Targeting the PI3K/Akt/mTOR signalling pathway in Cystic Fibrosis.

Deletion of phenylalanine 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) is a major cause of cystic fibrosis (CF), one of the most common inherited childhood diseases. ΔF508 CFTR is a trafficking mutant that is retained in the endoplasmic reticulum (ER) and unable to reach the plasma membrane. Efforts to enhance exit of ΔF508 CFTR from the ER and improve its trafficking are of utmost importance for the development of treatment strategies. Using protein interaction profiling and global bioinformatics analysis we revealed mammalian target of rapamycin (mTOR) signalling components to be associated with ∆F508 CFTR. Our results demonstrated upregulated mTOR activity in ΔF508 CF bronchial epithelial (CFBE41o-) cells. Inhibition of the Phosphatidylinositol 3-kinase/Akt/Mammalian Target of Rapamycin (PI3K/Akt/mTOR) pathway with 6 different inhibitors demonstrated an increase in CFTR stability and expression. Mechanistically, we discovered the most effective inhibitor, MK-2206 exerted a rescue effect by restoring autophagy in ΔF508 CFBE41o- cells. We identified Bcl-2-associated athanogene 3 (BAG3), a regulator of autophagy and aggresome clearance to be a potential mechanistic target of MK-2206. These data further link the CFTR defect to autophagy deficiency and demonstrate the potential of the PI3K/Akt/mTOR pathway for therapeutic targeting in CF.

Estrogen receptor α regulates non-canonical autophagy that provides stress resistance to neuroblastoma and breast cancer cells and involves BAG3 function.

Breast cancer is a heterogeneous disease and approximately 70% of newly diagnosed breast cancers are estrogen receptor (ER) positive. Out of the two ER types, α and ß, ERα is the only ER that is detectable by immunohistochemistry in breast cancer biopsies and is the predominant subtype expressed in breast tumor tissue. ER-positive tumors are currently treated with anti-hormone therapy to inhibit ER signaling. It is well known that breast cancer cells can develop endocrine resistance and resistance to anti-hormone therapy and this can be facilitated via the autophagy pathway, but so far the description of a detailed autophagy expression profile of ER-positive cancer cells is missing. In the present study, we characterized tumor cell lines ectopically expressing ERα or ERß as well as the breast cancer-derived MCF-7 cell line endogenously expressing ERα but being ERß negative. We could show that ERα-expressing cells have a higher autophagic activity than cells expressing ERß and cells lacking ER expression. Additionally, for autophagy-related gene expression we describe an ERα-specific 'autophagy-footprint' that is fundamentally different to tumor cells expressing ERß or lacking ER expression. This newly described ERα-mediated and estrogen response element (ERE)-independent non-canonical autophagy pathway, which involves the function of the co-chaperone Bcl2-associated athanogene 3 (BAG3), is independent of classical mammalian target of rapamycin (mTOR) and phosphatidylinositol 3 kinase (PI3K) signaling networks and provides stress resistance in our model systems. Altogether, our study uncovers a novel non-canonical autophagy pathway that might be an interesting target for personalized medicine and treatment of ERα-positive breast cancer cells that do not respond to anti-hormone therapy and classical autophagy inhibitors.

Holo-APP and G-protein-mediated signaling are required for sAPPα-induced activation of the Akt survival pathway.

