Phase 1 dose-escalation study of a siRNA targeting the RTP801 gene in age-related macular degeneration patients.

BACKGROUND: To evaluate the safety, tolerability, pharmacokinetics, and dose-limiting toxicities of a single intravitreal (IVT) injection of PF-04523655, a 19-nucleotide, O-methyl stabilized, double-stranded small interfering ribonucleic acid targeting the RTP801 gene in patients with neovascular age-related macular degeneration (AMD). METHODS: Prospective, phase 1, clinical multicentre trial, enrolled 27 patients with neovascular AMD unresponsive to prior treatment and best corrected visual acuity (BCVA) ≤ 20/200 in the study eye in stratum 1: (dose-escalating, open-label: 50 to 3000 µg of PF-04523655) and 27 patients who had potential to benefit from therapy and BCVA of ≤ 20/100 and ≥ 20/800 in stratum 2 (parallel, masked study of 1000, 1500, 2250, and 3000 µg of PF-04523655). The primary outcome was safety and tolerability assessment as well as pharmacokinetic profiling following a single IVT injection of PF-04523655. RESULTS: Doses of PF-04523655 ≥ 400 µg were generally detectable in the plasma at 1, 4, and 24 h post-injection. And all doses were below the lowest level of quantification by day 14. A single IVT injection of 50 to 3000 µg of PF-045237655 was generally safe and well tolerated over 24 months. There were no dose-limiting toxicities. CONCLUSION: A single IVT injection of PF-0523655 ≤ 3000 µg seems safe and well tolerated in eyes with neovascular AMD.

Effect of DOV 102,677 on the volitional consumption of ethanol by Myers' high ethanol-preferring rat.

BACKGROUND: Inhibitors of monoamine neurotransmitter transporters are well established as antidepressants. However, the evidence that single (serotonin) or dual (serotonin-norepinephrine) neurotransmitter uptake inhibitors can treat ethanol abuse, either as a comorbidity with depression or as a separate entity, is inconsistent. Drugs that have, in addition, the ability to inhibit dopamine uptake may have an advantage in the treatment of alcohol abuse. Therefore, the inhibitor of norepinephrine, serotonin and dopamine uptake, DOV 102,677, was tested for its effects on the volitional consumption of ethanol by an ethanol-preferring rat strain. METHODS: Myers' high ethanol-preferring rats were screened by a 10-day, 3 to 30% step-up test and then given free access to the preferred concentration of ethanol in a 3-bottle choice task. Consumption of ethanol (g/kg), water, food, and body weight were measured daily during a 3-day predrug treatment period, a 3-day treatment period, and a 3-day posttreatment period. Additional Sprague-Dawley rats were observed for 24 hours for the behavioral effects of 2.0 mg/kg s.c. reserpine after a 30-minute pretreatment with different doses of DOV 102,677. RESULTS: The triple monoamine uptake inhibitor DOV 102,677 dose-dependently decreased the volitional consumption of ethanol by as much as 71.2% (20 mg/kg i.p., b.i.d.) over 3 days of administration. This effect carried over into the posttreatment period. Similarly, the proportion of ethanol to total fluids consumed declined by 66.2% (20 mg/kg s.c., b.i.d.), while food consumption and body weight were unaltered. In contrast, amperozide (2 mg/kg i.p., b.i.d.) suppressed the amount of ethanol consumed by 56%, while naltrexone (5 mg/kg i.p., b.i.d.) was without effect. DOV 102,677 (40 mg/kg s.c.) inhibited reserpine-induced akinesia and ptosis, but not hypothermia in Sprague-Dawley rats, consistent with its transient inhibition of serotonin transport, and more long-lived inhibition of norepinephrine and dopamine uptake. CONCLUSIONS: DOV 102,677 significantly decreased the volitional consumption of ethanol with minimal alterations in the intake of food or on body weight in an ethanol-preferring rat strain, suggesting that triple reuptake inhibitors may find utility in treating alcohol abuse.

Glutamate-induced over-expression of GAD is down-regulated by acetyl-L-carnitine in rat islet cells.

