3D-QSAR pharmacophore modeling and in silico screening of phospholipase A2α inhibitors

Cytosolic phospholipase A_2α have emerged as an attractive target for the development of analgesic and anti-inflammatory drugs. 3D-QSAR pharmacophore model was developed, based on previously reported 28 indole-5-carboxylic acids, and used to understand the structural factors affecting the inhibitory potency of these derivatives. Hydrogen bond acceptor, negative charge, and aromatic effects contribute to the inhibitory activity. The model was employed as a 3D search query to screen SPECS database to select new scaffolds. Finally, docking studies lead to the identification of fourteen potential phospholipase A_2α inhibitors. Subsequent ADME studies revealed the pharmacokinetic efficiency of these compounds.     

Exposure to the n-3 polyunsaturated fatty acid docosahexaenoic acid impairs α1-adrenoceptor-mediated contractile responses and inositol phosphate formation in rat cardiomyocytes

The beneficial effects of n-3 polyunsaturated fatty acids of fish oil in the prevention of fatal arrhythmias in myocardial ischemia were suggested to be at least in part mediated by a modulation of dihydropyridine-sensitive L-type calcium channels. As cardiac ₁-adrenoceptor stimulation has been suggested to have no significant effect on L-type calcium channels, the aim of this study using cultured neonatal rat cardiomyocytes was to investigate whether chronic n-3 polyunsaturated fatty acid exposure may have an influence on ₁-adrenoceptor-induced positive inotropic effects and induction of arrhythmias. Pretreatment of the rat cardiomyocytes for 3 days in the presence of the n-3 polyunsaturated fish oil-derived fatty acid docosahexaenoic acid (60 mol/l) markedly decreased ₁-adrenoceptor-stimulated increase in contraction velocity and induction of arrhythmias. The increase in contraction velocity of the cardiomyocytes induced by the -adrenoceptor agonist isoprenaline was also markedly reduced by the n-3 fatty acid pretreatment. Basal contractile amplitude and spontaneous beating frequency of the cardiomyocytes were not significantly altered by the docosahexaenoic acid exposure. The pretreatment of the rat cardiomyocytes for 3 days in the presence of docosahexaenoic acid (60 mol/l) decreased ₁-adrenoceptor-stimulated formation of the calcium-mobilizing second messenger IP₃ and its metabolites IP₂ and IP₁ by 55%. The depression of IP₃ formation by docosahexaenoic acid treatment was not mediated by a decreased uptake of myo-inositol into the cardiomyocytes nor by a decreased synthesis of phosphatidylinositol bisphosphate (PIP₂), the substrate of phospholipase C. The level of glycerol-3-phosphate, an important substrate of the phosphoinositide cycle, was unaltered by the docosahexaenoic acid pretreatment. Receptor binding studies revealed that the dissociation constant and maximal binding capacity of the ₁-adrenoceptor antagonist (³H)prazosin was unchanged by the n-3 polyunsaturated fatty acid exposure. -Adrenoceptor-and forskolin-stimulated adenylyl cyclase activities were not diminished by the docosahexaenoic acid pretreatment. Chronic exposure of the cardiomyocytes to the n-6 polyunsaturated fatty acid arachidonic acid (60 mol/l) did neither significantly alter ₁-adrenoceptor-induced inositol phosphate formation nor ₁-adrenoceptor-stimulated increase in contraction velocity. The results presented show that chronic n-3 polyunsaturated fatty acid pretreatment of rat cardiomyocytes leads to a marked impairment of ₁-adrenoceptor-induced positive inotropic effects and induction of arrhythmias concomitant with a n-3 fatty acid-induced decrease in IP₃ formation. This derangement of the phosphonositide pathway by chronic n-3 fatty acid exposure may, thus, contribute to the beneficial effects of fish oil-derived fatty acids in the prevention of fatal arrhythmias in myocardial ischemia.     

