Analyses of lysophosphatidic acids by gas chromatography mass spectrometry without hydrolytic pretreatment

Lysophosphatidic acids having different fatty acyl moieties were directly analysed by gas chromatography/mass spectrometry after silylation. Trimethylsilyl derivatives of various LPAs escaped thermal degradation, and electron impact and chemical ionization of these derivatives yielded many ions which were useful for the structural determination. It was found by this new method that five species of lysophosphatidic acids were formed in rat plasma during incubation at 37 degrees C.     

Free-radical degradation of high-molar-mass hyaluronan induced by ascorbate plus cupric ions: testing of stobadine and its two derivatives in function as antioxidants

Stobadine·2HCl and its two hydrophilic derivatives SM1dM9dM10·2HCl and SME1i-ProC2·HCl were tested in the function of antioxidants on hyaluronan (HA) degradation induced by the Weissberger oxidative system [ascorbate plus Cu(II)]. As a primary method, rotational viscometry was applied, where the substance tested was added before or 1 h after the initiation of HA degradation. The most effective scavengers of ?OH and peroxy-type radicals were recorded to be stobadine·2HCl and SME1i-ProC2·HCl, respectively. The most effective scavenger, determined by applying the ABTS assay, was stobadine·2HCl.     

Placental transfer of stobadine in rabbits

Stobadine, a pyridoindole antioxidant, was investigated for its placental transfer and distribution in New Zealand white rabbits on the 27th day of gestation. The concentrations of stobadine were determined in maternal and foetal organs (plasma, brain, heart) at 30, 60, 120, and 360 minutes after oral administration of the drug in a dose of 5 mg/kg. The results obtained proved that after oral stobadine intake by rabbits at the stage of advanced pregnancy both maternal and foetal organs were under a certain drug level which could act protectively against oxidative stress--frequently occurring during late organogenesis, foetal stages and delivery, as well as during early postnatal development.     

Aggregation of human blood platelets in the presence of the pyridoindole stobadine

The antiarrhythmic and cardioprotective drug stobadine, possessing antioxidant and neuroprotective properties, was studied as to its in vitro effect on aggregation of human blood platelets. Pretreatment of platelets with stobadine for 30 s inhibited stimulated platelet aggregation in a dose-dependent way. Depending on the aggregation stimulus used, the minimal effective concentrations of the drug were 1 micromol/l (adrenaline), 200 micromol/l (ADP), and 1,000 micromol/l (PMA). Aggregation induced with thrombin or Ca2+-ionophore A23187 was not changed in the presence of stobadine even in the concentration of 1,000 micromol/l. Addition of stobadine 30 s after adrenaline was also effective and terminated aggregation (100 and 1,000 micromol/l) or prolonged onset of its second phase (10 micromol/l). The presented experiments showed stobadine as a potent inhibitor of adrenaline-induced aggregation, indicating its involvement in the observed antithrombotic and cytoprotective activity.     

Lipid peroxidation of phosphatidylcholine liposomes depressed by the calcium channel blockers nifedipine and verapamil and by the antiarrhythmic-antihypoxic drug stobadine

Nifedipine, verapamil and stobadine were tested and compared with butylated hydroxytoluene (BHT) as possible free radical scavengers inhibiting lipid peroxidation in phosphatidylcholine liposomes. Liposomes were peroxidized by incubation in air at 50 degrees C. Verapamil less than nifedipine less than BHT less than stobadine depressed the lipid peroxidation as detected spectroscopically for conjugate diene and thiobarbituric acid product formation. Verapamil and stobadine were tested as OH radical scavengers in a Fenton-type reaction against spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO), as detected by ESR spectroscopy. The tested drugs competed with DMPO in trapping OH radicals, with stobadine being more effective than verapamil. ESR spectra of nifedipine in the incubated liposomes revealed that nifedipine could be involved in free radical reactions in the liposomes leading to nifedipine-stable radical(s) which were immobilized in the membrane. The obtained results suggest that some of the beneficial effects of the studied drugs can be mediated in disease by their ability to scavenge free radicals and by their protective effect on lipid peroxidation.     

