Stress affects brain serotonin (5HT) and dopamine (DA) function, and the effectiveness of 5HT and DA to regulate stress and emotional responses. However, our understanding of the long-term impact of early life adversity (ELA) on primate brain monoaminergic systems during adolescence is scarce and inconsistent. Filling this gap in the literature is critical, given that the emergence of psychopathology during adolescence has been related to deficits in these systems. Here, we use a translational nonhuman primate (NHP) model of ELA (infant maltreatment by the mother) to examine the long-term impact of ELA on adolescent 5HT1A, 5HT2A and D2 receptor systems. These receptor systems were chosen based on their involvement in stress/emotional control, as well as reward and reinforcement. Rates of maternal abuse, rejection, and infant’s vocalizations were obtained during the first three postnatal months, and hair cortisol concentrations obtained at 6 months postnatal were examined as early predictors of binding potential (BP) values obtained during adolescence using positron emission tomography (PET) imaging. Maltreated animals demonstrated significantly lower 5HT1A receptor BP in prefrontal cortical areas as well as the amygdala and hippocampus, and lower 5HT2A receptor BP in striatal and prefrontal cortical areas. Maltreated animals also demonstrated significantly lower D2 BP in the amygdala. None of the behavioral and neuroendocrine measurements obtained early in life predicted any changes in BP data. Our findings suggest that early caregiving experiences regulate the development of brain 5HT and DA systems in primates, resulting in long-term effects evident during adolescence.

PDF file, 591K, Supplementary Figure 4. KCN1 inhibits the growth of pre-established human glioblastoma s.c. xenografts. Supplementary Figure 5. Effect of KCN1 treatment on mice weight. Supplementary Figure 6. Chronic systemic KCN1 administration shows little effect on major organs, except liver. Supplementary Figure 7. Effect of KCN1 on the survival of mice with orthotopic (intracranial) brain tumor models. Supplementary Figure 8. Permeability coefficients (PC) of KCN1 calculated from the transfer across monolayers of brain endothelial cells.

Background Despite observed sex differences in the prevalence of stress-related psychiatric conditions, most preclinical and translational studies have only included male subjects. Therefore, it has not been possible to effectively assess how sex interacts with other psychosocial risk factors to impact the etiology and maintenance of stress-related psychopathology. One psychosocial factor that interacts with sex to impact risk for stress-related behavioral and physiological deficits is social dominance. The current study was designed to assess sex differences in the effects of social status on socioemotional behavior and serotonin neurochemistry in socially housed rhesus monkeys. We hypothesized that sex and social status interact to influence socioemotional behaviors as well as serotonin 1A receptor binding potential (5HT1AR-BP) in regions of interest (ROIs) implicated in socioemotional behavior. Methods Behavioral observations were conducted in gonadally intact adult female ( n = 14) and male ( n = 13) rhesus monkeys. 5HT1AR-BP was assessed via positron emission tomography using 4-(2ʹ-Methoxyphenyl)-1-[2ʹ-( N -2ʺ-pyridinyl)- p [ 18 F]fluorobenzamido]ethylpiperazine ([ 18 F]MPPF). Results Aggression emitted was greater in dominant compared to subordinate animals, regardless of sex. Submission emitted was significantly greater in subordinate versus dominant animals and greater in females than males. Affiliative behaviors emitted were not impacted by sex, status, or their interaction. Anxiety-like behavior emitted was significantly greater in females than in males regardless of social status. Hypothalamic 5HT1AR-BP was significantly greater in females than in males, regardless of social status. 5HT1AR-BP in the dentate gyrus of the hippocampus was significantly impacted by a sex by status interaction whereby 5HT1AR-BP in the dentate gyrus was greater in dominant compared to subordinate females but was not different between dominant and subordinate males. There were no effects of sex, status, or their interaction on 5HT1AR-BP in the DRN and in the regions of the PFC studied. Conclusions These data have important implications for the treatment of stress-related behavioral health outcomes, as they suggest that sex and social status are important factors to consider in the context of serotonergic drug efficacy.