Inhibitory effects of ω-3 polyunsaturated fatty acids on receptor-mediated non-selective cation currents in rat A7r5 vascular smooth muscle cells

1. The effects of ω-3 polyunsaturated fatty acids on receptor-mediated non-selective cation current (I_cat) and K⁺ current were investigated in aortic smooth muscle cells from foetal rat aorta (A7r5 cells). The whole-cell voltage clamp technique was employed.
2. With a K⁺-containing solution, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA, 30 μM) produced an outward current at a holding potential of −40 mV. This response was inhibited by tetraethylammonium (20 mM) or Cs⁺ in the patch pipette solution, and the reversal potential of the EPA-induced current followed the K⁺ equilibrium potential in a near Nernstian manner.
3. Under conditions with a Cs⁺-containing pipette solution, both vasopressin and endothelin-1 (100 nM) induced a long-lasting inward current at a holding potential of −60 mV. The reversal potential of these agonist-induced currents was about +0 mV, and was not significantly altered by the replacement of the extracellular or intracellular Cl⁻ concentration, suggesting that the induced current was a cation-selective current (I_cat).
4. La³⁺ and Cd²⁺ (1 mM) completely abolished these agonist-induced I_cat, but nifedipine (10 μM) failed to inhibit it significantly.
5. ω-3 polyunsaturated fatty acids (3100 μM), EPA, DHA and docosapentaenoic acids (DPA), inhibited the agonist-induced I_cat in a concentration-dependent manner. The potency of the inhibitory effect was EPA>DHA>DPA, and the half maximal inhibitory concentration (IC₅₀) of EPA was about 7 μM.
6. Arachidonic and linoleic acids (10, 30 μM) showed a smaller inhibitory effect compared to ω-3 fatty acids. Also, oleic and stearic acids (30 μM) did not show a significant inhibitory effect on I_cat.
7. A similar inhibitory action of EPA was observed when I_cat was activated by intracellularly applied GTPγS in the absence of agonists, suggesting that the site of action of ω-3 fatty acids is not located on the receptor.
8. These results demonstrate that ω-3 polyunsaturated fatty acids can activate a K⁺ current and also effectively inhibit receptor-mediated non-selective cation currents in rat A7r5 vascular smooth muscle cells. Thus, the data suggest that ω-3 fatty acids may play an important role in the regulation of vascular tone.

     

The regulation by phosphorylation of ‘priming' of phospholipase A2 activity in the neutrophil model system,differentiated HL60 cells

1. Differentiated HL60 cells have been utilized as a model system to examine the ‘priming'' of neutrophil phospholipase A₂ activity. In control cells activation of phospholipase A₂ by a 5 min stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (100 nM) was essentially undetectable. When cells were primed by preincubation with 5 μM cytochalasin B for 5 min arachidonate release, a measure of phospholipase A₂ activation, was observed within 20 s.
2. Priming by cytochalasin B did not involve or require a change in intracellular free calcium concentration.
3. Priming was associated with an increase in general protein tyrosine phosphorylation and could also be induced by the receptor tyrosine kinase agonist granulocyte macrophage colony-stimulating factor (GM-CSF, 20 ng ml⁻¹) and be mimicked by treatment with the phosphotyrosine phosphatase inhibitor perhydrovanadate (0.5 mM). However, an increase in MAP kinase activity was not involved in the priming process.
4. Western blot analysis demonstrated that phospholipase A₂ was phosphorylated in both control and primed cells, but that an increase in the amount of membrane associated enzyme was found in the primed cells.
5. Thus priming appears to be due to membrane association of the phospholipase and this may be regulated by tyrosine kinase activities.

     

Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1

Little is known about whether trans-isoferulic acid (TIA) regulates the production of lipopolysaccharide (LPS)-induced proinflammatory mediators. Therefore, we examined the effect of TIA isolated from Clematis mandshurica on LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in BV2 microglial cells. We found that TIA inhibited the production of LPS-induced NO and PGE₂ without accompanying cytotoxicity in BV2 microglial cells. TIA also downregulated the expression levels of specific regulatory genes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing LPS-induced NF-κB activity via dephosphorylation of PI3K/Akt. In addition, we demonstrated that a specific NF-κB inhibitor PDTC and a selective PI3K/Akt inhibitor, LY294002 effectively attenuated the expression of LPS-stimulated iNOS and COX-2 mRNA, while LY294002 suppressed LPS-induced NF-κB activity, suggesting that TIA attenuates the expression of these proinflammatory genes by suppressing PI3K/Akt-mediated NF-κB activity. Our results showed that TIA suppressed NO and PGE₂ production through the induction of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent heme oxygenase-1 (HO-1). Taken together, our data indicate that TIA suppresses the production of proinflammatory mediators such as NO and PGE₂, as well as their regulatory genes, in LPS-stimulated BV2 microglial cells, by inhibiting PI3K/Akt-dependent NF-κB activity and enhancing Nrf2-mediated HO-1 expression.     