Glutamic acid decarboxylase (GAD65 and GAD67) in pancreatic beta cells is the target of autoantibodies and autoreactive T cells in insulin-dependent diabetes mellitus (IDDM). Regulating expression of GAD perhaps is a practical approach to treat IDDM. In this study, we established an in vitro system, in which GAD was expressed and glutamate treatment produced over-expression of GAD67 and GAD65 in rat islet cells. By using the system we were able to demonstrate basal level of expression of GAD and effects of glutamate and the antioxidant, acetyl-L-carnitine (ALC) on expression of GAD. We found that GAD67 expressed in 10% of islets cells, whereas GAD65 was localized in only 4% of the cells. Glutamate treatment resulted in significant over-expression of GAD67, but not GAD65. Such glutamate-induced overexpression of GAD67 was attenuated by pretreatment with ALC (100 microM). These findings suggest that the over-expression of GAD67 induced by glutamate in islet cells of rat may act as a suitable cellular model to study GAD autoreactivity during the development of IDDM. Meanwhile, it indicates that ALC, an ester of the trimethylated amino acid, can block glutamate-induced over-expression of GAD67, a key beta-cell autoantigen, suggesting a therapeutic potential of ALC in IDDM.

IgG isolated from LP-BM5 infected mouse brain activates ionotropic glutamate receptors.

Biochemical and immunological studies have shown that mice infected with LP-BM5 virus develop antibodies to ionotropic glutamate receptors. Here, IgG isolated from brain of infected mice has been tested electrophysiologically on cultured rat cortical and hippocampal neurons. The IgG elicited glycine-independent currents that reversed at approximately 0 mV. Equivalent concentrations of IgG from uninfected mice were inactive. The glycine-independent currents were less influenced by DNQX and GYKI-52466 than currents elicited by AMPA and KA. The IgG also elicited glycine-dependent currents that reversed at -10 mV and were blocked by dl-AP5, 5,7-DCKA, and polyamine amides. Glycine-dependent and -independent currents were unaffected by tetrodotoxin, strychnine, the transmembrane Cl- gradient or d-tubocurare. Although part of the glycine-independent current remains uncharacterized, these results confirm that a virus-induced immunopathology produces IgG clones that activate ionotropic glutamate receptors and that could, thereby, contribute to the excitotoxic neurological syndrome observed in LP-BM5-infected mice.

The hypnotic actions of the fatty acid amide, oleamide.

Oleamide is an endogenous fatty acid amide which can be synthesized de novo in the mammalian nervous system, and has been detected in human plasma. It accumulates in the CSF of rats after six hours of sleep deprivation and induces sleep in naive rats and mice. Inhibition of the primary catabolic enzyme of oleamide (fatty acid amide hydrolase) by trifluoromethyl-octadecenone reduces sleep latency and increases total sleep time when given centrally to rats and peripherally to mice. While the mechanism of action of oleamide is unclear, it has been demonstrated to increase the amplitude of currents gated by 5-HT2a, 5HT2c and GABAa receptors. Moreover, the action of oleamide most relevant to sleep induction involves, in part, cannabinergic pathways, as evidenced by the ability of the cannabinoid antagonist SR 141716 to inhibit the hypnotic actions of OA. Nonetheless, enhancement of cannabinergic function may not be the only mechanism by which OA alters sleep, as it can act synergistically with subthreshold doses of triazolam (0.125 microg) to reduce sleep latency. These findings raise the possibility that OA may be representative of a group of compounds which might be developed into clinically-used hypnotics, and are discussed in the context of fatty acid derivatives as modulators of neuronal function.

Behavioral evidence for the interaction of oleamide with multiple neurotransmitter systems.

