Norepinephrine Transporter Protein Research Articles

Norepinephrine transporter and vesicular monoamine transporter 2 tumor expression as a predictor of response to 131 I-MIBG in patients with relapsed/refractory neuroblastoma.

Prior studies suggest that norepinephrine transporter (NET) and vesicular monoamine transporter 2 (VMAT2) mediate meta-iodobenzylguanidine (MIBG) uptake and retention in neuroblastoma tumors. We evaluated the relationship between NET and VMAT2 tumor expression and clinical response to 131 I-MIBG therapy in patients with neuroblastoma. Immunohistochemistry (IHC) was used to evaluate NET and VMAT2 protein expression levels on archival tumor samples (obtained at diagnosis or relapse) from patients with relapsed or refractory neuroblastoma treated with 131 I-MIBG. A composite protein expression H-score was determined by multiplying a semi-quantitative intensity value (0-3+) by the percentage of tumor cells expressing the protein. Tumor samples and clinical data were available for 106 patients, of whom 28.3% had partial response (PR) or higher. NET H-score was not significantly associated with response (≥PR), though the percentage of tumor cells expressing NET was lower among responders (median 80% for ≥PR vs. 90% for <PR; p=.0014). VMAT2 H-score was not significantly associated with PR or higher versus less than PR, though patients with PR or higher had lower VMAT2 staining intensity (p=.005). VMAT2 H-score was significantly lower in patients with complete response (median 40 vs. 210 for patients with <complete response; p=.0049). VMAT2 H-scores were significantly higher in ganglioneuroblastoma (vs. neuroblastoma; p=.037), differentiated/poorly differentiated tumors (vs. undifferentiated; p=.0047), and tumors lacking MYCN amplification (vs. MYCN amplified; p=.0011). Markers of lower NET and VMAT2 protein expression are associated with higher likelihood of response to 131 I-MIBG therapy in patients with relapsed/refractory neuroblastoma. Increased VMAT2 protein expression is associated with a more differentiated disease phenotype.

Variable Response of Norepinephrine Transporter to Traumatic Stress and Relationship to Hyperarousal.

The noradrenergic systems play a key role in stress triggered disorders such as post-traumatic stress disorder (PTSD). We hypothesized that traumatic stress will alter expression of norepinephrine transporter (NET) in locus coeruleus (LC) and its target brain regions which could be related to hyperarousal. Male Sprague-Dawley rats were subjected to single prolonged stress (SPS) and several weeks later the LC was isolated. NET mRNA levels in LC, determined by RT-PCR, displayed variable response with high and low responsive subgroups. In different cohort, acoustic startle response (ASR) was measured 2 weeks after SPS and levels of NET mRNA and protein in LC determined. The high NET responsive subgroup had greater hyperarousal. Nevertheless, NET protein levels, as determined by western blots, were lower than unstressed controls in LC, ventral hippocampus and medial prefrontal cortex and displayed considerable variability. Hypermethylation of specific CpG region in promoter of SLC6A2 gene, encoding NET, was present in the low, but not high, NET mRNA responsive subgroup. Taken together, the results demonstrate variability in stress elicited changes in NET gene expression and involvement of epigenetic changes. This may underlie mechanisms of susceptibility and resilience to traumatic stress triggered neuropsychiatric symptoms, especially hyperarousal.

Pharmacological Characterization of Toludesvenlafaxine as a Triple Reuptake Inhibitor.

Toludesvenlafaxine hydrochloride dihydrate is a novel chemical entity and a potential triple monoamine reuptake inhibitor. This study characterized the in vitro triple reuptake inhibition activity, antidepressant-like activity in animals, and pharmacokinetic profiles in rats of toludesvenlafaxine. Binding affinity was determined using human serotonin transporter (SERT) protein, norepinephrine transporter (NET) protein and dopamine transporter (DAT) protein, and the reuptake inhibition was determined using Chinese hamster ovary cells expressing human SERT, NET and DAT. The antidepressant-like activity was examined in rat chronic unpredictable mild stress model and olfactory bulbectomized model. In rats, the tissue distribution and pharmacokinetic parameters were determined. Toludesvenlafaxine had high binding affinity on SERT, NET and DAT, and significantly inhibited the reuptake of serotonin (IC50 = 31.4 ± 0.4 nM), norepinephrine (IC50 = 586.7 ± 83.6 nM) and dopamine (IC50 = 733.2 ± 10.3 nM) in vitro. Toludesvenlafaxine demonstrated significant antidepressant-like effects in rat models at 8–16 mg/kg. In addition, toludesvenlafaxine significantly reduced serum corticosterone and significantly increased testosterone levels in rats. Toludesvenlafaxine was quickly absorbed and converted to O-desvenlafaxine (ODV) after oral administration, both of which were selectively distributed into the hypothalamus with high concentration. Plasma ODV exposure was proportionally related to the doses after oral dosing. These results suggest that toludesvenlafaxine is a triple reuptake inhibitor with relatively fast-acting antidepressant-like activity and good therapeutic profile including improvement of anhedonia and sexual function.