Engineering of an ω-3 polyunsaturated fatty acid-containing nanoemulsion system for combination C6-ceramide and 17β-estradiol delivery and bioactivity in human vascular endothelial and smooth muscle cells

Delayed endothelial cell (EC) regeneration and the medial vascular smooth muscle cells (VSMCs) proliferation contribute to arterial restenosis. Although ω-3-polyunsaturated fatty acids (PUFAs), 17β-estradiol (17-βE) and C6-ceramide (CER) have shown therapeutic promise in addressing restenosis, extensive protein binding and lipophilicity complicate their (co-)delivery to cellular targets. We report engineering of an ω-3-PUFA-rich oil-in-water nanoemulsion formulation that effectively delivers 17-βE and CER cargo to cultured vascular cells. The cargo-free, ω-3-PUFA-rich nanoemulsion itself typically reduced growth factor-stimulated cellular proliferation, as did nanoemulsion-delivered CER alone, through enhanced pro-apoptotic caspase 3/7 activity. 17-βE loaded nanoemulsion inhibited VSMC proliferation and supported EC proliferation, responses associated with the mitogen-activated-protein-kinase (MAPK) signaling. Co-administration of 17-βE and CER loaded nanoemulsions exerted an anti-proliferative effect more pronounced on VSMCs than ECs. These therapeutically beneficial responses to ω-3-PUFA, CER, and/or 17-βE in our nanoemulsion formulation invite evaluation of this novel approach in animal models of restenosis and other occlusive vasculopathies.From the Clinical EditorThis team of investigators report the engineering of an ω-3-PUFA-rich oil-in-water nanoemulsion formulation that effectively delivers 17-βE and C6-ceramide cargo to cultured vascular cells in an effort to address vascular restenosis. Further preclinical studies will be needed in animal models before this approach could be considered for clinical trials.     

The effects of recombinant rat μ-opioid receptor activation in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase

1. The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca²⁺]_i and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃), [Ca²⁺]_i and adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor.
2. Opioid receptor binding was assessed with [³H]-diprenorphine ([³H]-DPN) as a radiolabel. Ins(1,4,5)P₃ and cyclic AMP were measured by specific radioreceptor assays. [Ca²⁺]_i was measured fluorimetrically with Fura-2.
3. Scatchard analysis of [³H]-DPN binding revealed that the B_max varied between passages. Fentanyl (10 pM–1 μM) dose-dependently displaced [³H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6±0.1), and was best fit to a two site model, with pK_i values (% of sites) of 9.97±0.4 (27±4.8%) and 7.68±0.07 (73±4.8%). In the presence of GppNHp (100 μM) and Na⁺ (100 mM), the curve was shifted to the right and became steeper (Hill slope=0.9±0.1) with a pK_i value of 6.76±0.04.
4. Fentanyl (0.1 nM–1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC₅₀=7.42±0.23), in a pertussis toxin (PTX; 100 ng ml⁻¹ for 24 h)-sensitive and naloxone-reversible manner (K_i=1.7 nM). Morphine (1 μM) and [D-Ala², MePhe⁴, gly(ol)⁵]-enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen², D-Pen⁵], enkephalin (DPDPE, 1 μM) did not.
5. Fentanyl (0.1 nM–10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P₃ formation, with a pEC₅₀ of 7.95±0.15 (n=5). PTX (100 ng ml⁻¹ for 24 h) abolished, whilst Ni²⁺ (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P₃ response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P₃ formation. Fentanyl (1 μM) also caused an increase in [Ca²⁺]_i (80±16.4 nM, n=6), reaching a maximum at 26.8±2.5 s. The increase in [Ca²⁺]_i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca²⁺]_i.
6. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca²⁺]_i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.

     

Influenza A virus replication is inhibited in IFN-λ2 and IFN-λ3 transfected or stimulated cells

Interferons lambda (IFN-λ) are the most recently defined members of the class III cytokine family. To investigate whether IFN-λ2 and IFN-λ3 displayed antiviral activity against influenza A virus (IAV), a number of cell lines induced with IFNs – as well as two established cell lines (A549-IFN-λ2 and A549-IFN-λ3) – were infected with IAV. Our results indicate that IFN-λ2 has statistically significant antiviral activity in A549-IFN-λ2 (P = 0.0028) although less so than IFN-λ3, which reduced viral titer to 10% (P < 0.0001). The reverse was observed for cells treated with IFNs, with IFN-λ2-treated A549 cells inhibiting IAV infection more efficiently than IFN-λ3-treated A549 cells. The antiviral effect on IFN-stimulated cells was most apparent on Vero cells (compared with MDCK and HeLa). Both IFNs significantly inhibited IAV replication and inhibition was observed in a dose-dependent manner, with an optimal IFN concentration of 20 ng/ml. IFN-λ2 was more potent than IFN-λ3 on Vero cells while IFN-λ3 appeared more efficient than IFN-λ2 on MDCK and HeLa cells.     