While the endogenous fatty acid amide oleamide has hypnotic properties, neither the breadth of its behavioral actions nor the mechanism(s) by which these behaviors may be mediated has been elucidated. Therefore, the effects of oleamide on the performance of rats in tests of motor function, analgesia, and anxiety were investigated. Oleamide reduced the distance traveled in the open field (ED50 = 14, 10-19 mg/kg, mean, 95% confidence interval), induced analgesia and hypothermia, but did not cause catalepsy. Moreover, a dose of oleamide without effect on motor function was anxiolytic in the social interaction test and elevated plus-maze. These actions of a single dose of oleamide lasted for 30 to 60 min. While rats became tolerant to oleamide following 8 days of repeated administration, oleamide is a poor inducer of physical dependence. Pretreatment with antagonists of the serotonin (5HT)1A, 5HT2C, and vanilloid receptors did not modify oleamide's effects. However, the cannabinoid receptor antagonist SR 141716A inhibited oleamide-induced analgesia in the tail-flick assay, the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline reversed the analgesia and hypothermia, and the dopamine D2 receptor antagonist L 741626 blocked oleamide's locomotor and analgesic actions. Interestingly, oleamide analogs resistant to hydrolysis by fatty acid amide hydrolase (FAAH) maintained but did not show increased behavioral potency or duration of action, whereas two FAAH inhibitors produced analogous behavioral effects. Thus, oleamide induces behaviors reminiscent of the actions of endogenous cannabinoids, but the involvement of GABAergic and dopaminergic systems, either directly or indirectly, in the actions of oleamide cannot be ruled out.

LP-BM5 virus-infected mice produce activating autoantibodies to the AMPA receptor.

Autoantibodies to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors may contribute to chronic hyperexcitability syndromes and neurodegeneration, but their origin is unclear. We examined LP-BM5 murine leukemia virus-infected mice, which manifest excitotoxic brain lesions and hypergammaglobulinemia, for the presence of AMPA-receptor Ab's. Endogenous IgG accumulated upon neurons in the neocortex and caudate/putamen of infected mice and interacted with native and recombinant AMPA-receptor subunits with the following relative abundance: GluR3 > or = GluR1 > GluR2 = GluR4, as determined by immunoprecipitation. In a radioligand assay, IgG preparations from infected mice specifically inhibited [(3)H]AMPA binding to receptors in brain homogenates, an activity that was lost after preadsorbing the IgG preparation to immobilized LP-BM5 virus. These IgGs also evoked currents when applied to hippocampal pyramidal neurons or to damaged cerebellar granule neurons. These currents could be blocked using any of several AMPA receptor antagonists. Thus, anti-AMPA-receptor Ab's can be produced as the result of a virus infection, in part through molecular mimicry. These Ab's may alter neuronal signaling and contribute to the neurodegeneration observed in these mice, actions that may be curtailed by the use of AMPA-receptor antagonists.

Mice transgenically overexpressing sulfonylurea receptor 1 in forebrain resist seizure induction and excitotoxic neuron death.

The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIalpha promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [(3)H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68-79% of pyramidal neurons in the CA1-3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.

Activation of microglia by secreted amyloid precursor protein evokes release of glutamate by cystine exchange and attenuates synaptic function.

Microglial activation as part of a chronic inflammatory response is a prominent component of Alzheimer's disease. Secreted forms of the beta-amyloid precursor protein (sAPP) previously were found to activate microglia, elevating their neurotoxic potential. To explore neurotoxic mechanisms, we analyzed microglia-conditioned medium for agents that could activate glutamate receptors. Conditioned medium from primary rat microglia activated by sAPP caused a calcium elevation in hippocampal neurons, whereas medium from untreated microglia did not. This response was sensitive to the NMDA receptor antagonist, aminophosphonovaleric acid. Analysis of microglia-conditioned by HPLC revealed dramatically higher concentrations of glutamate in cultures exposed to sAPP. Indeed, the glutamate levels in sAPP-treated cultures were substantially higher than those in cultures treated with amyloid beta-peptide. This sAPP-evoked glutamate release was completely blocked by inhibition of the cystine-glutamate antiporter by alpha-aminoadipate or use of cystine-free medium. Furthermore, a sublethal concentration of sAPP compromised synaptic density in microglia-neuron cocultures, as evidenced by neuronal connectivity assay. Finally, the neurotoxicity evoked by sAPP in microglia-neuron cocultures was attenuated by inhibitors of either the neuronal nitric oxide synthase (N(G)-propyl-L-arginine) or inducible nitric oxide synthase (1400 W). Together, these data indicate a scenario by which microglia activated by sAPP release excitotoxic levels of glutamate, probably as a consequence of autoprotective antioxidant glutathione production within the microglia, ultimately causing synaptic degeneration and neuronal death.

