OKN-007 decreases tumor necrosis and tumor cell proliferation and increases apoptosis in a preclinical F98 rat glioma model.

BACKGROUND: Glioblastoma is a malignant World Health Organization (WHO) grade IV glioma with a poor prognosis in humans. New therapeutics are desperately required. The nitrone OKN-007 (2,4-disulfophenyl-PBN) has demonstrated effective anti-glioma properties in several rodent models and is currently being used as a clinical investigational drug for recurrent gliomas. We assessed the regional effects of OKN-007 in the tumor necrotic core and non-necrotic tumor parenchyma. METHODS: An F98 rat glioma model was evaluated using proton magnetic resonance spectroscopy ((1) H-MRS), diffusion-weighted imaging (DWI), morphological T2-weighted imaging (T2W) at 7 Tesla (30 cm-bore MRI), as well as immunohistochemistry and microarray assessments, at maximum tumor volumes (15-23 days following cell implantation in untreated (UT) tumors, and 18-35 days in OKN-007-treated tumors). RESULTS: (1) H-MRS data indicates that Lip0.9/Cho, Lip0.9/Cr, Lip1.3/Cho, and Lip1.3/Cr ratios are significantly decreased (all P < 0.05) in the OKN-007-treated group compared with UT F98 gliomas. The Cho/Cr ratio is also significantly decreased in the OKN-007-treated group compared with UT gliomas. In addition, the OKN-007-treated group demonstrates significantly lower ADC values in the necrotic tumor core and the nonnecrotic tumor parenchyma (both P < 0.05) compared with the UT group. There was also an increase in apoptosis following OKN-007 treatment (P < 0.01) compared with UT. CONCLUSION: OKN-007 reduces both necrosis and tumor cell proliferation, as well as seems to mediate multiple effects in different tumor regions (tumor necrotic core and nonnecrotic tumor parenchyma) in F98 gliomas, indicating the efficacy of OKN-007 as an anti-cancer agent and its potential clinical use.

Toll-like receptor 3 and geographic atrophy in age-related macular degeneration.

BACKGROUND: Age-related macular degeneration is the most common cause of irreversible visual impairment in the developed world. Advanced age-related macular degeneration consists of geographic atrophy and choroidal neovascularization. The specific genetic variants that predispose patients to geographic atrophy are largely unknown. METHODS: We tested for an association between the functional toll-like receptor 3 gene (TLR3) variant rs3775291 (involving the substitution of phenylalanine for leucine at amino acid 412) and age-related macular degeneration in Americans of European descent. We also tested for the effect of TLR3 Leu and Phe variants on the viability of human retinal pigment epithelial cells in vitro and on apoptosis of retinal pigment epithelial cells from wild-type mice and Tlr3-knockout (Tlr3(-/-)) mice. RESULTS: The Phe variant (encoded by the T allele at rs3775291) was associated with protection against geographic atrophy (P=0.005). This association was replicated in two independent case-control series of geographic atrophy (P=5.43x10(-4) and P=0.002). No association was found between TLR3 variants and choroidal neovascularization. A prototypic TLR3 ligand induced apoptosis in a greater fraction of human retinal pigment epithelial cells with the Leu-Leu genotype than those with the Leu-Phe genotype and in a greater fraction of wild-type mice than Tlr3(-/-) mice. CONCLUSIONS: The TLR3 412Phe variant confers protection against geographic atrophy, probably by suppressing the death of retinal pigment epithelial cells. Since double-stranded RNA (dsRNA) can activate TLR3-mediated apoptosis, our results suggest a role of viral dsRNA in the development of geographic atrophy and point to the potential toxic effects of short-interfering-RNA therapies in the eye.

Impaired flexibility in decision making in rats after administration of the pharmacological stressor yohimbine.

RATIONALE: Stress-induced disruption of decision making has been hypothesized to contribute to drug-seeking behaviors and addiction. Noradrenergic signaling plays a central role in mediating stress responses. However, the effects of acute stress on decision making, and the role of noradrenergic signaling in regulating these effects, have not been well characterized. OBJECTIVE: To characterize changes in decision making caused by acute pharmacological stress, the effects of yohimbine (an α2-adrenergic antagonist) were examined in a delay discounting task. Noradrenergic contributions to decision making were further characterized by examining the effects of propranolol (a ß antagonist), prazosin (an α1 antagonist), and guanfacine (an α2 agonist). METHODS: Sprague-Dawley rats were administered drugs prior to performance on a delay discounting task, in which the delay preceding the large reward increased within each session (ascending delays). To dissociate drug-induced changes in delay sensitivity from behavioral inflexibility, drug effects were subsequently tested in a modified version of the discounting task, in which the delay preceding the large reward decreased within each session (descending delays). RESULTS: Yohimbine increased choice of the large reward when tested with ascending delays but decreased choice of the same large reward when tested with descending delays, suggesting that drug effects could be attributed to perseverative choice of the lever preferred at the beginning of the session. Propranolol increased choice of the large reward when tested with ascending delays. Prazosin and guanfacine had no effect on reward choice. CONCLUSIONS: The stress-like effects of yohimbine administration may impair decision making by causing inflexible, perseverative behavior.

