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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.

Purpose: The hypoxia-inducible factor-1 (HIF-1) plays a critical role in tumor adaptation to hypoxia, and its elevated expression correlates with poor prognosis and treatment failure in patients with cancer. In this study, we determined whether 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1, the lead inhibitor in a novel class of arylsulfonamide inhibitors of the HIF-1 pathway, had antitumorigenic properties in vivo and further defined its mechanism of action.Experimental Design: We studied the inhibitory effect of systemic KCN1 delivery on the growth of human brain tumors in mice. To define mechanisms of KCN1 anti-HIF activities, we examined its influence on the assembly of a functional HIF-1α/HIF-1β/p300 transcription complex.Results: KCN1 specifically inhibited HIF reporter gene activity in several glioma cell lines at the nanomolar level. KCN1 also downregulated transcription of endogenous HIF-1 target genes, such as VEGF, Glut-1, and carbonic anhydrase 9, in a hypoxia-responsive element (HRE)-dependent manner. KCN1 potently inhibited the growth of subcutaneous malignant glioma tumor xenografts with minimal adverse effects on the host. It also induced a temporary survival benefit in an intracranial model of glioma but had no effect in a model of melanoma metastasis to the brain. Mechanistically, KCN1 did not downregulate the levels of HIF-1α or other components of the HIF transcriptional complex; rather, it antagonized hypoxia-inducible transcription by disrupting the interaction of HIF-1α with transcriptional coactivators p300/CBP.Conclusions: Our results suggest that the new HIF pathway inhibitor KCN1 has antitumor activity in mouse models, supporting its further translation for the treatment of human tumors displaying hypoxia or HIF overexpression. Clin Cancer Res; 18(24); 6623–33. ©2012 AACR.

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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.

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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 20mg/kg fluoxetine or vehicle two-hours prior to a test with a non-aggressive intruder. Female rhesus monkeys (n=14) housed were administered fluoxetine (2.8mg/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

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 BP(ND)) 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 (E(2)) 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-[(18) F]MPPF was performed to determine the levels of 5-HT1A BP(ND) under a non-E(2) and a 3-week E(2) treatment condition. The short variant (s-variant) 5-HTTLPR genotype produced a significant reduction in 5-HT1A BP(ND) in the mPFC regardless of social status, and subordinate s-variant females showed a reduction in 5-HT1A BP(ND) within the ACC. Both these effects of 5-HTTLPR were unaffected by E(2). Additionally, E(2) reduced 5-HT1A BP(ND) in the dorsal raphe of all females irrespective of psychosocial stress or 5-HTTLPR genotype. Hippocampal 5-HT1A BP(ND) was attenuated in subordinate females regardless of 5-HTTLPR genotype during the non-E(2) condition, an effect that was normalised with E(2). Similarly, 5-HT1A BP(ND) in the hypothalamus was significantly lower in subordinate females regardless of 5-HTTLPR genotype, an effect reversed with E(2). Taken together, the data indicate that the effect of E(2) on modulation of central 5HT1A BP(ND) may only occur in brain regions that show no 5-HTTLPR genotype-linked control of 5-HT1A binding.

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.

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.

The hypoxia inducible factor (HIF) pathway is an attractive target for cancer, as it controls tumor adaptation to growth under hypoxia and mediates chemotherapy and radiation resistance. We previously discovered 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide as a novel, small-molecule HIF-1 pathway inhibitor in a high-throughput cell-based assay, but its in vivo delivery is hampered by poor aqueous solubility (0.009 μM in water; log P7.4 = 3.7). Here we describe the synthesis of 12 N-alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)methyl]heteroarylsulfonamides, which were designed to possess optimal lipophilicities and aqueous solubilities by in silico calculations. Experimental log P7.4 values of 8 of the 12 new analogs ranged from 1.2–3.1. Aqueous solubilities of three analogs were measured, among which the most soluble N-[(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)methyl]-N-(propan-2-yl)pyridine-2-sulfonamide had an aqueous solubility of 80 μM, e.g., a solubility improvement ...

We have discovered that 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, a novel small molecule HIF-1 pathway inhibitor, can antagonize tumor growth in animal models of cancer, but the treatment necessitates its delivery in a formulation, due to poor water solubility (15 μg/mL; pH 7.4), evidencing that the chemotype needs further exploration of its amenability to additional chemical modifications for ultimate optimization of function and pharmacology. As a first step towards this goal we investigated the structure-activity relationships of 15 lipophilic 2,2-dimethyl-2H-chromene based arylsulfonamide analogs of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide to find out strategies of modification. A 3,4-dimethoxybenzenesulfonyl group in region 1 showed the strongest inhibition among five arylsulfonyl groups tested. The presence of propan-2-amine in region 2 conferred the strongest inhibitory effect of the compound on HIF-1 activated transcription in a reporter assay. These findings are important as they help define the structural motifs where the 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide can be chemically modified to improve its pharmacological properties towards development as a cancer therapeutic.