Accumulating evidence indicates that loss of physiologic amyloid precursor protein (APP) function leads to reduced neuronal plasticity, diminished synaptic signaling and enhanced susceptibility of neurons to cellular stress during brain aging. Here we investigated the neuroprotective function of the soluble APP ectodomain sAPPα (soluble APPα), which is generated by cleavage of APP by α-secretase along the non-amyloidogenic pathway. Recombinant sAPPα protected primary hippocampal neurons and SH-SY5Y neuroblastoma cells from cell death induced by trophic factor deprivation. We show that this protective effect is abrogated in neurons from APP-knockout animals and APP-depleted SH-SY5Y cells, but not in APP-like protein 1- and 2- (APLP1 and APLP2) depleted cells, indicating that expression of membrane-bound holo-APP is required for sAPPα-dependent neuroprotection. Trophic factor deprivation diminished the activity of the Akt survival pathway. Strikingly, both recombinant sAPPα and the APP-E1 domain were able to stimulate Akt activity in wild-type (wt) fibroblasts, SH-SY5Y cells and neurons, but failed to rescue in APP-deficient neurons or fibroblasts. The ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) inhibitor GI254023X exacerbated neuron death in organotypic (hippocampal) slice cultures of wt mice subjected to trophic factor and glucose deprivation. This cell death-enhancing effect of GI254023X could be completely rescued by applying exogenous sAPPα. Interestingly, sAPPα-dependent Akt induction was unaffected in neurons of APP-ΔCT15 mice that lack the C-terminal YENPTY motif of the APP intracellular region. In contrast, sAPPα-dependent rescue of Akt activation was completely abolished in APP mutant cells lacking the G-protein interaction motif located in the APP C-terminus and by blocking G-protein-dependent signaling with pertussis toxin. Collectively, our data provide new mechanistic insights into the physiologic role of APP in antagonizing neurotoxic stress: they suggest that cell surface APP mediates sAPPα-induced neuroprotection via G-protein-coupled activation of the Akt pathway.

Spatial learning and expression patterns of PP1 mRNA in mouse hippocampus.

BACKGROUND: Synaptic plasticity is believed to be the major cellular basis for learning and memory. Protein phosphorylation is a key process involved in changes in the efficacy of neurotransmission. In long-term changes synaptic plasticity is followed by structural plasticity and protein de novo synthesis. Such mechanisms are believed to build the basis of hippocampal learning and memory investigated in the Morris water maze (MWM) task. To examine the role of dephosphorylation during that model for spatial learning, we analyzed protein phosphatase 1 (PP1) expression in the hippocampus of mice at various stages of the task and in two groups with different learning abilities. METHODS: Mice were trained for 4 days with four trials each day in the MWM. For gene expression hippocampi were prepared 1, 6 and 24 h after the last trial of each day. PP1 and brain-derived neurotrophic factor (BDNF) mRNA levels were determined by quantitative real-time PCR. RESULTS: The task requirements themselves affected expression levels of both PP1 and BDNF. In contrast to BDNF, PP1 was differentially expressed during learning. Poorly and well performing mice differed significantly. When performance was poor the expression level of PP1 was higher. CONCLUSION: Present results add further in vivo evidence that not only phosphorylation but also dephosphorylation is a major mechanism involved in learning and memory. Therefore, inhibition of hippocampal phosphatase activity might improve learning and memory.

The selective beta1-adrenoceptor antagonist nebivolol is a potential oestrogen receptor agonist with neuroprotective abilities.

BACKGROUND AND PURPOSE: Nebivolol, a selective beta(1)-adrenoceptor antagonist mediating rapid vasodilating effects, is used clinically to treat hypertension. Recently, it was reported that nebivolol also acts as an oestrogen receptor (ER) agonist. To investigate the neuroprotective potential of oestrogens, we assessed the oestrogenic effects of nebivolol in several in vitro neuronal models. EXPERIMENTAL APPROACH: Human neuroepithelioma SK-N-MC cells stably transfected with human ER alpha and beta, and mouse N2A neuroblastoma cells expressing human APP695(SWE)[N2Aswe, stably transfected with the Swedish mutation form of the Alzheimer-associated amyloid precursor protein (APPswe, K670M/N671L)] were incubated with different concentrations of nebivolol and 17beta-oestradiol (E2) for 24-48 h. ER activation was detected in a specific reporter assay, and ER-dependent gene expression was measured by quantitative real-time PCR (qRT PCR). Furthermore, cell survival rates were determined, and oxidative stress was induced by hydrogen peroxide and paraquat. Amyloid beta protein precursor (APP) processing was investigated, and the cleavage fragments sAPPalpha and Abeta were quantified via alpha-, beta- and gamma-secretase activity assays. Alterations of secretase expression levels were determined by qRT PCR. KEY RESULTS: Nebivolol induces oestrogen-dependent gene transcription, and protects neuronal cells against oxidative stress even at low and physiological concentrations (10(-8) M). Moreover, nebivolol modulates processing of APP in mouse neuronal N2Aswe cells by increasing alpha-secretase activity, ultimately leading to enhanced release of soluble non-amyloidogenic sAPPalpha. CONCLUSIONS AND IMPLICATIONS: We showed that nebivolol acts as ER agonist in neuronal cell lines, and suggest oestrogen-like neuroprotective effects mediated by nebivolol.