PDF file, 285K, Supplementary Figure 1. Synthetic scheme and purity of KCN1 3,4-dimethoxy-N-(2,2-dimethyl-2H-chromen-6-yl)methyl-N-phenylbenzenesulfonamide. Supplementary Figure 2. KCN1 is chemically stable in cell culture medium under normoxic or hypoxic conditions. Supplementary Figure 3. Effect of KCN1 in the NCI-60 Tumor Cell Line Screen.

Social subordination increases risk for psychiatric disorders, while dominance increases resilience to these disorders. Fluoxetine, a selective serotonin (5HT) reuptake inhibitor whose actions are mediated in part by the 5HT1A receptor (5HT1AR), has sex- and social status-specific effects on socioemotional behavior and aggressive behavior. However, the impact of social status on these sex-specific effects remains unclear. The current study evaluated the impact of acute fluoxetine treatment and social status on dominance-related behaviors in female and male hamsters, and the impact of chronic fluoxetine treatment on socioemotional behavior and 5HT1AR binding potential (5HT1AR(BP)) in female rhesus macaques. We hypothesized that sex differences in the effects of fluoxetine on aggression in hamsters would be diminished in dominant and enhanced in subordinate males and that aggression in female hamsters would be enhanced in dominants and diminished in subordinates. In female rhesus macaques, we hypothesized that chronic fluoxetine would alter socioemotional behaviors and site-specific 5HT1AR(BP) in a status-dependent manner. Male (n = 46) and female (n = 56) hamsters were paired with conspecifics for three days to establish social rank. Hamsters received a single dose of 20 mg/kg fluoxetine or vehicle two-hours prior to a test with a non-aggressive intruder. Female rhesus monkeys (n = 14) housed were administered fluoxetine (2.8 mg/kg/day) or vehicle injections chronically for 14-days, separated by a three-week washout period. On Day 15, positron emission tomography neuroimaging for 5HT1AR(BP) was conducted. Fluoxetine treatment decreased aggression in subordinate female monkeys and subordinate female hamsters but not in dominant females of either species. Fluoxetine decreased aggression in dominant but not in subordinate male hamsters. Fluoxetine also reduced and increased prefrontal 5HT1AR(BP) in dominant and subordinate females, respectively. Taken together, these results provide cross-species evidence that social status and sex impact how increased 5HT modulates agonistic behavior.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, and patients that develop metastases (~50%) survive <1 year, highlighting the urgent need for new therapies. TCGA has recently revealed that a hypoxia gene signature is associated with poor UM patient prognosis. Here we show that expression of hypoxia-regulated collagen prolyl-4-hydroxylase genes P4HA1 and P4HA2 is significantly upregulated in UM patients with metastatic disease and correlates with poor prognosis, suggesting these enzymes might be key tumor drivers. We targeted hypoxia-induced expression of P4HA1/2 in UM with KCN1, a hypoxia inducible factor-1 (HIF-1) pathway inhibitor and found potent inhibition of primary and metastatic disease and extension of animal survival, without overt side effects. At the molecular level, KCN1 antagonized hypoxia-induced expression of P4HA1 and P4HA2, which regulate collagen maturation and deposition in the extracellular matrix. The treatment decreased prolyl hydroxylation, induced proteolytic cleavage and rendered a disordered structure to collagen VI, the main collagen produced by UM, and reduced UM cell invasion. Together, these data demonstrate that extracellular collagen matrix formation can be targeted in UM by inhibiting hypoxia-induced P4HA1 and P4HA2 expression, warranting further development of this strategy in patients with uveal melanoma.