Lesions of the lateral habenula increase voluntary ethanol consumption and operant self-administration, block yohimbine-induced reinstatement of ethanol seeking, and attenuate ethanol-induced conditioned taste aversion.

The lateral habenula (LHb) plays an important role in learning driven by negative outcomes. Many drugs of abuse, including ethanol, have dose-dependent aversive effects that act to limit intake of the drug. However, the role of the LHb in regulating ethanol intake is unknown. In the present study, we compared voluntary ethanol consumption and self-administration, yohimbine-induced reinstatement of ethanol seeking, and ethanol-induced conditioned taste aversion in rats with sham or LHb lesions. In rats given home cage access to 20% ethanol in an intermittent access two bottle choice paradigm, lesioned animals escalated their voluntary ethanol consumption more rapidly than sham-lesioned control animals and maintained higher stable rates of voluntary ethanol intake. Similarly, lesioned animals exhibited higher rates of responding for ethanol in operant self-administration sessions. In addition, LHb lesion blocked yohimbine-induced reinstatement of ethanol seeking after extinction. Finally, LHb lesion significantly attenuated an ethanol-induced conditioned taste aversion. Our results demonstrate an important role for the LHb in multiple facets of ethanol-directed behavior, and further suggest that the LHb may contribute to ethanol-directed behaviors by mediating learning driven by the aversive effects of the drug.

Regression of glioma tumor growth in F98 and U87 rat glioma models by the Nitrone OKN-007.

BACKGROUND: Glioblastoma multiforme, a World Health Organization grade IV glioma, has a poor prognosis in humans despite current treatment options. Here, we present magnetic resonance imaging (MRI) data regarding the regression of aggressive rat F98 gliomas and human U87 glioma xenografts after treatment with the nitrone compound OKN-007, a disulfonyl derivative of α-phenyl-tert-butyl nitrone. METHODS: MRI was used to assess tumor volumes in F98 and U87 gliomas, and bioluminescence imaging was used to measure tumor volumes in F98 gliomas encoded with the luciferase gene (F98(luc)). Immunohistochemistry was used to assess angiogenesis (vascular endothelial growth factor [VEGF] and microvessel density [MVD]), cell differentiation (carbonic anhydrase IX [CA-IX]), hypoxia (hypoxia-inducible factor-1α [HIF-1α]), cell proliferation (glucose transporter 1 [Glut-1] and MIB-1), proliferation index, and apoptosis (cleaved caspase 3) markers in F98 gliomas. VEGF, CA-IX, Glut-1, HIF-1α, and cleaved caspase 3 were assessed in U87 gliomas. RESULTS: Animal survival was found to be significantly increased (P < .001 for F98, P < .01 for U87) in the group that received OKN-007 treatment compared with the untreated groups. After MRI detection of F98 gliomas, OKN-007, administered orally, was found to decrease tumor growth (P < .05). U87 glioma volumes were found to significantly decrease (P < .05) after OKN-007 treatment, compared with untreated animals. OKN-007 administration resulted in significant decreases in tumor hypoxia (HIF-1α [P < .05] in both F98 and U87), angiogenesis (MVD [P < .05], but not VEGF, in F98 or U87), and cell proliferation (Glut-1 [P < .05 in F98, P < .01 in U87] and MIB-1 [P < .01] in F98) and caused a significant increase in apoptosis (cleaved caspase 3 [P < .001 in F98, P < .05 in U87]), compared with untreated animals. CONCLUSIONS: OKN-007 may be considered as a promising therapeutic addition or alternative for the treatment of aggressive human gliomas.

Bioimaging of copper alterations in the aging mouse brain by autoradiography, laser ablation inductively coupled plasma mass spectrometry and immunohistochemistry.

Copper may play an important role in the brain in aging and neurodegenerative diseases. We compare the active Cu uptake, Cu-containing enzyme levels, and total Cu distribution in the brains of young and aging mice. (67)Cu was administered intravenously to 2, 7-9, and 14 month old mice. Active uptake of (67)Cu in the brain was measured at 24 h by digital phosphor autoradiography. Cerebral superoxide dismutase-1 (SOD-1) and cytochrome-C oxidase subunit-1 (CCO-1) levels were analyzed by immunohistochemistry. The total Cu distribution in brain section was determined by imaging laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). In aging mice, active (67)Cu uptake and SOD-1 levels were significantly decreased in the brain, whereas blood (67)Cu and CCO-1 levels were similar for all mice, irrespective of age. Paradoxically, global Cu cerebral content was increased in aged mice, suggesting that regulation of active Cu uptake by the brain may be linked to total Cu levels in an attempt to maintain Cu homeostasis. However, focal areas of both decreased Cu uptake and Cu content were noted in the striatum and ventral cortex in aging mice. These focal areas of Cu deficit correspond to the regions of greatest reduction in SOD-1 in the aged mice. In aging, dysregulated Cu homeostasis may result in decreased SOD-1 levels, which may contribute to oxidative vulnerability of the aging brain. This study illustrates the importance of a multi-modality approach in studying the biodistribution and homeostasis of Cu in the brain.