Several classes of drugs bind to the dopamine transporter (DAT) with high affinity, but some are weaker positive reinforcers than cocaine, suggesting that affinity for and occupancy of the DAT is not the only determinant of a drug's reinforcing effectiveness. Other factors such as the rate of onset have been positively and strongly correlated with the reinforcing effects of DAT inhibitors in nonhuman primates. In the current studies, we examined the effects of acute systemic administration of cocaine and three cocaine analogs (RTI-150, RTI-177, and RTI-366) on binding to DAT in squirrel monkey brain using positron emission tomography (PET) neuroimaging. During the PET scan, we also measured drug effects on dopamine (DA) levels in the caudate using in vivo microdialysis. In general, our results suggest a lack of concordance between drug occupancy at DAT and changes in DA levels. These studies also indicate that acute cocaine administration decreases the availability of plasma membrane DAT for binding, even after cocaine is no longer blocking DA uptake as evidence by a return to basal DA levels.

Using the known peroxide oxidation of N‐benzyl‐3‐phenylseleno‐2‐pyrrolidinone with 30% hydrogen peroxide at −5°C after 3.5 h, we prepared the expected N‐benzyl‐3‐pyrrolin‐2‐one. However, reaction with 30% hydrogen peroxide for 12 h (−5°C→ambient) gave the unexpected product N‐benzyl‐5‐hydroxy‐3‐pyrrolidin‐2‐one in 84% isolated yield. This procedure is a new synthetic route to N‐benzyl‐5‐hydroxy‐3‐pyrrolidin‐2‐one.

2-Methoxy-3,17β-estradiol, an endogenous estrogen metabolite, showed cytotoxicity in various cancer cell lines and also has antiangiogenic and proapoptotic activities. Clinical I and II trials of 2-methoxy-3,17β-estradiol for multiple myeloma, advanced solid tumors, metastatic breast and prostate cancer are underway. We prepared 2-[11C]methoxy-3,17β-estradiol to measure the pharmacokinetics and organ distribution of 2-methoxy-3,17β-estradiol in clinical trials. 2-[11C]Methoxy-3,17β-estradiol was synthesized from a precursor, 2-hydroxy-3,17β-O-bis(methoxymethyl)estradiol, in two steps with over 99% radiochemical purity. The overall reaction time was 45 min and the decay-corrected radiochemical yield was 32.9%. The distribution coefficient (logP7.4) of 2-[11C]methoxy-3,17β-estradiol at pH 7.4 was measured as 2.95. Copyright © 2006 John Wiley & Sons, Ltd.

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/m(2) 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 [(11)C]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 [(18)F]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 [(11)C]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.

Purpose: The hypoxia-inducible factor-1 (HIF-1) plays a critical role in tumor adaptation to hypoxia, and its elevated expression correlates with poor prognosis and treatment failure in patients with cancer. In this study, we determined whether 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1, the lead inhibitor in a novel class of arylsulfonamide inhibitors of the HIF-1 pathway, had antitumorigenic properties in vivo and further defined its mechanism of action. Experimental Design: We studied the inhibitory effect of systemic KCN1 delivery on the growth of human brain tumors in mice. To define mechanisms of KCN1 anti-HIF activities, we examined its influence on the assembly of a functional HIF-1α/HIF-1β/p300 transcription complex. Results: KCN1 specifically inhibited HIF reporter gene activity in several glioma cell lines at the nanomolar level. KCN1 also downregulated transcription of endogenous HIF-1 target genes, such as VEGF, Glut-1, and carbonic anhydrase 9, in a hypoxia-responsive element (HRE)-dependent manner. KCN1 potently inhibited the growth of subcutaneous malignant glioma tumor xenografts with minimal adverse effects on the host. It also induced a temporary survival benefit in an intracranial model of glioma but had no effect in a model of melanoma metastasis to the brain. Mechanistically, KCN1 did not downregulate the levels of HIF-1α or other components of the HIF transcriptional complex; rather, it antagonized hypoxia-inducible transcription by disrupting the interaction of HIF-1α with transcriptional coactivators p300/CBP. Conclusions: Our results suggest that the new HIF pathway inhibitor KCN1 has antitumor activity in mouse models, supporting its further translation for the treatment of human tumors displaying hypoxia or HIF overexpression. Clin Cancer Res; 18(24); 6623–33. ©2012 AACR.