The serotonin 5-HT2C receptor (5-HT2CR) is abundantly expressed throughout the central nervous system, and involved in a variety of neuroendocrine and neurobehavioral processes. The development of a selective radioligand that will enable in vivo imaging and quantification of 5-HT2CR densities represents a significant technological advancement in understanding both the normal function and pathophysiology of the 5-HT2CR. Four 7-halogen-2-phenyl isoindolones (7-F, Cl, Br, I) were synthesized and displayed high affinities for 5-HT2CR and high selectivity over 5-HT2A and 5-HT2B. [11C]7-Chloro-2-[4-methoxy-3-[2-(4-methylpiperidin-1-yl)ethoxy]phenyl]isoindolin-1-one (6) and [11C]7-iodo-2-[4-methoxy-3-[2-(4-methylpiperidin-1-yl)ethoxy]phenyl]isoindolin-1-one (9) were synthesized in high radiochemical yield of 37–44% [n = 10, decay corrected from end of (11C)CH3I synthesis] with high radiochemical purity via O-methylation with [11C]CH3I, respectively. MicroPET imaging studies in male rats with or without 5-HT2C antagonist SB-242084 showed that [11C]6 and [11C]9 display specific bindings to 5-HT2CR in the choroid plexus and hippocampus. In vivo microPET brain imaging studies in rhesus monkeys demonstrated that [11C]6 and [11C]9 exhibit excellent blood-brain barrier penetration. The contrast of bindings to the choroid plexus and hippocampus compared to the cerebellum peaked at 2.7 and 1.6, respectively, for [11C]6, and 3.7 and 2.7, respectively, for [11C]9, which were reduced by administration of a dose of SB-242084. Our results support the candidacy of [11C]6 and [11C]9 for further study as radioligands for in vivo quantitation of 5-HT2C sites by PET.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults with a survival time less than 1 year for patients that develop metastases (about 50%). HIF-1 plays a critical role in UM adaptation to the hypoxic microenvironment, treatment failure, and metastasis. In this study, we evaluated biodistribution and anti-cancer efficacy of arylsulfonamide KCN1, a lead compound in a novel class of small-molecule inhibitors of the hypoxia-inducible factor (HIF-1) pathway in UM models. PET with 11C-labeled KCN1 showed that KCN1 preferentially localizes to the eye and liver, the organ where UM preferentially metastasizes. In survival studies, UM cells were injected into the suprachoroidal space of the right murine eye using a transscleral technique and the eye was enucleated 7-9 days post-inoculation. Starting on Day 1, KCN1 or vehicle only (cremophor EL/ethanol 1:1, diluted with sterile PBS 1:5) were administered daily i.p., 5 x week, until animals reached the endpoint. Main organs were collected and evaluated for metastatic load. We found that KCN1 potently inhibited the growth of primary eye tumors, VEGF expression, angiogenesis in the primary tumor, the number and size of hepatic metastases, and extended mice survival. KCN1 inhibited invasion of UM cells in in vitromodels, such as scratch-wound and Boyden chamber migration assays. To identify possible mediators of hypoxia-HIF-induced metastatic processes inhibited by KCN1 in UM, we tested a series of 20 candidates by qRT-PCR. Among the most consistently HIF-induced/KCN1-inhibited genes were prolyl-4-hydroxylases P4HA1 and 2 that modulate physical properties of the tumor microenvironment by post-translationally modifying collagen. Expression of P4HA1 and 2 is significantly higher in UM patients with metastatic disease and high level of expression correlates with poor prognosis. In summary, our studies suggest KCN1 exerts anti UM activity by inhibiting the primary tumor growth, reducing vascular density in the primary tumor, and the number and size of the hepatic metastases; KCN1 has therefore therapeutic potential for treatment of UM and further clinical development. Citation Format: Stefan Kaluz, Qing Zhang, Hua Yang, Satoru Osuka, Jiyoung Mun, Narra S. Devi, Mark Goodman, Hans E. Grossniklaus, Erwin G. Van Meir. Arylsulfonamide KCN1 suppresses primary and metastatic growth of uveal melanoma through anti-angiogenic and anti-invasion mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1296.