Rhopressa-induced corneal edema: a case report

BackgroundRhopressa (netarsudil) has recently been added to the arsenal of treatment for open-angle glaucoma. It is an effective norepinephrine transporter and Rho-associated protein kinase (ROCK) inhibitor used to decrease intraocular pressure (IOP), with the most common side effect being conjunctival hyperemia.Case presentationWe report a unique case of Rhopressa-induced corneal edema in a 79-year-old African-American woman, which resolved after discontinuation. She had a history of smoking one cigarette per day and did not consume alcohol. She had no history of corneal edema or uveitis.ConclusionsPrevious case reports have documented patients with Rhopressa-induced corneal edema; however, they have all had a preexisting history of corneal edema or uveitis. We believe that this is a unique case of Rhopressa-induced corneal edema in a relatively healthy eye. While Rhopressa is effective in managing glaucoma, there may be effects of treatment that are still unknown. We will discuss clinical findings of our case, along with a review of previous literature on Rhopressa and novel ROCK inhibitors. We hope that we can add to the existing body of literature and invite further investigation of Rhopressa and ROCK inhibitors and their effects on the cornea.

Norepinephrine transporter expressed on mammary epithelial cells incorporates norepinephrine in milk into the cells

Mammary epithelial cells synthesize and secrete norepinephrine (NE) into breast milk to regulate β-casein expression through the adrenergic β2 receptor. We investigated the expression, localization, and roles of NE transporter (NET) in the mammary epithelium during lactation. mRNA and protein levels of NET were determined in primary normal human mammary epithelial cells (pHMECs) and non-malignant human mammary epithelial MCF-12A cells. In nursing CD1 mice, NET localized to the apical membranes of the mammary epithelium. The intracellular NE content of pHMECs incubated with NE increased. Although the β-casein concentration in milk was slightly higher at day 10 than at day 2 of lactation, the NE concentration and lactation-related proteins were only slightly changed on days 2–10. Restraint stress increased the NE concentration in milk from nursing mice and NET protein levels were significantly higher than in non-stressed nursing mice. NET is expressed on the apical membrane of mammary epithelial cells and incorporates NE in milk into cells, potentially regulating the NE concentration in milk.

Dexmedetomidine reduces inflammation in mice with acute pancreatitis by inhibiting NLRP3 inflammasome and sympathetic nerve activity

Purpose: To study the anti-inflammatory influence of dexmedetomidine (DEX) in mice with acute pancreatitis (AP), and to determine the underlying mechanism.Methods: A total of 75 healthy ICR male mice were randomly divided into control, mild acute pancreatitis (MAP), MAP+DEX, severe acute pancreatitis (SAP), and SAP+DEX groups, with 15 mice/group. Blood levels of inflammatory factors (TNF-α and IL-1β) and norepinephrine were assayed ineach group. Western blotting was used to assay the protein expressions of NLRP3 and norepinephrine transporter (NET) in the pancreatic tissue of each group.Results: The levels of inflammatory factors in the MAP+DEX group were markedly lower than those in the MAP group after 10 h of MAP induction (p &lt; 0.01). Mice in MAP+DEX group had significantly lower expression of NLRP3 in pancreatic tissue, and significantly higher NET protein level, relative to the MAP mice. Following 10 h of SAP, concentrations of the inflammatory factors and the pancreatic expression of NLRP3 were lower in SAP+DEX-treated mice than in SAP mice, while NET protein was significantly higher in SAP mice (p &lt; 0.01).Conclusion: DEX reduces the expressions of inflammation-related factors TNF-α and IL-1β, and inhibits inflammatory response in mice with AP via downregulation of NET protein expression via inhibition of NLRP3 and early sympathetic events.&#x0D; Keywords: Dexmedetomidine, NLRP3 inflammasome, Sympathetic nerve, Acute pancreatitis, Inflammatory response

