Development of the new group of indole-derived neuroprotective drugs affecting oxidative stress.

1. The role of oxidative stress, and accordingly uncontrolled reactive oxygen species generation/action, have been widely documented in a number of different neuronal pathologies. However, the concept of pharmacological interventions in prevention and therapy of oxidative stress-related diseases has not found adequate application in clinical practice. This may be due to the insufficient efficacy of drugs available, their unsuitable pharmacokinetics, side effects, toxicity, etc. 2. Based on stobadine, (--)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido[4,3-b]indole, a well-known antioxidant, free radical scavenger, and neuroprotectant, it was attempted to develop new stobadine derivatives with improved pharmacodynamic and toxicity profiles, on applying molecular design, synthesis and adequate tests. Stobadine molecule was modified mostly by electron donating substitution on the benzene ring and by alkoxycarbonyl substitution at N-2 position. A total of >70 derivatives were prepared. 3. In a mice model of head trauma, some of the new stobadine derivatives administered i.v. immediately after the trauma, significantly improved sensomotoric outcome in the animals assessed 1 h later. Accordingly, decrease in brain edema was proved histologically as well as by brain wet weight assessment. 4. Putative neuroprotective action of the compounds was confirmed on rat hippocampal slices exposed to reversible 6 min hypoxia/low glucose by analysis of synaptic transmission in CA1 region neurons. Irreversible impairment of neurotransmission resulting from the hypoxia was significantly reduced by the presence of SMe1EC2, one of the new compounds, in concentration range 0.03-10.0x10(-6) mol l(-1). Both the neuroprotective and antioxidant effect of the compound closely resembled those of stobadine, melatonin, 21-aminosteroids, alpha-phenyl-tert-butylnitrone and others, all well-established antioxidants, except the range of effective concentrations was by 1-2 orders lower in SMe1EC2. 5. A remarkable antioxidant efficacy was observed in the new compounds in rat brain homogenates exposed to iron/ascorbate system by protection of lipids and creatine kinase against the oxidative impairment. A link between the neuroprotective and antioxidant/ scavenger properties in the compounds can be assumed. 6. Acute toxicity of some of the new pyridoindoles was diminished compared to stobadine. That might be due to the virtually full elimination of stobadine's undesired alpha (1)-adrenolytic activity attained by appropriate modifications of its molecule. 7. The new pyridoindoles extend the range of available neuroprotectants interfering with oxidative stress in neuronal tissue.

In vitro physiological evidence of enhanced antioxidant and neuroprotective action of 2,3-dihydromelatonin, a melatonin analogue.

As the capacity of the endogenous antioxidative system is limited, pharmacological treatment with antioxidants may help to protect neuronal tissue against increased amount of reactive oxygen species produced during oxidative stress. We attempted to improve resistance of rat hippocampal slices exposed to ischaemia in vitro (hypoxia (HYP) accompanied with decreased glucose concentration followed by reoxygenation (ROX)) and thus to diminish the impairment of synaptic transmission after HYP/ROX. We compared the protective features of the melatonin analogue 2,3-dihydromelatonin (2,3-DHM) with melatonin itself. In preliminary experiments, the compound 2,3-DHM compared to melatonin revealed enhanced antilipoperoxidation action in rat brain homogenates exposed to Fe/ascorbate system (-logIC(50) = 4.76 +/- 0.01 versus -logIC(50) = 2.51 +/- 0.02, respectively). In this study, 2,3-DHM (from 0.3 to 10 micromol l(-1)) applied at 30 min before the beginning of HYP and remaining all over the 6-min HYP as well as 20-min ROX, exerted a protective effect demonstrated by improvement of the population spike amplitude (PoS) recovery during ROX, with the maximum effect at 3 micromol l(-1). In accordance with this, the ratio of irreversibly damaged slices after HYP/ROX was decreased in the groups treated with 2,3-DHM. Moreover, a significant delay of PoS decay during HYP (expressed as half time, t(0.5)) was revealed at 2,3-DHM concentration 1 and 3 micromol l(-1)). An equipotent effect of melatonin and 2,3-DHM was achieved by a 100-times lower concentration of the latter (0.3 and 1 micromol l(-1)) compared to that of melatonin (30 and 100 micromol l(-1)). Further, compared to the highest effect of 2,3-DHM in the concentration 3 micromol l(-1) on the percentage of irreversibly damaged slices (only 20%), melatonin did not exert such pronounced effect, either in the concentration 30 or 100 micromol l(-1) (67 and 50%, respectively). We conclude that hydrogenation of the melatonin molecule significantly improved its antihypoxic effect in our model of rat hippocampal slices exposed to ischaemic conditions in vitro, similarly as it enhanced the antilipoperoxidation action of 2,3-DHM in our previous studies. These findings suggest that 2,3-DHM deserves more attention concerning its neuroprotective effect in oxidative stress-associated tissue damage.

In vitro inhibition of lens aldose reductase by (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid in enzyme preparations isolated from diabetic rats.

(2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (compound 1), a novel aldose reductase inhibitor, was assayed for efficacy and selectivity to inhibit rat lens aldose reductase under in vitro conditions by using enzyme preparations obtained from diabetic animals. The inhibitory efficiency was characterized by IC(50) in micromolar region. Enzyme kinetics analysis revealed uncompetitive type of inhibition, both in relation to the D,L-glyceraldehyde substrate and to the NADPH cofactor. In testing for selectivity, comparisons to rat kidney aldehyde reductase, an enzyme with the highest homology to aldose reductase, was used. The inhibition selectivity of the compound tested was characterized by selectivity factor around 20 and was even slightly improved under conditions of prolonged experimental diabetes. These findings were identical with those in the control rats. To conclude, the inhibitory mode, efficacy and selectivity of compound 1, a novel aldose reductase inhibitor, was preserved even under the conditions of prolonged STZ-induced experimental diabetes of rats.