Synthesis of antioxidants for prevention of age-related macular degeneration.

Photooxidation of A2E may be involved in diseases of the macula, and antioxidants could serve as therapeutic agents for these diseases. Inhibitors of A2E photooxidation were prepared by Mannich reaction of the antioxidant quercetin. These compounds contain water-solubilizing amine groups, and several were more potent inhibitors of A2E photooxidation than quercetin.

Inhibition of MPP+-induced mitochondrial damage and cell death by trifluoperazine and W-7 in PC12 cells.

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of trifluoperazine and W-7 on the MPP+-induced mitochondrial damage and cell death in undifferentiated PC12 cells. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) at 0.5-1 microM significantly reduced the loss of cell viability in PC12 cells treated with 500 microM MPP+. Trifluoperazine and W-7 (0.5-1 microM) inhibited the nuclear damage, the loss of the mitochondrial transmembrane potential followed by cytochrome c release, and the elevation of intracellular Ca2+ levels due to MPP+ in PC12 cells and attenuated the formation of reactive oxygen species and the depletion of GSH. Calmodulin antagonists at 5-10 microM exhibited a cytotoxic effect on PC12 cells, and compounds at 10 microM did not attenuate cytotoxicity of MPP+. Calmodulin antagonists (0.5-1 microM) significantly reduced rotenone-induced mitochondrial damage and cell death, whereas they did not attenuate cell death and elevation of intracellular Ca2+ levels due to H2O2 or ionomycin. The results show that trifluoperazine and W-7 exhibit a differential inhibitory effect against cytotoxicity of MPP+ depending on concentration. Both compounds at the concentrations less than 5 microM may attenuate the MPP+-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering the intracellular Ca2+ levels.

Inhibitory effects of cigarette smoke on glial inducible nitric oxide synthase and lack of protective properties against oxidative neurotoxins in vitro.

Epidemiological studies consistently report an inverse correlation between cigarette smoking and associated risk for Parkinson's disease (PD). The degeneration of dopaminergic neurons may involve the toxic metabolic products of glial cell monoamine oxidase (MAO) and inducible nitric oxide synthase (iNOS). This study evaluates the direct protective effects of cigarette smoke (CS) against potential neurotoxic products of MAO, such as 1-methyl-4-phenylpyridinium (MPP+), 6-hydroxydopamine (6-OHDA) and hydrogen peroxide (H2O2) in brain neuroblastoma. Moreover, the effects of CS were also evaluated on endotoxin/cytokine activated glioma iNOS protein expression and MAO enzyme activity. Cigarette smoke condensates (CSCs) were acquired from Marlboro 20 Class A and Kentucky 2R4F reference research (2R4F) cigarettes. The CSCs did not protect against 6-OHDA or H2O2 toxicity in neuroblastoma, and exhibited a very mild protective effect [approximately 10%] against MPP+. Neither CSC demonstrated antioxidant capability, but conversely contained high concentration of NO2-. Paradoxically, in glioma cells, iNOS protein expression and endogenous enzymatic NO2- production were significantly blocked by both CSCs. Both CSCs also inhibited glioma MAO-A and MAO-B [1.4.3.4]. Kinetic analysis indicated that 2R4F-CSC displayed competitive inhibition and the Marlboro-CSC exerted potent competitive and non-competitive inhibition. In conclusion, these data suggest that cigarette smoke does not appear to directly protect against the toxicity of the selected neurotoxins. In contrast, CS exerts pronounced effects on glia, whereby its presence can simultaneously attenuate cytokine induction of iNOS and MAO.

Ascorbic acid reduces the dopamine depletion induced by methamphetamine and the 1-methyl-4-phenyl pyridinium ion.

The neurotoxic actions of methamphetamine and the 1-methyl-4-phenyl pyridinium ion (MPP+) have been ascribed, at least in part, to the generation of free radicals. This hypothesis was evaluated by attempting to reduce the toxic actions of these compounds by pretreatment with an antioxidant, ascorbic acid. The intrastriatal administration of methamphetamine caused a 37% depletion of dopamine. Treatment with 100 or 1000 mg/kg of ascorbic acid significantly reduced the methamphetamine-induced dopamine depletion (by 21 and 27%, respectively). The intrastriatal administration of MPP+ caused an 88% depletion of dopamine. Treatment with 100 or 1000 mg/kg of ascorbic acid significantly reduced the MPP+-induced dopamine depletion (by 22 and 45%, respectively). Thus, free radicals may mediate the toxic actions of these compounds.