Preclinical models of hepatic encephalopathy.

Hepatic encephalopathy is a multifactorial neuropsychiatric syndrome accompanying acute or chronic liver failure. Techniques for developing animal models of hepatic encephalopathy associated with acute or chronic liver failure, or vascular shunting are illustrated. In addition, the behavioral and biochemical characteristics of these models are described.

Accelerated development of neurochemical and behavioral deficits in LP-BM5 infected mice with targeted deletions of the IFN-gamma gene.

Mice homozygous for a germline deletion of the interferon-gamma gene (IFN-gamma (-/-)) were infected with the LP-BM5 (BM5) retrovirus mixture to determine if the inability to produce IFN-gamma reduces collateral CNS damage associated with chronic neuroinflammation. Virus burdens in spleens and brains of infected mice were comparable, but spatial memory deficits were manifested earlier and to a greater extent in BM5/IFN-gamma (-/-) mice. The mice with spatial memory deficits showed considerable degradation of axons and microtubules, along with apoptosis of striatal neurons. These lesions were accompanied by extensive infiltration of perivascular spaces and ventricles by iNOS-positive leukocytes, and a 17-fold increase in CSF glutamate levels. Despite high levels of VCAM and ICAM expression on cerebral vasculature endothelia, the serum levels of soluble ICAM-1 were significantly decreased in BM5/IFN-gamma (-/-) mice, which may contribute to the enhanced leukocyte infiltration and subsequent neuronal damage. These results suggest that the presence of IFN-gamma is necessary at some points in the inflammatory process to protect against neurodegeneration.

Suppression of neurocognitive damage in LP-BM5-infected mice with a targeted deletion of the TNF-alpha gene.

Brain levels of TNF-alpha increase in many inflammatory conditions, including HIV-1 infection, and may contribute to neurodegenerative processes. The paucity of agents that can selectively and potently block TNF-alpha processing or its receptors has led us to investigate the role of TNF-alpha in chronic neurodegeneration associated with retroviral infection using mice with targeted deletions of the TNF-alpha gene. Infection of wild-type C57BL/6 mice with the LP-BM5 murine leukemia retrovirus mixture leads to the development of a severe immunodeficiency as well as cognitive deficits and neuronal damage. TNF-alpha-(-/-) mice infected with LP-BM5 developed a systemic immunopathology indistinguishable in severity from that observed in contemporaneously infected wild-type mice. In contrast, the performance of infected TNF-alpha-(-/-) mice in the Y-maze and Morris water maze was not different from that of uninfected TNF-alpha-(-/-) mice. The extent of glial activation in the striatum, as indicated by the increase in density of peripheral benzodiazepine receptors, was equivalent in both groups of LP-BM5-infected mice. However, the decrease in striatal MAP-2 expression, a marker of neurodegeneration observed in infected wild-type mice, was not found in infected TNF-alpha-(-/-) mice. While the loss of TNF-alpha appeared to have no effect on the course or severity of the central or peripheral immunopathology resulting from LP-BM5 infection, the behavioral and biochemical manifestations were substantially curtailed in the TNF-alpha-(-/-) mice. These findings directly support a role for TNF-alpha in the neurodegenerative processes associated with viral infections such as HIV-1.

The hypnotic actions of oleamide are blocked by a cannabinoid receptor antagonist.