Evidence that Sympathetic Activity is Increased Selectively to the Heart in a Mouse Model of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM), the most common genetic heart disease, is characterized by cardiac hypertrophy, fibrosis, and increased risk of arrhythmias and sudden death, even in the absence of heart failure. We have studied young (&lt;20 wks) transgenic mice with cardiac‐specific expression of mutated alpha‐tropomyosin (Glu180Gly), an established model of HCM, to investigate autonomic mechanisms. In early experiments, abnormal heart rate and blood pressure responses to β‐adrenergic receptor blockade and ganglionic blockade indicated high cardiac accompanied by low vasomotor sympathetic tone in HCM vs. wild‐type (WT) mice. The aim of this study was to further characterize regional sympathetic activity in HCM vs. WT littermates by measuring expression/activity of tyrosine hydroxylase (TH, rate‐limiting enzyme for norepinephrine (NE) synthesis) and the norepinephrine transporter (NET) in sympathetic ganglia (stellate, superior cervical, celiac/mesenteric), and expression of β1 receptors in left ventricle (LV). Results (below) are expressed relative to WT (n=4 male mice each group). TH protein expression (Western blot) (standardized to neuronal content with PGP9.5) was significantly higher in stellate (cardiac) ganglia in HCM mice (HCM 162±12 vs. WT 100±7; p&lt;0.05), but not significantly different in superior cervical (HCM 82±4 vs. WT 100±14; p&gt;0.05) or celiac‐mesenteric (HCM 95±7 vs. WT 100±4; p&gt;0.05) ganglia. TH/PGP9.5 protein expression was also higher in the LV of HCM (135±15) vs. WT (100±1) mice (p&lt;0.05). LV β1 receptor protein expression was significantly lower in HCM (60±1) vs. WT (100±4) mice (p&lt;0.05), consistent with increased local NE levels. NET protein (immunofluorescence) was lower in stellate ganglia from HCM mice vs. WT (n=3 mice per group, 3 sections/mouse, p&lt;0.05). NET activity was assessed by measuring the rate of uptake of a fluorescent NET substrate (Molecular Devices) into individual sympathetic neurons isolated from stellate and superior cervical ganglia. NET activity measured in stellate neurons was significantly lower in HCM (4.5±0.3 a.u.*min−1, n=115 neurons, from 4 mice) vs. WT (5.3±0.4 a.u.*min−1, n=98 neurons, from 4 mice). NET activity in superior cervical ganglia did not differ between genotypes. In conclusion, increased TH expression (enhanced ‐NE synthesis) and decreased NET expression/activity (reduced NE reuptake) in stellate ganglia are consistent with increased cardiac sympathetic activity in HCM, whereas the lack of differences between HCM and WT in other sympathetic ganglia are consistent with normal sympathetic activity to non‐cardiac target organs in HCM.Support or Funding InformationNIH F32HL140880

Role of Neuro-Immune Cross-Talk in the Anti-obesity Effect of Electro-Acupuncture.

There is evidence to show that electro-acupuncture (EA) has a promotive effect on both lipolysis and thermogenesis, and that these mechanisms underlie the anti-obesity effect of EA. The sympathetic nervous system (SNS) is known to play a role in thermogenesis. Additionally, obesity is characterized by a chronic low-grade inflammatory state. Based on these findings, the aim of the present study is to investigate the potential neuro-immune mechanisms underlying the therapeutic effect of EA in obesity. In the experiment, we used a high fat diet (HFD) rats model to study the effect of EA in reducing body weight. EA increases the activity of sympathetic nerves in inguinal white adipose tissue (iWAT), especially in the HFD group. Compared to HFD rats, EA can decrease sympathetic associated macrophage (SAM) and the level of norepinephrine transporter protein (Slc6a2). The relative uncoupling protein 1 expression shows EA increases thermogenesis in iWAT, and increases β3 receptors. Interestingly, injecting β antagonist in iWAT increases Slc6a2 protein levels. Additionally, the SNS-macrophage cross-talk response to EA showed in iWAT but not in epididymis white adipose tissue. The results of the present study indicate that EA exerts its anti-obesity effect via three mechanisms: (1) inhibition of SAMs and the norepinephrine transporter protein SlC6a2, (2) promoting SNS activity and thermogenesis, and (3) regulating immunologic balance.

Spatial Inhomogeneity of Cardiac Norepinephrine Transport Protein and Meta-[123I]Iodobenzylguanidine Uptake in Swine Myocardial Tissue.

The aim of the present study was to evaluate the expression of the cardiac norepinephrine transporter (NET) in the left ventricle (LV) of healthy pigs and its relationship with regional meta-[123I]iodobenzylguanidine ([123I]MIBG) myocardial uptake. Experiments were performed on animals injected with [123I]MIBG and acquired 2h later using an ultrafast CZT gamma camera to assess the regional myocardial uptake. After image acquisition, animals were euthanized; the heart was quickly excised and underwent to an ex vivo single photon emission tomography (SPECT) imaging. Four small samples of tissue were then harvested from mid-walls and apex of the left ventricle; NET densities were evaluated and further normalized for protein loading per cardiac region. Three variants of NET protein with different molecular weights were detected. The expression of NET was not homogenous in the LV, with the highest density in the inferior wall and the lowest one in the apical area. The regional in vivo [123I]MIBG uptake revealed an analogous trend, showing a good linear relationship with NET expression. Parallel results were obtained from the ex vivo study. This study elucidates the expression of three different variants of NET proteins into the left ventricular myocardium of a healthy pig. NET expression into the LV was not homogeneous and paralleled by differences in regional [123I]MIBG uptake. Moreover, the correlation and the agreement between measurements of regional expression of NET variants and [123I]MIBG uptake represent a relevant finding for inferences about NET expression in the context of clinical imaging.