L-dopa reverses the effects of MPP+ toxicity.

The behavioral and neurochemical consequences of the intrastriatal administration of 1-methyl-4-phenylpyridinium ion (MPP+), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), were evaluated using the shuttlebox avoidance paradigm. MPP+ caused a significant depletion of striatal dopamine and significant disruption of shuttlebox performance. L-Dopa administration reversed both the dopamine depletion and the behavioral deficits. These observations are discussed in reference to the rodent-MPTP model of Parkinson's disease.

Differential effects of carboxyfullerene on MPP+/MPTP-induced neurotoxicity.

The effects of carboxyfullerene on a well-known neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenyl-pyridinium (MPP+) were investigated. In chloral hydrate-anesthetized rats, cytosolic cytochrome c was elevated in the infused substantia nigra 4 h after an intranigral infusion of MPP+. Five days after local application of MPP+, lipid peroxidation (LP) was elevated in the infused substantia nigra. Furthermore, dopamine content and tyrosine hydroxylase (TH)-positive axons were reduced in the ipsilateral striatum. Concomitant intranigral infusion of carboxyfullerene abolished the elevation in cytochrome c and oxidative injuries induced by MPP+. In contrast, systemic application of carboxyfullerene did not prevent neurotoxicity induced by intraperitoneal injection of MPTP. In mice, systemic administration of MPTP induced a dose-dependent depletion in striatal dopamine content. Simultaneous injection of carboxyfullerene (10 mg/kg) actually potentiated MPTP-induced reduction in striatal dopamine content. Furthermore, systemic administration of carboxyfullerene (30 mg/kg) caused death in the MPTP-treated mice. An increase in the striatal MPP+ level and reduction in hepatic P450 level were observed in the carboxyfullerene co-treated mice. These data showed that systemic application of carboxyfullerene appears to potentiate MPTP-induced neurotoxicity while local carboxyfullerene has been suggested as a neuroprotective agent. Furthermore, an increase in striatal MPP+ level may contribute to the potentiation by carboxyfullerene of MPTP-induced neurotoxicity.

GM1 ganglioside partially rescues cultured dopaminergic neurons from MPP(+)-induced damage: dependence on initial damage and time of treatment.

GM1 ganglioside is believed to be important in promoting the recovery of neurons from injury. The present study assesses the ability of GM1 to repair or prevent the damage of dopamine neurons caused by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Treatment of mesencephalic cell cultures with 2.5 microM MPP+ resulted in the loss of 30% of tyrosine hydroxylase (TH) immunoreactive neurons. In contrast, cultures administered 100 microM GM1 ganglioside for 3 days after toxin treatment contained nearly control numbers of TH+ neurons (97%). This reparative effect of GM1 was reflected in parallel increases in TH enzyme activity, dopamine and dopac levels. Cultures sustaining greater insult from higher doses of MPP+ (5.0-10.0 microM) did not benefit from ganglioside treatment, suggesting that rescue by GM1 depended on the degree of initial damage to cells. Moreover, the timing of ganglioside treatment was critical; pretreatment with GM1 alone did not prevent or attenuate the damage caused by subsequent incubation in 2.5 microM MPP+.

The neurotoxic actions of 1-methyl-4-phenylpyridine (MPP+) are not prevented by deprenyl treatment.

1-Methyl-4-phenylpyridine (MPP+) injected into the cerebral ventricles (ICV) of mouse caused depletions of striatal dopamine (DA)(-42%), 3,4-dihydroxyphenylacetic acid (DOPAC) (-34%) and homovanillic acid (HVA) (-16%) content without significant reductions in levels of noradrenaline (NA), serotonin (5-HT) or 5-hydroxyindoleacetic acid (5-HIAA). When deprenyl was administered before MPP+, striatal DA and its metabolites were further depleted, and striatal NA and 5-HT levels also were reduced. Further, whilst ICV MPP+ alone failed to influence the biochemistry of the limbic areas (nucleus accumbens plus tuberculum olfactorium), in the presence of deprenyl MPP+ caused 20-40% reductions in levels of limbic NA, DA, DOPAC, HVA, 5-HT and 5-HIAA. Therefore, deprenyl treatment does not prevent the neurotoxic actions of MPP+; indeed, a more extensive neurotoxicity for MPP+ is revealed in the presence of this monoamine oxidase inhibitor.

