1.5T to better characterize WM abnormal- Background: Irreversible ligands, derived from tropane-based ities in CD and to test whether they were reversible. Diffusion ten- dopamine uptake inhibitors, have been designed to covalently bind to sor imaging (DTI) was used for measurements in WM. DTI is an discrete points of attachment on the dopamine transporter (DAT), via MRI method that provides measures (fractional anisotropy, FA) a photoactivated azido group. We previously have demonstrated that that are sensitive to WM pathology but do not provide insight into depending on the position of the azido substitution on the tropane ring underlying mechanisms, as well as measures, such as axial (Dax) system, covalent attachment can occur in different transmembrane re- and radial (Dra) diffusivity, which can reflect specific microstruc- gions suggesting that 1) the position of the azido attachment on the tural changes. tropane ring dictates transmembrane domain labeling and/or 2) not all Methods: Subjects were patients in a therapeutic community that tropane-based DAT inhibitors bind to the same recognition site(s) on closely monitored ongoing abstinence with random toxicology test- the DAT. Based on these early studies, we hypothesized that appending ing. Patients were subgrouped by duration of abstinence- <6 weeks azido groups at numerous positions on different tropane-based DAT (COC-S, n=15), 2-10 months (COC-M, n=15), > 1 year (COC-L, inhibitors (3-phenyl tropanes and benztropines) would provide the n=16)- and matched with non-using controls (CON). 8-direction opportunity to identify points of attachment on the DAT, elucidate 3D- DTI was acquired with a double spin echo, pulsed-gradient echo- arrangement of the transmembrane domains, and direct future drug planar imaging method that minimized distortion due to eddy cur- design. Herein, we report our progress in these pursuits. rents. DTI maps were analyzed using both voxelwise methods and Methods: All photoaffinity labels were designed to have azido groups region of interest (ROI) approaches in separate analyses. Thresh- attached to the N-, 2- or 3-position of the tropane ring of either the olding in voxelwise tests used a two-stage seed-cluster method that benztropine or 3-phenyltropane parent molecule. Synthetic strategies was designed to minimize false positive detections. Briefly absti- were undertaken based on modifications of previously published nent subjects (n=7) also were scanned longitudinally to test work (Agoston et al., 1997, Zou et al., 2001, 2003, Lever et al., 2005). whether cross-sectional differences were supported by changes in Radiolabel displacement studies using [3H]WIN 35,428 in rat cau- individuals. date putamen tissue were used to assess IC50 values of all final prod- Results: Subjects were well-matched on demographic variables. Num- ucts and their immediate precursors. ber of years of cocaine use was similar in user subgroups and averaged Results: We initially prepared an N-substituted benztropine-based ~10 years. When the whole CD cohort was compared with CON in photoaffinity label [125I]GA 2-34, which covalently attached to the voxelwise t-tests, patients showed mainly lower FA in most regions in 1-2 transmembrane spanning region of the DAT. This was compared which differences were found. In contrast, COC-S subjects showed and found to be in contrast to the 4-7 transmembrane spanning re- more WM voxels with higher than lower FA, though these were con- gion labeled by a 2-substituted-3-phenyltropane-based photoaffinity centrated in the inferior frontal WM. Whereas all patients showed sig- label, [125I]RTI 82 (Vaughan et al. 1999). Based on this early study nificantly higher FA in frontal WM regions bilaterally, COC-S subjects we then synthesized a derivative of RTI 82 that had the same N-sub- also showed higher FA in ventromedial prefrontal WM regions that stituent as GA 2-34 (Zou et al. 2001). The resulting [125I]MFZ 2-24 was not present in COC-L. COC-S subjects also showed higher FA in (Lever et al 2005) has now been discovered to covalently link to a dis- WM of the internal capsule adjacent to the right caudate nucleus that crete domain in transmembrane domain 1-2 of the DAT (Vaughan et were not present in COC-L or when all CD patients were compared al 2005). In an attempt to provide a 