Transgenic overexpression of corticotropin releasing hormone provides partial protection against neurodegeneration in an in vivo model of acute excitotoxic stress.

Corticotropin releasing hormone (CRH) is the central modulator of the mammalian hypothalamic-pituitary-adrenal (HPA) axis. In addition, CRH affects other processes in the brain including learning, memory, and synaptic plasticity. Moreover, CRH has been shown to play a role in nerve cell survival under apoptotic conditions and to serve as an endogenous neuroprotectant in vitro. Employing mice overexpressing murine CRH in the CNS, we observed a differential response of CRH-overexpressing mice (CRH-COEhom-Nes) to acute excitotoxic stress induced by kainate compared with controls (CRH-COEcon-Nes). Interestingly, CRH-overexpression reduced the duration of epileptic seizures and prevented kainate-induced neurodegeneration and neuroinflammation in the hippocampus. Our findings highlight a neuroprotective action of CRH in vivo. This neuroprotective effect was accompanied by increased levels of brain-derived neurotrophic factor (BDNF) in CRH-COEhom-Nes mice, suggesting a potential role for BDNF in mediating CRH-induced neuroprotective actions against acute excitotoxicity in vivo.

Effects of neuron-specific ADAM10 modulation in an in vivo model of acute excitotoxic stress.

A disintegrin and metalloprotease (ADAM) 10 is the main candidate enzyme for the alpha-secretase processing of the amyloid precursor protein (APP). Neuron-specific ADAM10 overexpression proved beneficial in the APP[V717I] mutant Alzheimer mouse model [Postina R, Schroeder A, Dewachter I, Bohl J, Schmitt U, Kojro E, Prinzen C, Endres K, Hiemke C, Blessing M, Flamez P, Dequenne A, Godaux E, van Leuven F, Fahrenholz F (2004) A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Invest 113:1456-1464]. Since Alzheimer patients have a high prevalence for epileptic seizures, we investigated the effects of ADAM10 modulation under conditions of experimentally induced epileptic seizures. In this context we also examined whether ADAM10 effects were influenced by APP levels. Therefore we compared severity of kainate-induced seizures, neurodegeneration and inflammation in double transgenic mice overexpressing functional ADAM10 or a dominant negative ADAM10 mutant in the APP[V717I] background with single transgenic ADAM10 modulated mice. Double transgenic dominant negative ADAM10dn/APP[V717I] mice suffered from stronger epileptic seizures, had a longer recovery period and showed more neurodegeneration and glial activation in the hippocampal region than double transgenic mice moderately overexpressing functional ADAM10 (ADAM10mo/APP[V717I]) and APP[V717I] mice with endogenous ADAM10 levels. This suggests that ADAM10 activity is necessary to provide neuroprotection against excitotoxicity in the APP[V717I] mouse model. Interestingly, increased expression of functional ADAM10 above the endogenous level did not correlate with a better protection against seizures and neurodegeneration. Furthermore, ADAM10 dominant negative mice without transgenic APP overexpression (ADAM10dn) were seizing for a shorter time and showed less neuronal cell death and neuroinflammation after kainate injection than wild-type mice, which shows beneficial effects of ADAM10 inhibition in context with neurodegeneration. In contrast, mice with a high ADAM10 overexpression showed more seizures and stronger neuronal damage and inflammation than wild-type mice and mice with moderate ADAM10 overexpression. Hence, additional cleavage products of ADAM10 may counterbalance the neuroprotective effect of alpha-secretase-cleaved APP in the defense against excitotoxicity. Our findings highlight the need of a careful modulation of ADAM10 activity for neuroprotection depending on substrate availability and on neurotoxic stress conditions.