Metabolism of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in a rat pheochromocytoma cell line, PC12h.

The uptake and metabolism of a neurotoxin, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were examined in a rat pheochromocytoma cell line, PC12h. These cells which contain only type A monoamine oxidase (MAO-A) oxidize MPTP into N-methyl-4-phenylpyridinium ion (MPP+). By kinetic analysis, the apparent Km value and the maximal velocity of the MPP+ production are 70.4 +/- 6.5 microM and 38.3 +/- 10.0 pmol/min/mg protein, respectively. After 7 days of culture in the presence of MPTP, the cells could oxidize from 25 to 50% of the MPTP added to the culture medium and could accumulate MPP+. The intracellular concentrations of MPTP were almost the same after 7 days of culture in the presence of MPTP from 10 nM to 100 microM. The cells could survive 7 days after exposure to up to 100 microM MPTP. Tyrosine hydroxylase (TH) and MAO activity were not affected by the presence of MPTP. Dopamine (DA) concentrations and a nonspecific enzyme, beta-galactosidase activity in the cells were not affected by the addition of MPTP. These data show that the uptake and oxidative conversion of MPTP take place in the cells having MAO-A alone, and that the neurotoxicity of MPP+ may not be due directly to its storage in subcellular compartments.

Comparative evaluation of neuromuscular blockade and reversibility of AH 8165 and pancuronium bromide in man.

The neuromuscular properties of AH 8165 were compared to those of pancuronium bromide in 66 patients during analgesic anesthesia procedures. Administration of 0.5 mg/kg AH 8165 and 0.04 mg/kg pancuronium bromide produced very similar degrees of non-depolarizing block. AH 8165 acted quicker and therefore gave better conditions for intubation. Spontaneous recovery of muscle twitch tension was slightly shorter for AH 8165 than for pancuronium bromide, whether small or larger amounts of drugs were used. Administration of neostigmine caused rapid and complete return of muscle twitch tension, well-maintained tetanus and insignificant posttetanic potentiation within 20 minutes, with a similar efficiency of competitive neuromuscular block reversal for both drugs.

[RPE melanosomes bind A2E fluorophore of lipofuscin granules and products of its photooxidation].

The ability of melanosomes from human, bovine and frog retinal pigment epithelium cells (RPE) to bind A2E fluorophore of RPE lipofuscin granules and products of A2E photooxidation is investigated. RPE melanosomes are found to bind A2E molecules themselves as well as the molecules formed after A2E irradiation by visible light. In our experiments single melanosome was able to bind up to 0.08 fmol A2E. Antioxidant activity of melanosomes is compared to antioxidant activity of their complexes with A2E. It is shown by luminal chemiluminescence quenching in the presence of hydrogen peroxide that in A2E/melanosomes complex the chemiluminescence quenching is not significantly reduced. Comparison of inhibitory activity of melanosomes and their complexes with A2E on UV-induced (light conditions) and Fe(2+)-ascorbate-induced (dark conditions) peroxidation of photoreceptor outer segments (POS) demonstrated that bound A2E does not affect inhibitory ability of melanosomes in both systems. Thus, binding of A2E to RPE melanosomes in concentrations from 0.01 to 0.1 fmol A2E per melanosome does not significantly alter their antioxidant properties. It is supposed that both A2E and hydrophilic products of its photooxidation could be bound by RPE melanosomes and, thus, it lost the ability to exhibit toxic properties.

Kinetic analysis of interactions between alkylene-linked bis-pyridiniumaldoximes and human acetylcholinesterases inhibited by various organophosphorus compounds.