Stobadine inhibits lysosomal enzyme release in vivo and in vitro

This study investigated the ability of stobadine, an effective cardioprotective drug with antiarrhythmic, antihypoxic and oxygen free radical scavenging properties, to protect cells against cyclophosphamide-induced toxic and cytotoxic damage in vivo and in vitro. Cyclophosphamide-induced toxic damage in female ICR mice was accompanied by marked increase in the activity of lysosomal enzymes in the spleen and kidney. Administration of stobadine prior to cyclophosphamide inhibited these biochemical changes. The in vivo protective effect of stobadine was comparable with its in vitro effect established in HeLa cells.     

Antioxidant activity of the pyridoindole stobadine in liposomal and microsomal lipid peroxidation.

Stobadine, a pyridoindole derivative, is an efficient inhibitor of lipid peroxidation in phosphatidylcholine liposomes and in rat liver microsomes treated with iron/ADP/NADPH as pro-oxidant. Accumulation of thiobarbituric acid-reactive substances (TBARS) or low-level chemiluminescence were taken as a measure of lipid peroxidation and 5 microM stobadine doubled the duration of the lag phase preceding the onset of rapidly increasing chemiluminescence. Inhibition of lipid peroxidation was not observed with tocopherol-deficient microsomes, suggesting that the antioxidant effect of stobadine depends on vitamin E in the membrane. The cis(-) isomer was most effective, with the cis(+) and trans(rac) as well as dehydro- or acetyl derivatives being less active. In liposomes, the presence of reductant (NADPH or ascorbate) protects from the loss of stobadine.     

Stobadine is a potent modulator of endogenous endothelin-1 in human fibroblasts

Binding of endothelin (ET) peptides to their respective receptors with resulting proliferation of vascular smooth muscle has been implicated in the pathogenesis of arterial hypertension and atherosclerosis. Recently it was hypothesized that endothelin- (ET-1) bound to its two membrane receptors (ET(A) and ET(B)) continues to activate signal transducing proteins in cells. It was also shown that pyridoindole stobadine stabilized lysosomal membranes in myocardium in early ischemia. Therefore we decided to study the effects of stobadine on specific, subtype-selective binding and subsequent degradation of human, synthetic [125I]-ET-1 in human fibroblasts (HF). Our results indicate that stobadine significantly potentiated ET-1 binding by reductive ET(B) selective degradation of ET-1 in HF. Hence, it is very plausible that stobadine may modulate endogenous endothelin and its intracellular mitogenic and chemotactic factors, principally by affecting two presumably related processes, participating in the proliferative and mitogenic response, (1) potentiation of signal trasduction from ET(A) receptors, and (2) subtype-ET(B) selective intracellular processing.     

Stobadine: bellwether of a broader view of drug actions

Stobadine was recognized early in its development as having antioxidant properties. A number of laboratories found associations between the antioxidant properties of stobadine and its potential beneficial effects. We found that stobadine acted as an antioxidant in a modification of an oxygen radical absorbance capacity (ORAC) assay. Similar results were observed with other drugs, including tirilazad and pramipexole. We suggest that stobadine and certain other drugs exhibit antioxidant properties in both hydrophilic and hydrophobic environments. Other drugs have been developed for their antioxidant properties and some currently marketed drugs have antioxidant properties. Although they may not have been explicitly sought during development, at least some of the beneficial effects may be related to antioxidant properties and/or scavenging of free radicals. Because stobadine was one of the first drugs for which useful properties were associated with its antioxidant actions, stobadine may be seen as a bellwether of a broader view of pharmacological actions--a view that encompasses antioxidant properties as a useful basis of therapeutic effects.     