Influenza A virus replication is inhibited in IFN-λ2 and IFN-λ3 transfected or stimulated cells

Interferons lambda (IFN-λ) are the most recently defined members of the class III cytokine family. To investigate whether IFN-λ2 and IFN-λ3 displayed antiviral activity against influenza A virus (IAV), a number of cell lines induced with IFNs - as well as two established cell lines (A549-IFN-λ2 and A549-IFN-λ3) - were infected with IAV. Our results indicate that IFN-λ2 has statistically significant antiviral activity in A549-IFN-λ2 (P=0.0028) although less so than IFN-λ3, which reduced viral titer to 10% (P<0.0001). The reverse was observed for cells treated with IFNs, with IFN-λ2-treated A549 cells inhibiting IAV infection more efficiently than IFN-λ3-treated A549 cells. The antiviral effect on IFN-stimulated cells was most apparent on Vero cells (compared with MDCK and HeLa). Both IFNs significantly inhibited IAV replication and inhibition was observed in a dose-dependent manner, with an optimal IFN concentration of 20 ng/ml. IFN-λ2 was more potent than IFN-λ3 on Vero cells while IFN-λ3 appeared more efficient than IFN-λ2 on MDCK and HeLa cells.     

Time course study of Aβ formation and neurite outgrowth disruption in differentiated human neuroblastoma cells exposed to H2O2: Protective role of autophagy

Here, we tried to elucidate the possible role of autophagy against H₂O₂ and Amyloid beta (Aβ) induced neurotoxicity using retinoic acid differentiated SH-SY5Y cells. We found that H₂O₂ disrupted neurite outgrowth concomitant with production of Aβ. Furthermore, we showed that H₂O₂ could increase the apoptotic factors such as Bax/Bcl-2 ratio, caspase-3 level, and PARP activity in a time course manner. These findings were confirmed by acridine orange/ethidium bromide and Hoechst staining. In addition, we observed that H₂O₂ led to conversion of LC3 protein from LC3I to LC3II and an increase in autophagy flux. Autophagy factors including LC3B, Atg7, and Atg12 increased and reached their highest level after 2 h of insulting and then dropped to a lower level. Our results showed that autophagy could internalize and degrade intra- and extracellular Aβ after 3 h treatment with H₂O₂. However, the remaining amount of Aβ accelerated morphological atrophy and, as a result, increased neuronal death (apoptosis). Inhibition of autophagy influx, using 3-methyl-adenine, increased intra- and extracellular levels of Aβ, providing more proof for a protective role of autophagy against oxidative stress. Further studies can shed light on the important role of autophagy by finding new pathways involved in Aβ degeneration.     

Activated O2(•−) and H2O2 mediated cell survival in SU11274-treated non-small-cell lung cancer A549 cells via c-Met-PI3K-Akt and c-Met-Grb2/SOS-Ras-p38 pathways

The pharmacological activity of SU11274 is primarily due to its inhibition of hepotocyte growth factor receptor (c-Met) kinase overexpression. In this study, we demonstrated that the pathway involved in SU11274-induced autophagy was presumably through inhibition of c-Met and its down-stream pathways, including phosphatidylinositol 3-kinases – Akt (PI3K–Akt) and the growth factor receptor bound protein-2 / son of sevenless – Ras – p38 MAPK (Grb2/SOS–Ras–p38) pathway. SU11274 time-dependently induced the generation of superoxide anion (O2(??)) and hydrogen peroxide (H2O2). There is a negative feedback loop between reactive oxygen species (ROS) induction and SU11274. Then, we investigated the role of ROS in protecting cells against SU11274-induced autophagic cell death in A549 cells. O2(??) and H2O2 generation activated c-Met–PI3K–Akt and c-Met–Grb2/SOS–Ras–p38 signaling pathways, which were suppressed by O2(??) scavenger superoxide dismutase (SOD) and H2O2 scavenger catalase. In conclusion, O2(??) and H2O2 evoked cell resistance to SU11274 via activating c-Met–PI3K–Akt and c-Met–Grb2/SOS–Ras–p38 pathways in A549 cells. SU11274 also induced ROS generation in Caenorhabditis elegans.     