1396 Objectives: Neurotensin receptor 1 (NTR1) is a therapeutic target1 and overexpressed in most pancreatic adenocarcinoma (PA) tissues.2,3 SR48692 (Meclinertant) is a selective small molecule antagonist for NTR1 and inhibited neurotensin-induced proliferation of NTR1-overexpressing human PA cells and xenografts in nude mouse models.3-7 We assessed the potential use of SR48692 as a PET tracer and anticancer drug by investigating the specific binding of [11C]SR48692 to NTR1-overexpressing human PA cells and its bio-distribution in nude mouse models bearing subcutaneous human PA xenografts. A dynamic microPET with [11C]SR48692 was performed for the same mouse model to confirm the bio-distribution data. Methods: [11C]SR48692 was synthesized by reacting the corresponding O-desmethyl precursor with [11C]CH3I and Cs2CO3 in DMF at 135oC for 10 minutes and a subsequent deprotection of the t-butyl ester with trifluoroacetic acid. NTR1 overexpression in two PA cell lines, MIA PaCa-2 and PDX, was measured by immunofluorescence imaging using a NTR1 antibody (sc-7598, Santa Cruz Biotechnology). [11C]SR48692 was incubated in 0.5 mL of 0.5 million cell suspensions for 10 and 20 minutes and the cell-binding radioactivities were compared with 1% of the total incubated activity. The specific binding was measured by co-incubating SR48692 (2 and 22 μM) for 20 minutes. A mouse model was constructed by subcutaneously injecting 1 million MIA PaCa-2 cells into the lower back of a 5 - 6 week old nude mouse (2 month growth, 0.5 - 1.0 cm length) and the other side (2 week growth, invisible). 3.7 - 7.4 MBq of [11C]SR48692 was injected into the tail vein of eight mice and four mice were sacrificed in 15 and 60 minutes after injection. Blood, muscle, tumors, and 9 organs were dissected and their radioactivities and masses were measured. The specific bindings of [11C]SR48692 in 60 minutes after injection were measured by co-injecting SR48692 (100 μL of 1 mg/mL solution). A dynamic microPET for 1 hour was performed by Siemens Inveon microPET/CT scanner for the same nude mouse model after injecting 3.7 MBq of [11C]SR48692 and the image was reconstructed by the ASIPro software. Results: NTR1 is overexpressed in MIA PaCa-2 and PDX cell line while its expression is under the detection limit in a normal pancreatic epithelial cell line. [11C]SR48692 bound to MIA PaCa-2 and PDX cells and its binding reduced by co-incubation of SR48692 in a concentration-dependent manner. [11C]SR48692 did not show specific binding to subcutaneous MIA PaCa-2 xenografts and showed high uptake in liver, pancreas, and small intestine, which was confirmed by microPET image. Conclusions: SR48692 showed specific binding to NTR1-overexpressing PA cells but it showed typical characteristics of a highly lipophilic compound for the in vivo bio-distribution. Its lipophilicity should be improved significantly to develop its analogs as PET tracers and anticancer drugs.

272 Objectives: The assessment of striatal dopaminergic function using 18F-DOPA (18F-fluoro-3,4-dihydroxy-L-phenylalanine) of high specific activity was evaluated for the test-retest reliability in five rhesus monkeys, which received two 18F-DOPA PET scans, 10 to 18 days apart. Methods: 18F-DOPA was synthesized by nucleophilic 18F- substitution and acidic hydrolysis from a precursor possessing a p-methoxyphenyl iodonium salt (ALPDOPA, Ground Fluor Pharmaceuticals, Inc.) by a modification of a published method1. The animal study was approved by the IACUC of Emory University. (S)-(-)-Carbidopa (a peripheral aromatic L-amino acid decarboxylase inhibitor, 3 mg/kg) was injected 1 hour before a scan, and 74 - 111 MBq of an 18F-DOPA dose solution was injected. An emission scan was performed for two hours using a Siemens MicroPET Focus 220 scanner (1.7 mm of reconstructed image resolution). 18F-DOPA utilization (ki, 18F-DOPA influx constant) at quasi-equilibrium in the left and right caudate nucleus and putamen were determined relative to the cerebellar reference region, using Patlak graphical analysis. The test-retest reliability was determined by the within-subject and between-subject coefficient of variation (%COV). Results: The specific activity of 18F-DOPA at injection was greater than 37 GBq/µmol, the radiochemical purity was greater than 99%, the enantiometric purity was greater than 99%, and 8 - 79 µg/kg of 6-hydroxy-L-DOPA in a dose solution was injected. The 18F-DOPA influx constant (ki, min-1) at quasi-equilibrium ranged from 0.0073 to 0.013 (exclusion of one outliner), 0.0087 to 0.012, 0.011 to 0.016, and 0.010 to 0.015 in the left and right caudate nucleus and putamen respectively. The within-subject and between-subject coefficient of variation ranged from 3.1% to 4.7% and 7.4% to 15% respectively. Conclusions: The test-retest reliability of 18F-DOPA of high specific activity in five rhesus monkeys indicated no significant differences within and between subjects in dopamine synthesis capability in caudate nucleus and putamen. 18F-DOPA of high specific activity could be used to assess striatal dopaminergic function for disease diagnosis and drug treatment efficacy monitoring.