Antiulcerogenic effect of pentacaine, chlorpromazine and stobadine on ethanol- and indomethacin-induced stomach lesions in rats.

The aim was to compare the antiulcerogenic effect of pentacaine, chlorpromazine and stobadine on indomethacin- and ethanol-induced stomach lesions in rats. Pentacaine effectively inhibited the formation of ethanol lesions, but in the given dose and under the given experimental conditions it was ineffective against indomethacin-induced damage. Chlorpromazine and stobadine effectively inhibited indomethacin erosions, but did not affect ethanol lesions significantly. The similarity of the effect of chlorpromazine and stobadine as compared with pentacaine, in the two stomach injury models, allows the assumption that stobadine has properties typical for an indirectly acting antiulcerogenic substance. This newly discovered property of stobadine extends the possibilities of its utilization to a further set of indications.     

Protective effect of stobadine on NCV in streptozotocin-diabetic rats: augmentation by vitamin E

Hyperglycaemia-induced oxidative stress makes an important contribution to the aetiology of diabetic neuropathy. Elevated reactive oxygen species (ROS) cause cumulative damage to neurons and Schwan cells, however, they also have a deleterious effect on nerve blood flow causing endoneurial hypoxia, which is responsible for early nerve conduction velocity (NCV) deficits and contributes to an increase in resistance to ischaemic conduction failure (RICF). We tested whether antioxidants - stobadine, vitamin E or the combination of these drugs, could prevent the early signs of neural dysfunction in animal model of diabetes in 8-9 weeks old male Wistar rats, made diabetic by streptozotocin (55 mg/kg i.v.) 4 months prior to testing. Neuropathy was evaluated electrophysiologically by measuring motor NCV and RICF of sciatic nerve in vitro. We observed that treatment with the combination of stobadine and vitamin E significantly (p < 0.001) reduced the NCV slowing in diabetic rats, although it did not fully prevent the NCV impairment. Significant effect (p < 0.05) was observed also in stobadine monotherapy. The RICF elevated in diabetic animals was not affected by any drug applied. This study confirmed that treatment with appropriate antioxidants, especially their combination could partially prevented the decrease in NCV in diabetic rats.     

sn-Glycerol-3-phosphate acyltransferase activity in particulate preparations from anaerobic, light-grown cells of Rhodopseudomonas spheroides. Involvement of acyl thiolester derivatives of acyl carrier protein in the synthesis of complex lipids.

Crude particulate preparations obtained from anaerobic, light-grown cells of Rhodopseudomonas spheroides have been shown to possess a significant level of sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity. In contrast to the enzyme from Escherichia coli, the R. spheroides glycerophosphate acyltransferase has a high specificity for acyl thiolester derivatives of acyl carrier protein (ACP) as acyl donors for the reaction. Only limited , nonlinear glycerophosphate incorporation into lipid occurs when acyl coenzyme A (CoA) derivatives are employed as acyl substrate. With oleyl-ACP as substrate, maximal enzyme activity was observed at 40 degrees, over a broad pH range (6.0 to 8.5) and did not require a divalent metal cation. The presence of dithiothreitol stimulated enzyme-activity 15 to 20%. When oleyl-ACP or palmityl-ACP was employed as sole acyl group donor, the major products recoverable from the reaction mixtures were lysophosphatidic acid, phosphatidic acid, and monoglyceride. Althouh oleyl-ACP and palmityl-ACP gave comparable maximal velocities in the initial acylation of glycerophosphate, the formation of phosphatidic acid occurred preferentially with the unsaturated acyl-ACP derivative.     