Antioxidants as a potential therapy against age-related neurodegenerative diseases: amyloid Beta toxicity and Alzheimer's disease.

Alzheimer's disease (AD) is a progressive age-related neurodegenerative disorder with distinct neuropathological features. Extracellular plaques, consisting of aggregated amyloid peptides of 39-43 amino acids are one of the most prominent pathological hallmarks of this disease. Although the exact neurochemical effector mechanism of Abeta aggregation is not yet elucidated, age-associated disturbances of metal ion metabolism have been proposed to promote the formation of aggregates from soluble Abeta. Oxidative stress is postulated to be a downstream effect of Abeta-metal ion interactions. Therefore, the modulation of brain metal metabolism and attenuation of oxidative stress by antioxidant molecules are proposed as a potential therapeutic intervention in AD. Here, we summarize the recent literature focused on APP/Abeta-metal ion interactions and the use of antioxidant metal chelators as potential therapy against AD.

From structural biochemistry to expression profiling: neuroprotective activities of estrogen.

Estrogens are neuromodulatory and neuroprotective hormones. Chemically, estrogens are steroid compounds and unfold most of their activities through the activation of nuclear receptors that bind to specific target genes and control their transcription. Two subtypes of estrogen receptors are known (estrogen receptor alpha and estrogen receptor beta) and they are expressed throughout the body including the CNS and in particular the brain. We employed large scale DNA-chip-analysis to display the gene expression pattern differentially regulated by both estrogen receptor subtypes in human neuronal cells. We identified different gene families regulated by estrogen receptors that complement the knowledge about the estrogen receptor target genes. Some of these genes may serve neuroprotective functions and may therefore mediate the overall neuroprotective activities of estrogens. In addition to estrogen receptor-dependent neuroprotective effects, estrogen (17beta-estradiol) itself is a compound with a phenolic structure that may display also direct and estrogen receptor-independent antioxidant activities which may be important for the defense against oxidative stress. In summary estrogen can display a wide range of neuroprotective activities through different types of mechanisms and we are only understanding part of the molecular control of these activities which may help to develop neuropreventive strategies against neurodegenerative diseases in the future.

Glycogen synthase kinase 3beta links neuroprotection by 17beta-estradiol to key Alzheimer processes.

Estrogen exerts many of its receptor-mediated neuroprotective functions through the activation of various intracellular signal transduction pathways including the mitogen activating protein kinase (MAPK), phospho inositol-3 kinase and protein kinase C pathways. Here we have used a hippocampal slice culture model of kainic acid-induced neurotoxic cell death to show that estrogen can protect against oxidative cell death. We have previously shown that MAPK and glycogen synthase kinase-3beta (GSK-3beta) are involved in the cell death/cell survival induced by kainic acid. In this model and other cellular and in vivo models we have shown that estrogen can also cause the phosphorylation and hence inactivation of GSK-3beta, a known mediator of neuronal cell death. The effect of estrogen on GSK-3beta activity is estrogen receptor mediated. Further, this estrogen/GSK-3beta interaction may have functional consequences in cellular models of some key pathogenic pathways associated with Alzheimer's disease. More specifically, estrogen affects the basal levels of tau phosphorylation at a site known to be phosphorylated by GSK-3beta. Taken together, these data indicate a novel molecular and functional link between estrogen and GSK-3beta and may have implications for estrogen receptor modulation as a target for the prevention of neurodegenerative disorders.