Oxytocin modulates the expression of norepinephrine transporter, β3-adrenoceptors and muscarinic M2 receptors in the hearts of socially isolated rats

Social stress produces behavioral alterations, and autonomic and cardiac dysfunction in animals. In addition to the well-known roles of oxytocin on birth and maternal bonding, recent evidence shows that this neuropeptide possesses cardio-protective properties. However less is known about its role in the regulation of the autonomic nervous system. The direct influence of oxytocin on the cardiac catecholamine synthesizing enzyme, transport beta-adrenoceptors and muscarinic receptors in animals exposed to chronic social isolation stress has not yet been studied. In this study, we examined the influence of peripheral chronic oxytocin treatment on anxiety-related behavior, the morphology and content of epinephrine and norepinephrine, mRNA and protein levels of tyrosine hydroxylase (TH), norepinephrine transporter (NET) and receptors <beta> 3 (β3-AR) and muscarinic 2 (M2 MR) in the right and left cardiac atrium and ventricle of chronically socially isolated male rats. Our results show that oxytocin treatment exhibits an anxiolytic effect, decreases the heart/body weight ratio and prevents the hypertrophy of cardiomyocytes in the wall of the left ventricle of stressed rats. Epinephrine and TH protein levels were unchanged after prolonged oxytocin treatment. Peripheral oxytocin administration led to the enhancement of gene expression of β3-AR in both atria, NET protein in the left ventricle and gene expression of M2 MR in the right atrium and the left ventricle of chronically socially isolated rats. The study provides evidence that oxytocin treatment in chronically socially isolated animals enhances norepinephrine uptake and expression of cardio-inhibitory receptors in cardiac tissues, which could have a beneficial effect on the cardiovascular system under the increased activity of the sympathoneural system.

P.3.027 - Norepinephrine transporter gene and protein expression of the human brain investigated with postmortem data and PET

IL-22 is an IL-10-family cytokine that regulates chronic inflammation. We investigated the role of IL-22 and its receptor, IL-22R1, in the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP).IL-22 and IL-22R1 protein and mRNA expression in NP and in uncinate tissues (UT) from CRS and non-CRS patients was examined using immunohistochemistry and real-time PCR, respectively. Dispersed NP and UT cells were cultured with the Staphylococcus aureus exotoxins, staphylococcal enterotoxin B and alpha-toxin, following which exotoxin-induced IL-22 levels and their association with clinicopathological factors were analyzed. Effects of IL-22 on MUC1 expression and cytokine release in NP cells were also determined.IL-22 and IL-22R1 in NP were mainly expressed in infiltrating inflammatory cells and in epithelial cells, respectively. IL-22 mRNA levels in NP were significantly higher than those in UTs from non-CRS patients whereas IL-22R1 levels were conversely lower in NPs. NP cells produced substantial amounts of IL-22 in response to exotoxins. Exotoxin-induced IL-22 production by NP cells significantly and negatively correlated with the degree of local eosinophilia and postoperative computed tomography (CT) score, whereas conversely it positively correlated with the forced expiratory volume in 1s (FEV1)/forced vital capacity (FVC) ratio. IL-22 significantly enhanced MUC1 mRNA expression in NP cells. IL-22-induced MUC1 mRNA levels were significantly and positively correlated with IL-22R1 mRNA levels in NPs.These data suggest that imbalance of IL-22/IL-22R1 signaling regulates the pathogenesis of CRSwNP, including local eosinophilia, via alteration of MUC1 expression.

Gestational stress, placental norepinephrine transporter and offspring fertility.

Chronic cold stress produces adrenergic overload that can affect fetal development. The placental norepinephrine transporter (NET) clears norepinephrine (NE) from both maternal circulation and the fetus during gestation. If this system fails, NE clearance can be reduced, leading to high fetal exposure to NE. The main aim of this study was to determine the changes in NET expression during gestation and their relationship with the functional capacity of NET to transport NE under stressful conditions. Additionally, this study correlated these findings with the reproductive capacity of 2nd-generation progeny. Pregnant rats were subjected to chronic cold stress at 4°C for 3 h each day throughout their pregnancies. We found that exposure of pregnant rats to sympathetic stress caused the following effects: increased NE and corticosterone levels throughout pregnancy, decreased capacity of the placenta to clear NE from the fetus to the mother's circulation, altered NET protein levels depending on the sex of the fetus and increased placental and body weights of pups. For the first time, we also described the disrupted fertility of progeny as adults. Increased NE plasma levels during pregnancy under sympathetic stress conditions correlated with decreased NET functionality that provoked changes in the development of progeny and their fertility in adulthood.