Mass spectrometric and kinetic studies on slow progression of papain-catalyzed polymerization of L-glutamic acid diethyl ester

Papain polymerizes L-glutamic acid diethyl ester (Glu-di-OEt) regioselectively, resulting in the formation of poly (gamma-ethyl alpha-L-glutamic acid) with various degrees of polymerization of less than 13. Reaction temperatures below 20 degrees C were appropriate for the reaction in terms of suppression of non-enzymatic degradation of Glu-di-OEt and an increase in the peptide yield, while the reaction was preceded by a pronounced induction period. Mass spectrometric analyses of the reaction conducted at 0 degrees C revealed that the accumulation of the initial dimerization product, L-glutamyl-L-glutamic acid triethyl ester (Glu-Glu-tri-OEt), was limited during the induction period, and that a sequential polymer derived from a further elongation of the dimer was the tetramer, but not the trimer. Kinetic analyses of acyl transfer reactions with Glu-di-OEt and Glu-Glu-tri-OEt as acyl acceptors and Nalpha-benzoyl-L-arginine ethyl ester as an acyl donor affirmed that Glu-Glu-tri-OEt bound more strongly than Glu-di-OEt both to the S- and S'-subsites of papain. Therefore, what occurred during the initial stage of the polymerization was interpreted as follows: the rate of the papain-catalyzed dimerization of Glu-di-OEt was extremely slow, once Glu-Glu-tri-OEt was initially synthesized it exclusively bound to the active site of papain, and then papain utilized the dimer in polymerization effectively rather than the monomer.     

Oxidative modification of serum albumin in an experimental glycation model of diabetes mellitus in vitro: effect of the pyridoindole antioxidant stobadine

Under conditions of an experimental in vitro glycation model, the pyridoindole antioxidant stobadine significantly inhibited glycation-related fluorescence changes of bovine serum albumin as well as the yield of 2,4-dinitrophenylhydrazine-reactive carbonyls with an efficacy comparable to that of the reference antioxidants Trolox C and 2-keto-4-methiolbutyric acid, and more efficiently than did aminoguanidine. Since stobadine did not affect the covalent binding of glucose, the protective effect may be explained by the ability of the drug to eliminate free radical intermediates of glyco-oxidation reactions, operative after the preceding glycation steps.     

Improved method for the synthesis of 1- or 3-acyl-sn-glycerols

Optically active 1- or 3-acyl-sn-glycerols were synthesized from 2,3- or 1,2-isopropylidene-sn-glycerols, respectively. The 2,3- or 1,2-isopropylidene-sn-glycerols were condensed with appropriate long saturated or unsaturated fatty acids and the resulting acyl isopropylidene compounds were treated with dimethylboronbromide at - 50 degrees C to give the title compounds. The ketal cleavage of acyl isopropylidene-sn-glycerols by dimethylboronbromide to produce the long 1- or 3-acyl-sn-glycerols was effective and gave good yields (70-90%). The reaction conditions were mild and there was no acyl migration, as shown by optical rotation of the monoacyl-sn-glycerols. The synthesis of 2,3-isopropylidene-sn-glycerol was improved to give an overall yield of 40% from L-arabinose. L-Arabinose was first converted to its 1,1'-diethylmercapto derivative and then condensed with 2-methoxypropene to yield 1,1'-diethyl-mercapto-4,5-isopropylidene-L-arabinose. Oxidation of this compound with sodium periodate followed by reduction with sodium borohydride under alkaline conditions yielded 2,3-isopropylidene-sn-glycerol [alpha]22D = -14.90 degrees, neat (Lit. 8 [alpha]22D = -14.5 degrees, neat; 14 [alpha]25D = -10.8 degrees; methanol C, 16.9). The optical purity of isopropylidene-sn-glycerols was determined as benzoyl derivatives on a high performance liquid chromatographic column packed with a chiral stationary phase.     

Development of the New Group of Indole-Derived Neuroprotective Drugs Affecting Oxidative Stress

Summary 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 0.03−10.0×10⁻⁶ 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.     

Inhibitory effect of stobadine on FMLP-induced chemiluminescence in human whole blood and isolated polymorphonuclear leukocytes.