CD4+ Foxp3+ regulatory T cells induced by TGF-β, IL-2 and all-trans retinoic acid attenuate obliterative bronchiolitis in rat trachea transplantation

Obliterative bronchiolitis (OB) is the major obstacle for long-term allograft survival in lung transplantation, and the underlying mechanism is still not well understood. Regulatory T cells (Tregs) have been shown to be essential in the maintenance of immune tolerance. In this study we investigated the role of Tregs in protecting OB in rat. We show that the combination of TGF-β, Interleukin (IL)-2, and all-trans retinoic acid (atRA) could induce naïve rat CD4⁺CD25⁻ T cells to differentiate into CD4⁺CD25⁺Foxp3⁺ T cells in vitro, and they acquired suppressive function. In a rat orthotopic tracheal transplantation OB model, the adoptive transfer of the induced Tregs reduced symptoms of airway obliteration and fibrication of grafts when compared with adoptive transfer of control cells without suppressive property. Moreover, recipients treated with the induced Tregs secreted high level of immunosuppressive cytokine TGF-β and IL-10, and low level of pro-inflammatory cytokines IL-17, IFN-γ, IL-6, and MCP-1, and had fewer effector T cells including Th17 cells and Th1 cells in the graft. Taken together, these findings suggest that in vitro induced Tregs by the combination of TGF-β, IL-2, and atRA are effective in protecting rat trachea allograft rejection through the inhibition of effector T cells and their function. These datas implicate new therapies to prevent OB and allograft rejection in human lung transplantation.     

Permeability of novel 4′-<Emphasis Type="Italic">N</Emphasis>-substituted (aminomethyl) benzoate-7-substituted nicotinic acid ester derivatives of scutellarein in Caco-2 cells and in an in vitro model of the blood-brain barrier

A series of 4′-N-substituted (aminomethyl) benzoate-7-substituted nicotinic acid ester derivatives of scutellarein was designed and synthesized. Evaluation of physiochemical properties showed that the newly designed compounds had greater chemical stability and aqueous solubility than scutellarin or scutellarein. The permeabilities (P _{app AP to BL}) of compounds 7b and 7e in Caco-2 cells were 5.9-fold and 3.7-fold higher than that of scutellarin, and 3.7-fold and 2.4-fold higher than that of scutellarein. The permeabilities (P _{app AP to BL}) of compounds 7b and 7e in an in vitro model of the blood–brain barrier were 9.7-fold and 5.9-fold higher than that of scutellarin, and 9.2-fold and 5.6-fold higher than that of scutellarein.     

Interruption of hepatocyte growth factor signaling augmented oridonin-induced death in human non-small cell lung cancer A549 cells via c-met-nuclear factor-κB-cyclooxygenase-2 and c-Met-Bcl-2-caspase-3 pathways

The aim of this study was to elucidate the molecular mechanisms mediating hepatocyte growth factor (HGF)-induced protection against oridonin-induced apoptosis in A549 cells. Oridonin induced decrease in Bcl-2/Bax ratio and activation of caspase-3, while these processes were reversed by HGF, suggesting that HGF played an anti-apoptotic role in oridonin-induced A549 cell death. HGF-induced protective effect was partially attributed to the activation of nuclear factor (NF)-κB and cyclooxygenase 2 (COX-2), since the protective effect was abolished by inhibition of NF-κB or interruption of COX-2. Then the activated COX-2 could prevent cells from initiating the apoptotic response by promoting prostaglandin E? (PGE?) release. Activation of NF-κB-COX-2 by HGF-treatment triggered the increase in Bcl-2/Bax ratio, inhibition of procaspase-3 cleavage, promotion of Ca2?-independent intracellular phospholipase A2 (iPLA2) expression and augmentation of PGE? release, leading to antagonizing oridonin-induced cell death in A549 cells. HGF-induced cell survival in response to oridonin administration was associated with the activation of c-Met-NF-κB-COX-2 and c-Met-Bcl-2-caspase-3 signaling pathways. iPLA2, downstream effector of caspase-3, also participated in these processes.     