MicroRNAs 29b and 181a down-regulate the expression of the norepinephrine transporter and glucocorticoid receptors in PC12 cells.

MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The norepinephrine transporter (NET) and glucocorticoid receptors are closely related to the homeostatic integration and regulation after stress. Our previous studies demonstrated that NET mRNA and protein levels in rats are regulated by chronic stress and by administration of corticosterone, which is mediated through glucocorticoid receptors. Whether miRNAs are intermediaries in the regulation of these proteins remains to be elucidated. This study was undertaken to determine possible regulatory effects of miRNAs on the expression of NET and glucocorticoid receptors in the noradrenergic neuronal cell line. Using computational target prediction, we identified several candidate miRNAs potentially targeting NET and glucocorticoid receptors. Western blot results showed that over-expression of miR-181a and miR-29b significantly repressed protein levels of NET, which is accompanied by a reduced [3 H] norepinephrine uptake, and glucocorticoid receptors in PC12 cells. Luciferase reporter assays verified that both miR-181a and miR-29b bind the 3'UTR of mRNA of NET and glucocorticoid receptors. Furthermore, exposure of PC12 cells to corticosterone markedly reduced the endogenous levels of miR-29b, which was not reversed by the application of glucocorticoid receptor antagonist mifepristone. These observations indicate that miR-181a and miR-29b can function as the negative regulators of NET and glucocorticoid receptor translation invitro. This regulatory effect may be related to stress-induced up-regulation of the noradrenergic phenotype, a phenomenon observed in stress models and depressive patients. This study demonstrated that miR-29b and miR-181a, two short non-coding RNAs that provide global regulation of gene expression, markedly repressed protein levels of norepinephrine (NE) transporter and glucocorticoid receptor (GR), as well as NE uptake by binding the 3'UTR of their mRNAs in PC12 cells. Also, exposure of cells to corticosterone significantly reduced miR-29b levels through a GR-independent way.

Dexmedetomidine does not interfere with meta-iodobenzylguanidine (MIBG) uptake at clinically relevant concentrations.

Neuroblastoma is a pediatric malignancy, and most tumor cells express the norepinephrine transporter (NET) enabling uptake of NET ligands. Meta-iodobenzylguanidine (MIBG) is a NET-specific ligand used as a highly specific imaging agent and targeted radiotherapeutic. Patients with neuroblastoma frequently require sedation during targeted radiotherapy. Dexmedetomidine has been increasingly used to achieve efficacious sedation. There are theoretical concerns that this highly selective alpha-2 adrenergic receptor agonist may interfere with active uptake of MIBG through the NET transporter. In this study, we analyzed the impact of [125-iodine]-labeled MIBG ([125 I]MIBG) uptake in the presence of dexmedetomidine in human neuroblastoma-derived cellular models. Carrier-free [125 I]MIBG was synthesized using UltraTrace® resin (Molecular Insight Pharmaceuticals, Inc., Tarrytown, NY) through radioiododestannylation from a tin precursor bound by a solid-state polymer. NET (SLC6A2) protein expression was determined in human neuroblastoma cell lines (BE2C, SKNSH and IMR5). [125 I]MIBG internalization studies were performed using [125 I]MIBG alone or in combination with either desipramine or dexmedetomidine. Dexmedetomidine and desipramine competitive inhibition studies were performed and concentration at 50% maximal inhibition was calculated. Finally, NET inhibitor dissociation studies were performed in which after pre-incubation with either desipramine or dexmedetomidine, cells were washed and [125 I]MIBG was added. We show dose-dependent inhibition of [125 I]MIBG uptake by dexmedetomidine, but at several logs lower potency than the known NET inhibitor desipramine. A review of pediatric dexmedetomidine pharmacokinetic data shows that the concentrations achieved in the serum are much lower than those required to block MIBG uptake. We conclude that dexmedetomidine will not interfere with therapeutic [131 I]MIBG efficacy.

Organic cation transporter 3 contributes to norepinephrine uptake into perivascular adipose tissue.