The chemiluminescence (CL) technique with luminol and isoluminol was used to characterize the effect of stobadine on reactive oxygen metabolites (ROM) generation in human whole blood and in isolated polymorphonuclear leukocytes (PMNL) stimulated with N-formyl-methionyl-leucyl-phenyl-alanine (FMLP). In whole blood and in isolated PMNL, stobadine in the concentrations of 1, 10 and 100 micromol/L significantly inhibited the CL signal after FMLP, which activated predominantly extracellular generation of ROM. The same concentrations of stobadine were effective on CL in a cell-free system. On the other hand, myeloperoxidase (MPO) liberation was decreased by stobadine only in the concentration of 100 micromol/L. The results showed stobadine to act as a potent inhibitor/scavenger of extracellularly produced ROM in human PMNL and indicated interference of stobadine with ROM as well as with signalling events resulting in NADPH-oxidase activation and MPO liberation.     

Stobadine inhibits doxorubicin-induced apoptosis through a caspase-9 dependent pathway in P815 mastocytoma cells

Doxorubicin (DOXO), a widely used chemotherapeutic agent, induces apoptosis in transformed and non-transformed cells. The apoptotic effect of DOXO has been linked to the generation of reactive oxygen species (ROS). Antioxidants may be effective in the prevention of DOX-induced apoptosis. In the present study we investigated the effects of stobadine, a pyridoindole antioxidant in a DOXO-induced apoptosis model of P815 cells by flow cytometric analyses and by measuring caspase-3 and caspase-9 activities. Pretreating cells with stobadine significantly increased cell viability and decreased apoptosis rate. Inhibition in apoptosis was observed at maximum levels following treatment of cells with 10(-7)M stobadine as evident from flow cytometric analyses. The antiapoptotic effect of stobadine was further confirmed by inhibition of caspase-3 and caspase-9 activities. We found that the antioxidative effects of stobadine were comparable to the effects of a well known antioxidant, N-acetyl l-cysteine (NAC).     

The effect of stobadine on the copper-induced peroxidation of egg yolk phosphatidylcholine in multilamellar liposomes

Stobadine, (-)- cis-2,8-dimethyl-2 ,3,4,4a,5,9b-hexahydro-1H-pyrido-[4,3b]-indole, is a pyridoindole derivative with antioxidant, antiarrhytmic, neuroprotective, local anesthetic, alpha-adrenolytic, antihistaminic, myorelaxant and other pharmacodynamic effects. The antioxidant properties were tested in a model system of egg yolk phosphatidylcholine (EYPC) multilamellar liposomes. The lipoperoxidation was induced by adding Cu2+ ions and tert-butylhydroperoxide. The course of EYPC peroxidation was monitored spectrophotometrically for conjugated diene formation. We found that stobadine prolonged the lag phase and decreased the rate of peroxidation during the lag phase in a dose-dependent manner. Surprisingly, an increase in the rate of peroxidation was observed at low stobadine concentration in the propagation phase. The possible cause of prooxidative action of stobadine is discussed.     

Preparation of soluble p-aminobenzoyl chitosan ester by Schiff's base and antibacterial activity of the derivatives

Schiff's base of chitosan (BCTS) was obtained by the reaction of chitosan (CTS) and benzaldehyde. Then BCTS reacted with acyl chloride which was synthesized by p-aminobenzoic acid and thionyl chloride to get N-benzoyl-O-aminobenzoyl chitosan ester (BABCTSE), removing the groups of amino protection of BABCTSE to get the final product (ABCTSE). The structures of the derivatives were characterized by FT-IR, (1)H NMR, (13)C NMR and elemental analysis. The elemental analysis results indicated that the degrees of substitution (DS) of the products were 16.8% and 40.4%. The synthesized compounds exhibited an excellent solubility in organic solvents. TG and DTG results showed that thermal stability of the derivatives was lower than that of chitosan. In addition, the existence of two different amido in the molecular structures contributed to forming more -NH(3)(+) in the acid solution which could make the derivatives have a greater advantage in the field of bacteriostasis.