A multicenter phase II trial of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine®) and gemcitabine in advanced non-small-cell lung cancer with pharmacokinetic evaluation using peripheral blood mononuclear cells

Summary Background: We tested the hypothesis that 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine?) may enhance response to re-treatment with gemcitabine by enhancing intracellular uptake of gemcitabine in a phase II study. Method: Patients who had prior exposure to gemcitabine as a first-line treatment of advanced non-small-cell lung cancer (NSCLC) were given weekly infusions of 3-AP and gemcitabine for 3 weeks followed by 1 week of rest, repeated every 28 days. Plasma and peripheral blood mononuclear cells (PBMCs) were collected to evaluate the effect of 3-AP on pharmacokinetics and intracellular uptake of gemcitabine. Result: Twelve patients were treated with a median of two treatment cycles without objective response, hence the study was terminated at interim analysis. Four patients had stable disease and the median time to progression was 3 months (95% confidence interval, CI: 1.7 to 9.1 months). Grade 3 toxicities included neutropenia (two patients), hypoxia (three patients) and dyspnea (one patient). Four patients developed reversible symptomatic methemoglobinemia during 3-AP infusion, with mild to moderately elevated methemoglobin levels that ranged from 7.8 to 17.6% of the total hemoglobin concentration. Limited pharmacokinetic data did not suggest any clinically relevant pharmacological influence of 3-AP on gemcitabine. Conclusion: 3-AP did not enhance clinical response to gemcitabine in this cohort of patients with prior exposure to gemcitabine for advanced NSCLC. Further development of 3-AP in lung cancer is challenged by its potential of causing methemoglobinemia and hypoxia, which could be problematic in patients with compromised pulmonary reserves.     

Different roles for type A and type B monoamine oxidase in regulating synaptic dopamine at D-1 and D-2 receptors associated with adenosine-3′,5′-cyclic monophosphate (cyclic AMP) formation

Summary The roles of multiple forms of monoamine oxidase (MAO) in regulating the synaptic concentration of dopamine, in the vicinity of dopamine receptors associated with cyclic AMP formation, was examined in striatal slices of the rat. d-Amphetamine (0.1 mol/l to 20 mol/l) caused a concentration-related increase in cyclic AMP formation, which correlated (in superfusion experiments) with the release of endogenously-formed dopamine. In the presence of (–)sulpiride (50 pmol/l), cyclic AMP formation was significantly increased at every concentration of d-amphetamine tested. At the same time, this concentration of (–)sulpiride had no effect on DA release. Inhibition of type A MAO with clorgyline (0.1 mol/l) significantly enhanced the increase in cyclic AMP formation seen after d-amphetamine. By contrast, inhibition of type B MAO with deprenyl (0.1 mol/l) was without effect on this action of d-amphetamine. At high concentrations of d-amphetamine (20 mol/l), however, deprenyl + clorgyline treatment enhanced cyclic AMP formation to a greater extent than with clorgyline alone. Similar results could be obtained at lower concentrations of d-amphetamine (5 mol/l), but only after inhibition of the dopamine neuronal reuptake system with nomifensine (30 gmol/1). Furthermore, in the presence of nomifensine, deprenyl alone was also able to significantly increase the cyclic AMP formation seen after d-amphetamine (5 gmol/l). In the presence of (–)sulpiride, relatively similar results were obtained following all MAO inhibitor treatments. These findings support the notion that type A MAO plays the primary role in regulating dopamine concentrations at D-1 and D-2 receptors within synapses of rat striatal tissue. However, a minor role for type B MAO can be demonstrated when high synaptic concentrations of dopamine are achieved or dopamine deamination is restricted to postsynaptic sites. Experiments with amezinium (10 mol/l) were conducted to further explore the importance of presynaptic and postsynaptic type A MAO in the regulation of synaptic concentrations of dopamine; total type A MAO inhibition was assessed with clorgyline (0.1 mol/l). caused a two-fold increase in the ability of d-amphetamine (5 mol/l) to increase cyclic AMP formation. This result represented 70% of the response obtained after total type A MAO inhibition with clorgyline. The values obtained with clorgyline, but not amezinium, were potentiated by inhibition of the dopamine neuronal reuptake system with nomifensine. These results support the idea that presynaptic type A MAO is highly instrumental in regulating synaptic dopamine concentrations; possibly by regulating the size of the pool of releasable dopamine. Postsynaptic type A MAO, however, can also play an effective role in the regulation of synaptic DA levels in instances where the dopamine neuronal reuptake system may become compromised.Abbreviations DA dopamine - CAMP cyclic AMP; adenosine-3,5-monophosphate - MAO monoamine oxidase Supported in part by the West Virginia University Medical Corporation, N. I. H. grant 5-T32-GM 07039, and a grant from the Fraternal Order of Eagles. Some of the findings were presented at the annual meeting of the Society for Neuroscience, Washington, DC (Azzaro and Liecione 1986) Send offprint requests to A. J. Azzaro