Perivascular adipose tissue (PVAT) reduces vasoconstriction to norepinephrine (NE). A mechanism by which PVAT could function to reduce vascular contraction is by decreasing the amount of NE to which the vessel is exposed. PVATs from male Sprague-Dawley rats were used to test the hypothesis that PVAT has a NE uptake mechanism. NE was detected by HPLC in mesenteric PVAT and isolated adipocytes. Uptake of NE (10 μM) in mesenteric PVAT was reduced by the NE transporter (NET) inhibitor nisoxetine (1 μM, 73.68 ± 7.62%, all values reported as percentages of vehicle), the 5-hydroxytryptamine transporter (SERT) inhibitor citalopram (100 nM) with the organic cation transporter 3 (OCT3) inhibitor corticosterone (100 μM, 56.18 ± 5.21%), and the NET inhibitor desipramine (10 μM) with corticosterone (100 μM, 61.18 ± 6.82%). Aortic PVAT NE uptake was reduced by corticosterone (100 μM, 53.01 ± 10.96%). Confocal imaging of mesenteric PVAT stained with 4-[4-(dimethylamino)-styrl]-N-methylpyridinium iodide (ASP(+)), a fluorescent substrate of cationic transporters, detected ASP(+) uptake into adipocytes. ASP(+) (2 μM) uptake was reduced by citalopram (100 nM, 66.68 ± 6.43%), corticosterone (100 μM, 43.49 ± 10.17%), nisoxetine (100 nM, 84.12 ± 4.24%), citalopram with corticosterone (100 nM and 100 μM, respectively, 35.75 ± 4.21%), and desipramine with corticosterone (10 and 100 μM, respectively, 50.47 ± 5.78%). NET protein was not detected in mesenteric PVAT adipocytes. Expression of Slc22a3 (OCT3 gene) mRNA and protein in PVAT adipocytes was detected by RT-PCR and immunocytochemistry, respectively. These end points support the presence of a transporter-mediated NE uptake system within PVAT with a potential mediator being OCT3.

Abstract 1610: Development of meta-[211At]astatobenzylguanidine ([211At]MABG) as an alpha particle emitting systemic targeted radiotherapeutic for neuroblastoma

Abstract BACKGROUND Neuroblastoma (NB) is a radiosensitive malignancy and NB cells express the norepinephrine transporter (NET) enabling uptake of NET ligands. The majority of relapsed NBs remain sensitive to radiation therapy even after extensive therapy. The currently employed targeted radiotherapeutic [131I]MIBG is a highly active β -particle emitting NET ligand useful for cytoreduction of bulk tumor. However, [131I]MIBG does not target isolated tumor cells, and thus targeted radiotherapy with [211At]MABG (α-particle emitter with higher biological effectiveness) can address this critical problem due to the short path length that should lead to the killing of isolated tumor cells. METHODS 211At was synthesized using a bismuth target via the 209Bi(α,2n)211At reaction and used for solid-phase radiosynthesis of [211At]MABG. Cellular models were created by first determining NET (SLC6A2) mRNA and protein expression in 35 human NB cell lines, and then creating isogenic pairs by forced overexpression of NET in 4 NB cell models. We then performed [211At]MABG uptake assays, as well as biodistribution experiments, using these models. We are defining the toxicity of [211At]MABG in mice via ongoing dose escalation and monitoring laboratory parameters every 3 weeks after dosing. A linear mixed effects model was used to determine the differences in the parameters between the treated groups and controls. Finally, clonogenic assays and therapeutic trials in mouse models comparing [131I]MIBG with [211At]MABG are ongoing. RESULTS We synthesized [211At]MABG (radiochemical yield of 25-50%, radiochemical purity &amp;gt; 99%) and showed NET-specific uptake in NB1691 cells. Specificity was demonstrated by competition assays with the blocking agent desipramine. NET-overexpressing cell lines showed 4-10 fold higher uptake of NET ligands than parental isogenic lines, and demonstrated tumor-specific [211At]MABG uptake in vivo with tumor-muscle ratios of 7.37. Toxicity studies have shown that doses of 10 and 25 uCi of [211At]MABG were well tolerated, but there was weight loss observed at higher doses (P = 0.01). There was also an expected trend towards thrombocytopenia (P = 0.06), but no detectable impact on white blood cell counts, (P = 0.74), hemoglobin (P = 0.41), bilirubin (P = 0.53) or creatinine (P = 0.10). Clonogenic assays show [211At]MABG to be potently cytotoxic (1000 fold higher than [131I]MIBG), and murine efficacy studies are ongoing. CONCLUSIONS We have synthesized 211At-MABG in quantities sufficient for our preclinical experiments, and will scale this up for clinical applications. The uptake and biodistribution of [211At]MABG is similar to the currently used radiotherapeutic [131I]MIBG, and to date there was no unanticipated toxicity. Preliminary results from clonogenic assays suggest that [211At]MABG may be an effective agent for salvage therapy for children with refractory/relapsed NB. Citation Format: Vandana Batra, Pietro Ranieri, Mehran Makvandi, Matthew Tsang, Catherine Hou, Yimei Li, Ganesan Vaidyanathan, Daniel A. Pryma, John M. Maris. Development of meta-[211At]astatobenzylguanidine ([211At]MABG) as an alpha particle emitting systemic targeted radiotherapeutic for neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1610. doi:10.1158/1538-7445.AM2015-1610