Estrogen-induced cell signalling in a cellular model of Alzheimer's disease.

Alzheimer's disease (AD) is characterised by deposition of a 4 kDa amyloid-beta peptide (Abeta) into senile plaques of the affected brain. Abeta is a proteolytic product of the membrane protein, amyloid precursor protein (APP). An alternative cleavage pathway involves alpha-secretase activity and results in secretion of a 100 kDa non-amyloidogenic APP (sAPPalpha) and therefore a potential reduction in Abeta secretion. We have shown that estrogen induces alpha-cleavage and therefore results in the secretion of sAPPalpha. This secretion is signalled via MAP-kinase and PI-3 kinase signal-transduction pathways. These pathways also have the potential to inhibit the activation of glycogen synthase kinase 3beta (GSK), a protein involved in cell death. Therefore, the aim of this work was to further elucidate the estrogen-mediated signaling pathways involved in APP processing, with particular emphasis on GSK activity. By stimulating rat hypothalamic neuronal GT1-7 cells with estradiol, we found that estrogen decreases the activation state of GSK via the MAP kinase pathway. Moreover, the inhibition of GSK activity by LiCl causes enhanced sAPPalpha secretion in a pattern similar to that seen in response to estrogen, suggesting a pivotal role for this deactivation in APP processing. Further, inactivation of GSK by estrogen can be confirmed in an in vivo model. Elucidation of the signaling pathways involved in APP processing may help to understand the pathology of AD and may also prove beneficial in developing therapeutic strategies to combat AD.

Human Nck-associated protein 1 and its binding protein affect the metabolism of beta-amyloid precursor protein with Swedish mutation.

Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system, and beta-amyloid precursor protein (betaAPP) plays a pivotal role in AD pathology. We previously reported that the suppression of human Nck-associated protein 1 (Nap1) whose expression was down-regulated in sporadic AD led to apoptosis in human neuroblastoma cells, and also its binding protein, hNap1BP was identified. Here, we examined whether these molecules were involved in the regulation of betaAPP metabolism. Human Nap1 and hNap1BP were found not to effect the amount of intracellular betaAPP but induced sAPPalpha secretion. Interestingly, they didn't reduce but slightly increased the extracellular level of Abeta. Furthermore, neither human Nap1 nor hNap1BP influenced the ratio of Abeta42/43 to total Abeta. Taken together, human Nap1 and hNap1BP may play a role in regulation of beta-secretase activity in the processing of betaAPP.

Estrogen induces a rapid secretion of amyloid beta precursor protein via the mitogen-activated protein kinase pathway.

The female sex hormone estrogen (17beta-estradiol; E2) may function as a neurohormone and has multiple neuromodulatory functions in the brain. Its potent neuroprotective activities can be dependent and independent of estrogen receptors (ERs). In addition, E2 influences the processing of the amyloid beta precursor protein (APP), one central step in the pathogenesis of Alzheimer's disease. Here, we show: (a) that physiological concentrations of E2 very rapidly cause an increased release of secreted nonamyloidogenic APP (sAPPalpha) in mouse hippocampal HT22 and human neuroblastoma SK-N-MC cells; and (b) that this effect is mediated through E2 via the phosphorylation of extracellular-regulated kinase 1 and 2 (ERK1/2), prominent members of the mitogen-activated protein kinase (MAPK) pathway. Furthermore, we show that the activation of MAPK-signaling pathway and the enhancement of the sAPP release is independent of ERs and could be induced by E2 to a similar extent in neuronal cells either lacking or overexpressing a functional ER.

Activation of guanylate cyclase by natriuretic peptides in mouse pituitary AtT20 cells is influenced by phosphorylation of ANP.