The therapeutic approach of organophosphorus compound (OP) intoxications is to reactivate the inhibited enzyme acetylcholinesterase (AChE). Numerous studies demonstrated a limited efficacy of standard oxime-based reactivators against different nerve agents such as tabun and cyclosarin. This emphasizes research for more effective oximes. In the present study, reactivation kinetics of tabun-, sarin-, cyclosarin-, VX- or paraoxon-ethyl-inhibited human AChE (hAChE) with a homologous series of bis-ortho-pyridiniumaldoximes, Ortho-4 - Ortho-9, was investigated with a robot-assisted setting, allowing determination of second-order reactivation rate constants as well as model calculations. The reactivation constants of Ortho-4 - Ortho-9 resulted in marked differences of affinity and reactivity depending on the OP structure and the linker length of the oximes. In general, the K(D) values decreased with increasing linker length. Reactivity increased from Ortho-4 to Ortho-6 for PXE- and VX-inhibited hAChE and from Ortho-4 to Ortho-7 for GA-inhibited hAChE and decreased again with Ortho-8 and Ortho-9. In contrast, k(r) decreased with increasing linker length for sarin- and cyclosarin-inhibited hAChE. In view of the pronounced decrease of K(D) from Ortho-4 to Ortho-9, the k(r2) values increased with all tested OP. Hence, the ratios of K(I)/K(D) and of K(I)/k(r2) showed that in almost all cases the affinity of Ortho-N to the native hAChE was higher than to OP-inhibited enzyme. Model calculations indicated that Ortho-6 - Ortho-9 could be superior to obidoxime in reactivating tabun-inhibited hAChE. Finally, these data emphasize the need to develop oximes with a higher selective affinity towards OP-inhibited hAChE in order to minimize possible side effects.

Protective effects of myricetin and related flavonols against A2E and light mediated-cell death in bovine retinal primary cell culture.

The present study was performed to investigate the effect of flavonols, namely myricetin and structurally related quercetin and kaempferol against A2E and blue light-induced photoreceptors death in primary retinal cell cultures. Primary retinal cell cultures were prepared from bovine retinas. Fourteen-day-old cultures were pretreated with different concentrations of myricetin, quercetin, kaempferol (1-40 microM) for 24 h, then treated with 30 microM of A2E or exposed to blue-actinic light for 20 h. Green nucleic acid stain assay was used to evaluate cell death. Photoreceptor and bipolar cells were immunolabeled with specific antibodies and were counted using automated microscope imaging and image-based cell counting software. Twenty hours exposure to blue light induced approximately 75% death of photoreceptors in bovine retinal cell cultures. Myricetin protected 100% of photoreceptors against blue-light-mediated damage with an EC(50) of 9+/-0.7 microM. Quercetin resulted in a maximum of 15% protection against light damage, and kaempferol was inactive. A2E induced photoreceptor and bipolar cell death in a concentration-dependent manner with EC(50) of 25 microM for photoreceptors and 31 microM for bipolar cells. Myricetin, quercetin and kaempferol protected against A2E-induced photoreceptors and bipolar cells death with EC(50) values of 2+/-0.3 microM, 2+/-0.3 microM, 5+/-0.09 microM and 0.8+/-0.07 microM, 0.44+/-0.06 microM, 1+/-0.4 microM, respectively. Caspase-3 inhibitor (Z-DEVD-fmk) protected 42% photoreceptors and 57% bipolar cells from A2E toxicity. In contrast, this inhibitor had no effect against light-induced photoreceptor damage. Despite the poor activity of quercetin and the inactivity of kaempferol against blue light, myricetin, quercetin and kaempferol exhibited approximately 100% protection against A2E toxicity. This suggests that light- and A2E-induced cell deaths are mediated through different pathways. These results suggest that myricetin functions as potent and effective neuroprotective agent for photoreceptor cells against A2E and light damage.

Fazadinium in anaesthesia.

The clinical use of the neuromuscular blocking agent fazadinium was investigated in a survey of 500 patients. Rapid conditions for tracheal intubation followed by excellent relaxation and easy antagonism were obtained. It is suggested that these findings commend it as an alternative to suxamethonium. Tachycardia appeared to be the only disadvantage associated with its use.

The neuromuscular blocking properties of AH 8165 during halothane anaesthesia.