Abstract B48: Preclinical development of meta-[211At] astatobenzylguanidine ([211At] MABG) targeted radiotherapy for neuroblastoma

Abstract Background: Neuroblastoma (NB) is a radiosensitive malignancy accounting for 10% of childhood cancer mortality. NB cells frequently express the norepinephrine transporter (NET) providing a specific mechanism for uptake of NET-ligands. Meta-[131I]iodobenzylguanidine ([131I]MIBG) is a NET-ligand radiotherapeutic that shows single-agent response rates in refractory NB of 40-50%. However, due to the long path lengths of 131 I beta (β)-emission, and low biological effectiveness compared to alpha (α)-emitting radionuclides, [131I]MIBG is generally not curative, perhaps due to non-targeting of isolated circulating tumor cells. Here we report our efforts to optimize NET-targeted radiotherapy by developing relevant preclinical models of refractory NB for α-particle therapeutic [211At] MABG therapy. Methods: We first determined NET (SLC6A2) mRNA and protein expression in 35 human NB cell lines using quantitative RT-PCR and western blotting. We then chose 5 lines with absent to intermediate levels of native NET expression (NB1691, SKNSH, IMR5, NLF and SKNBE2) for dual forced overexpression of human NET and luciferase cDNAs. We used [125I]MIBG for cell-based uptake assays in all isogenic pairs and biodistribution experiments in athymic mice bearing three separate NET-transduced xenografts (N=5 per cell line). These cell lines were also treated with [131I]MIBG and/or external beam radiation (XRT) followed by multi-log cytotoxicity assays. Therapeutic trials of [131I]MIBG (25 mCi/kg) in NB1691 subcutaneous xenograft and metastatic mouse models were also conducted. In parallel, [211At] MABG was synthesized by: (i) cyclotron-production of 211 At via 209 Bi(α,2n)211At reaction (ii) distillation of 211 At from the target, and (iii) solid phase no-carrier-added synthesis of [211At] MABG by radioastato-destannylation. [211At] MABG uptake studies were performed in isogenic NB cell lines. Results: Unlike primary human NBs, NET expression was low in the majority of 35 cell-lines studied (median normalized expression value = 0.145; range 0.000-1.005), but all transduced lines showed significant overexpression (0.860-1.107) comparable to human primary tumors. Transduced lines showed 4-10 fold higher uptake of [125I]MIBG than non-transduced isogenic parental cell lines in vitro, and demonstrated significant tumor-specific uptake and retention in vivo with tumor-muscle ratios ranging from 13.80 to 29.48. In vitro cytotoxicity experiments using [131I]MIBG showed NET-expressing cell lines to be more susceptible to treatment compared to non-NET expressing pairs (IC50 of 2.937nCi vs. 15.99 nCi). Treatment of mice bearing NB1691-NET xenografts with [131I]MIBG showed tumor growth delay (p=0.0065), but no significant impact on survival, likely due to de novo radioresistance (1200 cGy of XRT had no impact on NB1691 proliferation; IMR-05 showed 97% decreased cell viability). Lastly, we successfully synthesized [211At] MABG, with radiochemical yields of ∼20% and showed NET specific uptake of [211At] MABG into 1691 NET transfected cells. Conclusions: Development of targeted radiotherapy for neuroblastoma has been limited by the lack of preclinical models and alternative therapeutics. Our development of multiple isogenic pairs with varying NET expression, documentation of de novo radiation sensitivity, and the production of [211At] MABG, will allow for rapid assessment of targeted radiotherapeutic strategies (including combination approaches) to support clinical development of alpha-particle therapeutics in a childhood cancer. Citation Format: V Batra, AM Chacko, M Gagliardi, C Hou, J L. Mikitsh, R H. Freifelder, A Kachur, B C. LeGeyt, A Schmitz, L Toto, G Vaidyanathan, M R. Zalutsky, K K. Matthay, W A. Weiss, W C. Gustafson, D Pryma, J M. Maris. Preclinical development of meta-[211At] astatobenzylguanidine ([211At] MABG) targeted radiotherapy for neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B48.

Effects of desipramine treatment on stress-induced up-regulation of norepinephrine transporter expression in rat brains.