The discovery of free and membrane-bound ectokinases raises the question whether phosphorylation is another mechanism to modulate the action of distinct neuropeptides. Atrial-natriuretic-peptide (ANP) which is widespread found in the central nervous system (CNS) and involved in the modulation of stress reactions and emotional states like anxiety contains a recognition-motif for cAMP-dependent protein kinase A. We investigated the effect of phosphorylation of ANP and C-type natriuretic peptide (CNP), a related peptide without phosphorylation site, on their ability to activate their receptors in mouse pituitary AtT20 cells by measuring the formation of cyclic guanosinmonophosphate (cGMP). Phosphorylation with protein kinase A inactivated ANP. Coincubation experiments adding adenosintriphosphate (ATP), ATP-analogues or inhibitors of protein kinases to the medium pointed to the presence of an intrinsic protein kinase A like ectokinase-activity on AtT20 cells. The activity of CNP was unaffected in these experiments. Phosphorylation by ectokinases may be a physiological mechanism to regulate the biological activity of ANP in different tissues, such as pituitary and CNS.

[Estrogens against Alzheimer disease? The female sexual hormone as a protective neurohormone]. / Ostrogene gegen Alzheimer? Das weibliche Sexualhormon als schützendes Neurohormon.

The female sex hormone estrogen has a wide range of activities besides the sex-associated functions. Estrogen is a neurohormon. Its effects on structure and function of nerve cells is in the focus of research of basic neuroscience. In the brain estrogens not exclusively act via estrogen receptors but also receptor-independently. Estrogen can function dependent but also independent from estrogen receptors. In humans estrogen improves cognitive functions, learning and memory. In addition, beneficial effects of a hormone replacement therapy in prevention of Alzheimer's disease have been demonstrated. But on the basis of the current knowledge, estrogen is not a drug for the treatment of an already ongoing Alzheimer's disease process.

Protective activity of aromatic amines and imines against oxidative nerve cell death.

Oxidative stress is a widespread phenomenon in the pathology of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Neuronal cell death due to oxidative stress may causally contribute to the pathogeneses of these diseases. Therefore, neuroprotective antioxidants are considered to be a promising approach to slow down disease progression. We have investigated different aromatic amine and imine compounds for neuroprotective antioxidant functions in cell culture, and found that these compounds possess excellent cytoprotective potential in diverse paradigms of oxidative neuronal cell death, including clonal cell lines, primary cerebellar neurons, and organotypic hippocampal slice cultures. Aromatic amines and imines are effective against oxidative glutamate toxicity, glutathione depletion, and hydrogen peroxide toxicity. Their mode of action as direct antioxidants was experimentally confirmed by electron spin resonance spectroscopy, cell-free brain lipid peroxidation assays, and intracellular peroxide measurements. With half-maximal effective concentrations of 20-75 nM in different neuroprotection experiments, the aromatic imines phenothiazine, phenoxazine, and iminostilbene proved to be about two orders of magnitude more effective than common phenolic antioxidants. This remarkable efficacy could be directly correlated to calculated properties of the compounds by means of a novel, quantitative structure-activity relationship model. We conclude that bridged bisarylimines with a single free NH-bond, such as iminostilbene, are superior neuroprotective antioxidants, and may be promising lead structures for rational drug development.

Testosterone stimulates rapid secretory amyloid precursor protein release from rat hypothalamic cells via the activation of the mitogen-activated protein kinase pathway.

The processing of the amyloid precursor protein (APP) has become a major focus of research into Alzheimer's disease (AD). Recently, repeated doses of testosterone have been shown to enhance the secretion of the product of the alpha-cleavage pathway of APP (sAPPalpha) over a period of days. Here, the time course of secretion of sAPPalpha after a single physiological dose of testosterone using an immortalized rat hypothalamic cell line (GT1-7) and the signalling pathways involved was analyzed. Testosterone was found to increase the amount of APP secretion rapidly after treatment without effecting the overall amount of cellular APP. The species of APP secreted was found to be predominantly the product of the non-amyloidogenic alpha-secretory pathway. Further, this event is regulated via aromatase-mediated conversion of testosterone to estrogen and the mitogen-activated protein kinase (MAP kinase) signalling pathway. Taken together these data partially elucidates the cellular cascade by which testosterone stimulates sAPP secretion.