2-substituted benztropine pho- with CON. All patients showed lower FA in the substantia nigra and toaffinity label, analogous to RTI 82, MFZ 4-40, (Zou, et al. 2003) was globus pallidus that was right-lateralized and lower FA in the deep prepared as well as an additional compound in which the chain WM of the temporal lobe that was left-lateralized. Moreover, frontal length at the 2-position was extended by two atoms. The resulting lig- WM FA correlated positively with years of cocaine use and correlated and, MFZ 6-107, showed lower DAT affinity (IC50= 131 nM) as com- negatively with duration of abstinence across all subjects. Comparison pared to MFZ 4-40 (IC50= 55.6 nM), suggesting that an extended 2- of COC-S and COC-L subjects showed predominantly higher frontal position substituent was not well tolerated. Thus, a third lobe FA in COC-S. Four regions with significant changes in FA in 2-substituted analogue was derived using the linker length of MFZ 4- COC-S subjects, compared with COC-L, were also found in compar- 40 and the ester linkage of MFZ 6-107, which provided an improved isons of FA in COC-S vs. CON, suggesting that these regions showed synthetic strategy. In addition, a 3-beta-(3-iodo-4-azido-bipheny- reversible deficits in CD. Longitudinal testing confirmed that in- lene)-analogue of RTI 82 was recently synthesized. This latest addi- creased FA in COC-S in frontal WM regions was reversible, but sug- tion to our arsenal of DAT photoaffinity labels, JHC 2-48 (IC50= 15.1 gested that changes were more widespread. Analysis of tensor compo- nM) demonstrated the highest DAT binding affinity of any photoli- nents showed that reversible changes were due to increased Dra over gand prepared to date, as well as moderate affinity for SERT (IC50= time. ROIs placed in frontal WM confirmed both the cross-sectional 109) suggesting its potential utility in labeling SERT for analogous and longitudinal findings in frontal WM. protein structure studies. Discussion: This study showed reversible as well as persistent in- Discussion: Radioiodination of these remaining photoaffinity labels creases in frontal WM FA which must be viewed as deficits given the will provide the necessary tools for elucidating binding domains on impaired functional status of patients tested. Elucidation of the basis DAT (and SERT) of the tropane-based dopamine uptake inhibitors of these changes may provide insight into the anatomical mecha- using immunoprecipitation and proteolysis strategies. nisms in CD that may inform treatment. ACNP 2005 Annual Meeting S211 3. Prediction of Alcohol Problems Using A Prospective our QTLs for the MA sensitivity (LOD=4.5), a region that has previ- Longitudinal Design Including Genotype ously been associated with sensitivity to the stimulant effects of co- John I Nurnberger*, R Wiegand, L Bierut, K Bucholz, T Foroud, H caine. These data suggested the existence of an allele that influences Edenberg, E T Meyer, J Kramer, D Dick, W Reich, S Kuperman, V MA sensitivity by altering expression of Csnk1e. Human resutls: The Hesselbrock, A Goate, B Porjesz and H Begleiter primary outcome measure, subjects’ ratings of whether they felt a drug effect (Drug Effects Questionnaire; DEQ), revealed a significant Psychiatry, Indiana University School of Medicine, Indianapolis, effect (p = 0.01) of a non-coding SNP (rs135745) on the subjective IN, USA response to AMPH. Subjects with one or two copies of the C allele of Background: 1022 subjects from 448 families in the Collaborative rs135745 were more sensitive to the 10 mg dose of AMPH (p = Study of the Genetics of Alcoholism (COGA) were assessed at two 0.001). Subjects with different genotypes at this SNP also differed on time points separated by an interval of 4-5 years. The age of subjects secondary outcomes, including the “euphoria” scale of the ARCI, at at Time 2 was 17.4 + 3.4 years. At Time 2, 230 subjects were diagnosed the 10 mg dose. with DSM IV alcohol dependence or abuse by personal interview Discussion: These findings demonstrate a powerful translational ap- and/or interview of a parent about the subject. proach to studying pharmacogenetics in mice and humans, and offer Methods: Characteristics at Time 1 were used to predict affected sta- a road-map for future investigations of genetically determined differ- tus at Time 2.