Many studies demonstrate down-regulation of the norepinephrine transporter (NET) by desipramine (DMI) in vitro and in stress-naive rats. Little is known regarding regulation of the NET in stressed animals. The present study was designed to investigate effects of DMI on the expression of NET and protein kinases in the stress rat. Adult Fischer 344 rats were subjected to chronic social defeat (CSD) for 4 weeks. DMI (10 mg/kg, intraperitoneal (i.p.)) was administered concurrently with CSD or 1 or 2 weeks after cessation of CSD. Sucrose consumption, NET expression, and protein kinases were measured. CSD significantly increased messenger RNA (mRNA) and protein levels of NET in the locus coeruleus, as well as NET protein levels in the hippocampus, frontal cortex, and amygdala. These effects were nearly abolished when DMI was administered concurrently with CSD. CSD-induced up-regulation of NET expression in the locus coeruleus, hippocampus, and amygdala lasted at least 2 weeks after cessation of CSD, an effect that was significantly attenuated by 1 or 2 weeks of DMI treatment starting from cessation of the CSD. Concurrent administration of DMI with CSD did not markedly interfere with CSD-induced decreases in protein levels of protein kinases A and C in these brain regions, but it did reverse the CSD-induced reduction in phosphorylated cAMP response element-binding (pCREB) protein levels in most brain regions. These findings suggest that NET regulation by DMI occurs in both stressed and behaviorally naive rats, and DMI-induced changes in pCREB may be involved.

Sympathetic Nervous System Moves Toward Center Stage in Cardiovascular Medicine

In 1664, the first anatomically correct depiction of the sympathetic nervous system came from Thomas Willis and his circle of London anatomists,1 included in The Anatomy of the Brain and Nerves , 1664 (Figure 1). This, the first work dedicated completely to the nervous system, also described the arterial loops at the base of the brain, which we now know as the Circle of Willis.1 Christopher Wren, an anatomist member of the group, was the principal illustrator1 before being asked by the City Fathers to turn his talents to town planning, architecture, and cathedral building after the 1666 Great Fire of London. Figure 1. An illustration of the human sympathetic nerves of the neck and thorax, from The Anatomy of the Brain and Sympathetic Nerves , published in 1664 by Thomas Willis and reproduced in Soul Made Flesh .1 Almost 2 centuries later, subsequent microscopic examination demonstrated that blood vessel walls were densely innervated, leading Stelling in 18402 to correctly conclude that these vasomotor fibers were in fact sympathetic nerves that were carried from the central nervous system to the blood vessels. In the mid-19th century, celebrated European physiologists, including Brown-Sequard, Waller, and Bernard,2 built on these observations, demonstrating vasoconstriction with electrical stimulation of the cut nerves and vasodilatation on nerve section, which indicated that the sympathetic fibers exerted a tonic, vasoconstrictor influence. The pressor nerves had gained recognition. Identification of the sympathetic neurotransmitter proved to be difficult. Claims for epinephrine3,4 and the hypothetical sympathins I and E confused the picture. Ulf von Euler compared bioassay responses of epinephrine, norepinephrine, and dihydroxy norephedrine with those of cattle splenic nerve extract, by testing blood pressure (BP) responses in the anesthetized cat and contractile responses in the isolated pregnant rabbit uterus, to definitively demonstrate the …

Cardiac sympathetic innervation and PGP9.5 expression by cardiomyocytes after myocardial infarction: effects of central MR blockade

Central mechanisms involving mineralocorticoid receptor (MR) activation contribute to an increase in sympathetic tone after myocardial infarction (MI). We hypothesized that this central mechanism also contributes to cardiac sympathetic axonal sprouting and that central MR blockade reduces cardiac sympathetic hyperinnervation post-MI. Post-MI, tyrosine hydroxylase (TH) and norepinephrine transporter protein content in the noninfarcted base of the heart remained unaltered. In contrast, protein gene product (PGP)9.5 protein was increased twofold in the base of the heart and sixfold in the peri-infarct area at 1 wk post-MI and was associated with increased ubiquitin expression. These changes persisted to a lesser extent at 4 wk post-MI and were no longer present at 12 wk. Cardiac myocytes rather than sympathetic axons were the main source of this elevated PGP9.5 expression. At 7-10 days post-MI, in the peri-infarct area, sympathetic hyperinnervation was observed with a fourfold increase in growth-associated protein 43, a twofold increase in TH, and a 50% increase in PGP9.5-positive fibers compared with the epicardial side of the left ventricle in sham rats. Central infusion of the MR blocker eplerenone markedly attenuated these increases in nerve densities but did not affect overall cardiac PGP9.5 and ubiquitin protein overexpression. We conclude that central MR activation contributes to sympathetic hyperinnervation, possibly by decreasing cardiac sympathetic activity post-MI, or by affecting other mechanisms, such as the expression of nerve growth factor. Marked PGP9.5 expression occurs in cardiomyocytes early post-MI, which may contribute to the increase in ubiquitin.