The unsaturated fatty acid amide oleamide (OA), which accumulates in the CSF of rats during sleep deprivation, induces electroencephalographically measured sleep when administered intracerebroventricularly. The mechanism of sleep induction by OA is unclear but may derive from enhancements of GABA or 5-HT receptor function, or alternatively from changes in the catabolism or uptake of the related fatty acid amide anandamide, an endogenous cannabinoid-1 (CB1) receptor ligand. The present study tests the latter hypothesis by administering OA alone and in combination with the CB1 receptor antagonist SR141716. As previously reported, 2.8 microg OA administered intracerebroventricularly significantly shortened electroencephalographic sleep latency. SR141716 in a dose of 3 microg had no effects on sleep by itself, but when co-administered with OA prevented its sleep-inducing effects. These data suggest that at least one aspect of the hypnotic action of OA involves interactions with the CB1 receptor system, possibly by blocking the metabolism of the endogenous CB1 receptor agonist anandamide.

Increased blood-brain barrier permeability in LP-BM5 infected mice is mediated by neuroexcitatory mechanisms.

Serum protein levels in LP-BM5 infected mouse brains were investigated to gain insight into the contribution of blood-brain barrier (BBB) patency to the pathogenesis of retroviral encephalopathy. Evans blue uptake by the forebrain and cerebellum was significantly increased between 8-12 weeks post inoculation. Immunohistochemistry revealed foci of albumin, transferrin, alpha(2)-macroglobulin and IgG transudation around blood vessels particularly in the cerebral cortex and cerebellar vermis. These leaks were often associated with astrocytosis and apoptotic cells. Unlike the other serum proteins, IgG immunoreactivity extended from the circumventricular organs and disseminated throughout the brain parenchyma, accumulating on the plasma membranes of hippocampal and cortical neurons. Consistent with the chronic elevation of free glutamate levels in LP-BM5 infected mice, the increase in Evans blue uptake into the forebrain was completely reversed following dizocilpine administration. Thus, the chronic increase in free glutamate levels in LP-BM5 infected mouse brain contributes to BBB disruption. Furthermore, the CNS accumulation of serum proteins, particularly IgG, observed in these mice may increase osmotic load, impair neuronal function, and cause white matter pallor. Administration of NMDA receptor antagonists may prove useful in managing BBB permeability in those neuropathologies, such as HIV-associated dementia/cognitive/motor complex, having a glutamatergic component.

Relevance of glutamate levels in the CSF of patients with HIV-1-associated dementia complex.

Chronic hyperactivation of excitatory amino acid pathways in the CNS of patients infected with HIV-1 may contribute to the pathogenesis of HIV-1-associated dementia complex. However, no correlation between the concentration of glutamate in CSF (mean 3.3 micromol/L) and either HIV-1 infection or HIV-1-associated dementia complex was observed. The results clarify several important issues regarding analysis of glutamate in the CSF and the role of excitotoxins in HIV-1-associated dementia complex.

Group II metabotropic glutamate receptor activation attenuates traumatic neuronal injury and improves neurological recovery after traumatic brain injury.

We examined the effects of modulating group II metabotropic glutamate receptors (mGluRs) on traumatic neuronal injury using both in vitro and in vivo models. Treatment with various selective group II mGluR agonists significantly decreased lactate dehydrogenase release, a marker of cell death, after traumatic injury to rat neuronal-glial cultures; injury-induced increases in cyclic AMP and glutamate levels were also significantly reduced by a group II agonist. The neuroprotective effects of group II agonists were markedly attenuated by coadministration of a group II antagonist or a membrane-permeable cyclic AMP analog and were additive to those provided by an N-methyl-D-aspartate receptor antagonist or a selective group I mGluR antagonist. Administration of a group II mGluR agonist 30 min after lateral fluid percussion-induced brain injury in rats significantly improved subsequent behavioral recovery as compared with vehicle-treated controls. Together these studies indicate that group II mGluR agonists protect against traumatic neuronal injury by attenuating glutamate release and cAMP levels and suggest a potential role for these agents in the treatment of clinical neurotrauma.

Characterization of the hypnotic properties of oleamide.