A long-acting, thermostable bacterial cocaine esterase (CocE) has been identified that rapidly degrades cocaine with a KM of 1.33+0.085 μM. In vivo evaluation of CocE has shown protection against convulsant and lethal effects of cocaine in rodents, confirming the therapeutic potential of CocE against cocaine overdose. However, the current study is the first to evaluate the effects of CocE on cocaine brain levels. Positron emission tomogrpahy neuroimaging of [11C]cocaine was used to evaluate the time course of cocaine elimination from brain in the presence and absence of CocE in nonhuman primates. Systemic administration of CocE eliminated cocaine from the rhesus-monkey brain approximately three times faster than control conditions via peripheral actions through attenuating the input function from blood plasma. The efficiency of this process is sufficient to alleviate or prevent adverse central nervous system effects induced by cocaine. Although the present study used tracer doses of cocaine to access brain clearance, these findings further support the development of CocE for the treatment of acute cocaine toxicity.

Neuroimaging studies in humans have demonstrated that inflammatory cytokines target basal ganglia function and presynaptic dopamine (DA), leading to symptoms of depression. Cytokine-treated nonhuman primates also exhibit evidence of altered DA metabolism in association with depressive-like behaviors. To further examine cytokine effects on striatal DA function, eight rhesus monkeys (four male, four female) were administered interferon (IFN)-α (20 MIU/m2 s.c.) or saline for 4 weeks. In vivo microdialysis was used to investigate IFN-α effects on DA release in the striatum. In addition, positron emission tomography (PET) with [11C]raclopride was used to examine IFN-α-induced changes in DA2 receptor (D2R) binding potential before and after intravenous amphetamine administration. DA transporter binding was measured by PET using [18F]2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane. Anhedonia-like behavior (sucrose consumption) was assessed during saline and IFN-α administration. In vivo microdialysis demonstrated decreased release of DA after 4 weeks of IFN-α administration compared with saline. PET neuroimaging also revealed decreased DA release after 4 weeks of IFN-α as evidenced by reduced displacement of [11C]raclopride following amphetamine administration. In addition, 4 weeks of IFN-α was associated with decreased D2R binding but no change in the DA transporter. Sucrose consumption was reduced during IFN-α administration and was correlated with decreased DA release at 4 weeks as measured by in vivo microdialysis. Taken together, these findings indicate that chronic peripheral IFN-α exposure reduces striatal DA release in association with anhedonia-like behavior in nonhuman primates. Future studies examining the mechanisms of cytokine effects on DA release and potential therapeutic strategies to reverse these changes are warranted.

The goal of the present study was to examine how social subordination stress and 5HTT polymorphisms affect the development of brain serotonin (5HT) systems during the pubertal transition in female rhesus monkeys. We also examined associations with developmental changes in emotional reactivity in response to a standardized behavioral test, the Human Intruder (HI). Our findings provide the first longitudinal evidence of developmental increases in 5HT1A receptor and 5HTT binding in the brain of female primates from pre- to peripuberty. The increase in 5HT1A BP(ND) in these socially housed female rhesus monkeys is a robust finding, occurring across all groups, regardless of social status or 5HTT genotype, and occurring in the left and right hemispheres of all prefrontal regions studied, as well as the amygdala, hippocampus, hypothalamus, and raphe nuclei. 5HTT BP(ND) also showed an increase with age in raphe, anterior cingulate cortex, and dorsolateral prefrontal cortex. These changes in brain 5HT systems take place as females establish more adult-like patterns of social behavior, as well as during the HI paradigm. Indeed, the main developmental changes in behavior during the HI (increase in freezing and decrease in submission/appeasement) were related to neurodevelopmental increases in 5HT1A receptors and 5HTT, because the associations between these behaviors and 5HT endpoints emerge at peripuberty. We detected an effect of social status on 5HT1A BP(ND) in the hypothalamus and on 5HTT BP(ND) in the orbitofrontal cortex, with subordinates showing higher BP(ND) than dominants in both cases during the pubertal transition. No main effects of 5HTT genotype were observed for 5HT1A or 5HTT BP(ND). Our findings indicate that adolescence in female rhesus monkeys is a period of central 5HT reorganization, partly influenced by exposure to the social stress of subordination, that likely functions to integrate adrenal and gonadal systems and shape the behavioral response to emotionally challenging social situations.