Differential induction of NF-kappaB activity and neural cell death by antidepressants in vitro.

Tricyclic antidepressants and selective serotonin reuptake inhibitors are here shown to induce cell death in a neural cell line. The exposure to these drugs led to increased generation of reactive oxygen species and a concomitant reduction of intracellular glutathione levels. Furthermore, these antidepressants induced DNA fragmentation and increased the transcriptional and DNA-binding activity of NF-kappaB. In contrast, treatment with type A and B monoamine oxidase inhibitors did not induce changes in NF-kappaB activity and did not exert a detrimental influence on cell viability. These results indicate that some antidepressant drugs may cause both oxidative stress and changes in cellular antioxidative capacity, resulting in altered NF-kappaB activity and, ultimately, cell death.

Dexamethasone-enhanced sensitivity of mouse hippocampal HT22 cells for oxidative stress is associated with the suppression of nuclear factor-kappaB.

Glucocorticoids (GCs) exacerbate various insults to the hippocampus but the exact molecular mechanisms of this GC activity is not known. GCs can suppress the activity of the redox-sensitive nuclear factor NF-kappaB, which potentially serves neuroprotective functions. Employing electrophoretic mobility shift assays and transfection assays using a NF-kappaB-dependent reporter plasmid, we demonstrate that the increased oxidative stress sensitivity of clonal mouse hippocampal HT22 cells caused by GCs is associated with the suppression of NF-kappaB. GCs increased the expression of IkappaBalpha, the physiological inhibitor of NF-kappaB. Downregulation of NF-kappaB activity after overexpression of a dominant-negative mutant form of IkappaBalpha results in an increased sensitivity to oxidative stress. We conclude that the suppression of the basal NF-kappaB activity contributes to the enhanced vulnerability of neuronal cells to oxidative stress caused by GCs.

Effects of pH and dose on nasal absorption of scopolamine hydrobromide in human subjects.

PURPOSE: The present study was conducted to evaluate the effects of formulation pH and dose on nasal absorption of scopolamine hydrobromide, the single most effective drug available for the prevention of nausea and vomiting induced by motion sickness. METHODS: Human subjects received scopolamine nasally at a dose of 0.2 mg/0.05 mL or 0.4 mg/0.10 mL, blood samples were collected at different time points, and plasma scopolamine concentrations were determined by LC-MS/MS. RESULTS: Following administration of a 0.2 mg dose, the average Cmax values were found to be 262+/-118, 419+/-161, and 488+/-331 pg/ mL for pH 4.0, 7.0, and 9.0 formulations, respectively. At the 0.4 mg dose the average Cmax values were found to be 503+/-199, 933+/-449, and 1,308+/-473 pg/mL for pH 4.0, 7.0, and 9.0 formulations, respectively. At a 0.2 mg dose, the AUC values were found to be 23,208+/-6,824, 29,145+/-9,225, and 25,721+/-5,294 pg x min/mL for formulation pH 4.0, 7.0, and 9.0, respectively. At a 0.4 mg dose, the average AUC value was found to be high for pH 9.0 formulation (70,740+/-29,381 pg x min/mL) as compared to those of pH 4.0 (59,573+/-13,700 pg x min/mL) and pH 7.0 (55,298+/-17,305 pg x min/mL) formulations. Both the Cmax and AUC values were almost doubled with doubling the dose. On the other hand, the average Tmax, values decreased linearly with a decrease in formulation pH at both doses. For example, at a 0.4 mg dose, the average Tmax values were 26.7+/-5.8, 15.0+/-10.0, and 8.8+/-2.5 minutes at formulation pH 4.0, 7.0, and 9.0, respectively. CONCLUSIONS: Nasal absorption of scopolamine hydrobromide in human subjects increased substantially with increases in formulation pH and dose.