While preliminary studies associated oleamide with sleep regulation, we now characterize the involvement of oleamide in sleep using a number of techniques. Peripheral administration of oleamide to rats dose dependently suppressed motor activity in the open field, with an ED50 of 17+/-1.5mg/kg for the decrease in distance traveled. Moreover, endogenous oleamide concentrations increased 3- to 4-fold in the cerebrospinal fluid of rats sleep-deprived for 6 h or longer. Oleamide also decreased sleep latency to 44-64% of control values without altering other sleep parameters. Unlike many putative endogenous sleep-inducing agents, oleamide potently induces behavioral and electroencephalographic manifestations of sleep. Moreover, its endogenous concentrations and temporal associations are consistent with previous reports of its enhancement of serotonergic and GABAergic neurotransmission, which may be involved in sleep induction.

Dizocilpine attenuates streptomycin-induced vestibulotoxicity in rats.

NMDA receptor mediated excitotoxicity contributes substantially to aminoglycoside antibiotic-induced cochlear damage. Since vestibular as well as cochlear hair cells have glutamatergic synapses, aminoglycoside-induced vestibulotoxicity may also have an excitotoxic component. This hypothesis was tested by examining the effects of the uncompetitive NMDA receptor antagonist dizocilpine on streptomycin-induced vestibulotoxicity. Streptomycin-treated rats exhibited almost complete destruction of sensory hair cells in the crista ampullaris, vestibular impairment in the drop test, and hyperkinesia. Concurrent treatment with dizocilpine not only rescued a substantial population of sensory hair cells in the cristae, but prevented the attendant hyperkinesis and vestibular impairments. These results indicate that excitotoxic mechanisms contribute to aminoglycoside-induced vestibulotoxicity and that NMDA antagonists may be useful in attenuating aminoglycoside ototoxicity.

A gas chromatographic-mass spectral assay for the quantitative determination of oleamide in biological fluids.

Oleamide is a putative endogenous sleep-inducing lipid which potently enhances currents mediated by GABAA and serotonin receptors. While a quantitative assay would aid in determining the role of oleamide in physiological processes, most of the available assays are lacking in sensitivity. We now describe a quantitative assay for measuring low nanogram amounts of oleamide in biological fluids using GC/MS in the selective ion-monitoring mode. The internal standard (13C18 oleamide) was added to known concentrations of oleamide, which were converted to the N-trimethylsilyl or N-tert-butyldimethylsilyl derivatives before analysis by GC/MS, yielding linear calibration curves over the range of 1-25 ng of oleamide when monitoring the m/z 338/356 fragments. Using this technique, oleamide levels were determined following solvent extraction of normal rat cerebrospinal fluid and plasma to be 44 and 9.9 ng/ml, respectively. This technique constitutes a sensitive and reliable method for determining low nanogram quantities of oleamide in biological fluids.

Glutamate augments retrovirus-induced immunodeficiency through chronic stimulation of the hypothalamic-pituitary- adrenal axis.

The mechanisms for activating the hypothalamic-pituitary-adrenal (HPA) axis and the roles glucocorticoids play in the pathogenesis of chronic infectious disease are largely undefined. Using the LP-BM5 model of retrovirus-induced immunodeficiency, we found alterations in HPA axis function, manifested as an increase in circulating levels of adrenocorticotropic hormone and corticosterone, beginning after only 3 mo of infection. These changes occurred contemporaneously with a shift in the profile of circulating cytokines from a Th1-dominant (IFN-gamma) to Th2-dominant (IL-4, IL-10) phenotype. No significant changes in either circulating IL-1beta, IL-6, or TNF-alpha levels were observed in infected mice. Administering the N-methyl-D-aspartate receptor antagonist MK-801 to infected mice normalized plasma adrenocorticotropic hormone and corticosterone levels, indicating that glutamate was a major activator of the HPA axis. Moreover, MK-801 treatment of late-stage mice also reversed the type 1 to type 2 cytokine shift to a degree comparable or superior to treatment with the glucocorticoid receptor antagonist RU-486. These findings indicate that HPA axis activation during LP-BM5 retrovirus infection is mediated by the chronic hyperactivation of glutamatergic pathways in the hypothalamus. Through this mechanism, the degree of peripheral immunodeficiency observed in the late-stage disease is profoundly augmented.