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.

Acute SSRI (selective serotonin reuptake inhibitor) treatment has been shown to attenuate the abuse-related effects of cocaine; however, SSRIs have had limited success in clinical trials for cocaine abuse, possibly due to neurobiological changes that occur during chronic administration. In order to better understand the role of serotonin (5HT) in cocaine abuse and treatment, we examined the effects of chronic treatment with the SSRI fluoxetine at clinically relevant serum concentrations on cocaine-related neurobiology and behavior. Rhesus macaques self-administering cocaine underwent a 6-week dosing regimen with fluoxetine designed to approximate serum concentrations observed in humans. Self-administration and reinstatement were monitored throughout the treatment and washout period. In vivo microdiaylsis was used to assess changes in dopaminergic and serotonergic neurochemistry. Positron emission tomography was used to assess changes in the 5HT transporter and 2A receptor binding potential (BP). Functional output of the 5HT system was assessed using prolactin levels. Cocaine-primed reinstatement and cocaine-elicited dopamine overflow were significantly suppressed following chronic fluoxetine treatment. 5HT2A receptor BP was increased in the frontal cortex following treatment while prolactin release was blunted, suggesting desensitization of the 5HT2A receptor. These effects persisted after a 6-week washout period. Measures of pre-synaptic serotonergic function and cocaine self-administration were unaffected. These data demonstrate that acute and chronic fluoxetine treatments exert different effects on cocaine-related behavior. Furthermore, chronic fluoxetine treatment causes alterations in 5HT2A receptors in the frontal cortex that may selectively disrupt cocaine-primed reinstatement. Fluoxetine may not be useful for treatment of ongoing cocaine abuse but may be useful in relapse prevention.

Hypoxia inducible factors (HIFs) are transcription factors that activate expression of multiple gene products and promote tumor adaptation to a hypoxic environment. To become transcriptionally active, HIFs associate with cofactors p300 or CBP. Previously, we found that arylsulfonamides can antagonize HIF transcription in a bioassay, block the p300/HIF-1α interaction, and exert potent anticancer activity in several animal models. In the present work, KCN1-bead affinity pull down, (14)C-labeled KCN1 binding, and KCN1-surface plasmon resonance measurements provide initial support for a mechanism in which KCN1 can bind to the CH1 domain of p300 and likely prevent the p300/HIF-1α assembly. Using a previously reported NMR structure of the p300/HIF-1α complex, we have identified potential binding sites in the p300-CH1 domain. A two-site binding model coupled with IC50 values has allowed establishment of a modest ROC-based enrichment and creation of a guide for future analogue synthesis.

Reboxetine analogues with methyl and fluoroalkyl substituents at position 2 of the phenoxy ring 1-4 were synthesized. In vitro competition binding with [(3)H]nisoxetine demonstrated that 1-4 have a high affinity for the norepinephrine transporter (NET) with K(i)'s = 1.02, 3.14, 3.68, and 0.30 nM, respectively. MicroPET imaging in rhesus monkeys showed that the relative regional distribution of [(11)C]1 and [(11)C]4 is consistent with distribution of the NET in the brain, while [(18)F]2 and [(18)F]3 showed only slight regional differentiation in brain uptake. Especially, the highest ratios of uptake of [(11)C]1 in NET-rich regions to that in caudate were obtained at 1.30-1.45 at 45 min and remained relatively constant over 85 min. Pretreatment of the monkey with the selective NET inhibitor, desipramine, decreased the specific binding for both [(11)C]1 and [(11)C]4. PET imaging in awake monkeys suggested that anesthesia influenced the binding potential of [(11)C]1 and [(11)C]4 at the NET.

Using the known peroxide oxidation of N‐benzyl‐3‐phenylseleno‐2‐pyrrolidinone with 30% hydrogen peroxide at −5°C after 3.5 h, we prepared the expected N‐benzyl‐3‐pyrrolin‐2‐one. However, reaction with 30% hydrogen peroxide for 12 h (−5°C→ambient) gave the unexpected product N‐benzyl‐5‐hydroxy‐3‐pyrrolidin‐2‐one in 84% isolated yield. This procedure is a new synthetic route to N‐benzyl‐5‐hydroxy‐3‐pyrrolidin‐2‐one.

A long-acting, thermostable bacterial cocaine esterase (CocE) has been identified that rapidly degrades cocaine with a K(M) 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 [(11)C]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.