Social subordination in female macaques represents a well‐described model of chronic psychosocial stress. Additionally, a length polymorphism (5‐HTTLPR) in the regulatory region of the serotonin (5‐HT) transporter (5‐HTT) gene (SLC6A4) is present in rhesus macaques, which has been linked to adverse outcomes similar to that described in humans with an analogous 5‐HTTLPR polymorphism. The present study determined the effects of social status and the 5‐HTTLPR genotype on 5‐HT1A receptor binding potential (5‐HT1A BPND) in brain regions implicated in emotional regulation and stress reactivity in ovariectomised female monkeys, and then assessed how these effects were altered by 17β‐oestradiol (E2) treatment. Areas analysed included the prefrontal cortex [anterior cingulate (ACC); medial prefrontal cortex (mPFC); dorsolateral prefrontal cortex; orbitofrontal prefrontal cortex], amygdala, hippocampus, hypothalamus and raphe nucleui. Positron emission tomography using p‐[18F]MPPF was performed to determine the levels of 5‐HT1A BPND under a non‐E2 and a 3‐week E2 treatment condition. The short variant (s‐variant) 5‐HTTLPR genotype produced a significant reduction in 5‐HT1A BPND in the mPFC regardless of social status, and subordinate s‐variant females showed a reduction in 5‐HT1A BPND within the ACC. Both these effects of 5‐HTTLPR were unaffected by E2. Additionally, E2 reduced 5‐HT1A BPND in the dorsal raphe of all females irrespective of psychosocial stress or 5‐HTTLPR genotype. Hippocampal 5‐HT1A BPND was attenuated in subordinate females regardless of 5‐HTTLPR genotype during the non‐E2 condition, an effect that was normalised with E2. Similarly, 5‐HT1A BPND in the hypothalamus was significantly lower in subordinate females regardless of 5‐HTTLPR genotype, an effect reversed with E2. Taken together, the data indicate that the effect of E2 on modulation of central 5HT1A BPND may only occur in brain regions that show no 5‐HTTLPR genotype‐linked control of 5‐HT1A binding.

2-Deoxy-2-[(18)F]fluoro-D-glucose (2-(18)FDG) has represented radiofluorinated carbohydrates as the most successful tracer for positron emission tomography (PET). 2-(18)FDG uptake depends on glucose metabolism, which is related to a disease progression. 2-(18)FDG has been widely used in oncology, neurology, cardiology, infectious diseases, and inflammation, to complement anatomical modalities such as CT and MRI. Followed by the success of 2-(18)FDG, various radiofluorinated carbohydrates have been evaluated as PET tracers, which include analogs of D-ribose, D-mannose, D-galactose, D-talose, D-fructose, D-allose, lactose, L-fucose, N-acetylneuraminic acid, and L-ascorbic acid. Among those radiofluorinated carbohydrates, several have implied potential for further development. 2-Deoxy-2-[(18)F]fluoro-D-galactose has been developed to assess liver function and diagnose hepatic carcinoma. 6-Deoxy-6-[(18)F]fluoro-D-fructose showed promising characteristics for diagnosis of breast cancer. Three radiofluorinated analogs of lactose have been designed as the substrates of the overexpressed hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein in peritumoral pancreatic tissue for early diagnosis of pancreatic cancer. The metabolism of 6-[(18)F]fluoro-L-fucose suggested that it is a bioactive analog of L-fucose in the synthesis of glycoconjugate macromolecules. 6-Deoxy-6-[(18)F]fluoro-L-ascorbic acid was evaluated to assess antioxidant function of L-ascorbic acid in rodent models of transient global ischemia and glutathione deficiency.