The time to onset, duration, time for reversal, and dose required to abolish twitch response for AH 8165 have been investigated. The drug is shown to be a rapidly acting agent, with a duration of action similar to pancuronium.

Initial clinical experience with fazadinium bromide during anaesthesia in Nigerians.

Fazadinium Bromide (Fazadon, AH 8165), a non depolarising muscle relaxant, was injected into one hundred unpremedicated Nigerian patients requiring endotracheal intubation and prolonged skeletal muscle relaxation. The dose of Fazadon used was 1.5 mg per kg. body weight. Endotracheal intubation was possible in all the patients within thirty seconds of the intravenous injection of Fazadon. The initial single dose of the drug provided muscle relaxation for an average of forty minutes without the addition of any volatile anaesthetic agent. Arterial blood pressure was well maintained, but there was a mean increase in pulse rate of 20 beats per minute. Return of skeletal muscle activity was observed within one minute of the injection of neostigmine, in all but one patient.

The use of fazadinium in children. An electromyographic study.

To evaluate the use fazadinium in children and to compare it with pancuronium, 64 children aged 6 months to 12 yr received fazadinium 0.75, 1.0, 1.25 mg kg-1 and pancuronium 0.1 mg kg-1 in groups of 16. Neuromuscular transmission was evaluated electromyographically using train-of-four stimuli. The times to first effect, maximum effect, first sign of recovery, reversal and full recovery after reversal were recorded and neuromuscular transmission was monitored continuously until complete recovery was obtained. Duration of satisfactory clinical relaxation and neuromuscular transmission when relaxation became inadequate were recorded. The speed, duration and depth of effect, and the duration and degree of neuromuscular blockade increased significantly depending on dose. Fazadinium was found to be a potent drug providing satisfactory relaxation in a dose of 1.0-1.25 mg kg-1 for operations of medium to long duration in children under light halothane anaesthesia. When relaxation became inadequate neuromuscular transmission still showed marked depression. Duration and degree of blockade of fazadinium and pancuronium were not age-dependent. In all groups the neuromuscular blockade was reversed easily. Apart from the speed of onset of action, pancuronium 0.1 mg kg-1 corresponded to a dose of fazadinium between 1.0 and 1.25 mg kg-1. Train-of-four ratio was found to be more sensitive than single twitch as an index of the recovery of transmission during recovery and in the period after operation.

[Inhibition by "essential" phospholipids of the bactericidal effect of cetylpyridinium chloride on common bacteria. Short communication]. / Durch "essentielle" Phospholipide induzierte Hemmung der bekteriziden Wirkung von Cetylpyridinium-chlorid auf banale Keime. Kurze Mitteilung

The authors examined the conditions under which the bactericidal action of the disinfectant cetylpyridinium chloride (CPC) is neutralized by a dispersion of "essential" phospholipids (EPL). Both gram-positive and gram-negative strains of bacteria were exposed to various concentrations of the quarternary ammonium over different periods of time. Three cultures were prepared for each contact model to check on the effect of either pre-treatment and after-treatment with phospholipids or of CPC-exposure without the addition of phospholipids. The results obtained demonstrate that EPL are able to "receive" gram-negative bacteria like E. coli, P. aerug. and Morax. glucidol. after exposure to lethal concentrations of CPC for 30 min. While the "phenomenon of restoration" depended on the dose of phospholipids applied, the time of expsure to phospholipids proved irrelevant. The stabilisation of bacterial membranes due to EPL is discussed as a possible explanation of the phenomenon of restoration.

Fructose prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced ATP depletion and toxicity in isolated hepatocytes.

The loss of viability of isolated rat hepatocytes exposed to either 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) was prevented by addition of fructose to the incubation medium. This protection was dependent on fructose concentration, being complete at 10 mM. Addition of fructose dramatically delayed MPTP- and MPP+-induced depletion of ATP and was accompanied by a significant accumulation of lactate, indicating the occurrence of enhanced glycolytic production of ATP. Glucose was much less effective against MPTP and MPP+ toxicity, probably because it is a relatively poor substrate for glycolysis in liver cells. We conclude that depletion of ATP is a critical event in MPTP cytotoxicity in our in vitro model system, and that the use of alternative sources of ATP production may represent an important protective device against the effects of this toxic agent.