R-deprenyl: pharmacological spectrum of its activity.

Deprenyl has been discovered by Knoll and co-workers. The R-enantiomer of deprenyl (selegiline) is a selective and irreversible inhibitor of the B-isoform of monoamine oxidase (MAO-B) enzyme. Due to its dopamine potentiating and possible neuroprotective properties it has an established role in the treatment of parkinsonian patients. By inhibiting MAO-B enzyme, R-deprenyl decreases the formation of hydrogen peroxide, alleviating the oxidative stress also reduced by increased expression of antioxidant enzymes (superoxide dismutases and catalase) reported during chronic treatment. It was shown to prevent the detrimental effects of neurotoxins like MPTP and DSP-4. R-Deprenyl elicits neuroprotective and neuronal rescue activities in concentrations too low to inhibit MAO-B. It is extensively metabolized and some of the metabolites possess pharmacological activities, thus their contribution to neuroprotective properties was also suggested. The recently identified deprenyl-N-oxide is extensively studied in our laboratory. Effects other than neuroprotection, like influencing cell adhesion and proliferation cannot be neglected.

Influence of acute and chronic administration of benzylamine on glucose tolerance in diabetic and obese mice fed on very high-fat diet

The combination of vanadate plus benzylamine has been reported to stimulateglucose transport in rodent adipocytes and to mimic other insulin actions in diversestudies. However, benzylamine alone activates glucose uptake in human fat cells andincreases glucose tolerance in rabbits. The aim of this work was to unravel the benzylamineantihyperglycemic action and to test whether its chronic oral administrationcould restore the defective glucose handling of mice rendered slightly obese anddiabetic by very high-fat diet (VHFD). When VHFD mice were i.p. injected withbenzylamine at 0.7 to 700 ìmol/kg before glucose tolerance test, they exhibitedreduced hyperglycemic response without alteration of insulin secretion. Whole bodyglucose turnover, as assessed by the glucose isotopic dilution technique, wasunchanged in mice perfused with benzylamine (total dose of 75 ìmol/kg). However,their in vivo glycogen synthesis rate was increased. Benzylamine appeared thereforeto directly facilitate glucose utilisation in peripheral tissues. When given chronicallyat 2000 or 4000 ìmol/kg/d in drinking water, benzylamine elicited a slightreduction of water consumption but did not change body weight or adiposity anddid not modify oxidative stress markers. Benzylamine treatment improved glucosa tolerance but failed to normalize the elevated glucose fasting plasma levels of VHFDmice. There was no influence of benzylamine ingestion on lipolytic activity, basal andinsulin-stimulated glucose uptake, and on inflammatory adipokine expression inadipocytes. The improvement of glucose tolerance and the lack of adverse effects onadipocyte metabolism, reported here in VHFD mice allow to consider orally givenbenzylamine as a potential antidiabetic strategy which deserves to be further studied in other diabetic models (AU)

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Influence of acute and chronic administration ofbenzylamine on glucose tolerance in diabetic andobese mice fed on very high-fat diet

The combination of vanadate plus benzylamine has been reported to stimulateglucose transport in rodent adipocytes and to mimic other insulin actions in diversestudies. However, benzylamine alone activates glucose uptake in human fat cells andincreases glucose tolerance in rabbits. The aim of this work was to unravel the benzylamineantihyperglycemic action and to test whether its chronic oral administrationcould restore the defective glucose handling of mice rendered slightly obese anddiabetic by very high-fat diet (VHFD). When VHFD mice were i.p. injected withbenzylamine at 0.7 to 700 ìmol/kg before glucose tolerance test, they exhibitedreduced hyperglycemic response without alteration of insulin secretion. Whole bodyglucose turnover, as assessed by the glucose isotopic dilution technique, wasunchanged in mice perfused with benzylamine (total dose of 75 ìmol/kg). However,their in vivo glycogen synthesis rate was increased. Benzylamine appeared thereforeto directly facilitate glucose utilisation in peripheral tissues. When given chronicallyat 2000 or 4000 ìmol/kg/d in drinking water, benzylamine elicited a slightreduction of water consumption but did not change body weight or adiposity anddid not modify oxidative stress markers. Benzylamine treatment improved glucose tolerance but failed to normalize the elevated glucose fasting plasma levels of VHFDmice. There was no influence of benzylamine ingestion on lipolytic activity, basal andinsulin-stimulated glucose uptake, and on inflammatory adipokine expression inadipocytes. The improvement of glucose tolerance and the lack of adverse effects onadipocyte metabolism, reported here in VHFD mice allow to consider orally givenbenzylamine as a potential antidiabetic strategy which deserves to be further studiedin other diabetic models (AU)

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Alteration of serum semicarbazide-sensitive amine oxidase activity in chronic renal failure.

Despite recent intensive investigations, physiological and pathological role of semicarbazide-sensitive amine oxidase (SSAO) is far from clear. In this study, serum SSAO activity was determined, radiochemically, in various groups of uremic patients: haemodialysed (HD), peritoneally dialysed (PD) and those receiving conservative treatment but still not dialysed (ND), as well as in controls. Reduced enzyme activity was found in HD uremic patients before and after dialysis treatment, compared to controls (5260 +/- 862 and 6011 +/- 958 pmol/h/ml vs. 8601 +/- 283 pmol/h/ml, p < 0.01 and p < 0.05, respectively). The activity was slightly lower in PD, and normal in ND patients. In HD patients SSAO activity was also determined by an assay based on the formation of hydrogen peroxide, and was found to be elevated compared to controls (2384 +/- 323 pmol/h/ml vs. 1437 +/- 72 pmol/h/ml, p < 0.05). The elevated serum SSAO activity measured through the detection of the enzyme-generated hydrogen peroxide in HD patients might indicate its contribution to the accelerated atherosclerotic disease observed in uremia.

Studies on the insulinomimetic effects of benzylamine, exogenous substrate of semicarbazide-sensitive amine oxidase enzyme in streptozotocin induced diabetic rats.

Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO) is believed to be a bifunctional membrane protein. It is localized extracellularly and preferentially oxidizes short chain primary amines to aldehydes, hydrogen peroxide and ammonia, but also functions as an adhesion molecule, which is involved in leukocyte migration. Serum SSAO activity is increased in diabetic patients and animals and the aldehydes formed in the enzyme reaction may contribute to vascular damage. However, administration of exogenous substrates has been shown to improve glucose tolerance and reduce hyperglycaemia in diabetic animals. Hydrogen peroxide and/or its vanadate complexes have been suggested responsible for these effects. Streptozotocin induced diabetic rats were treated with benzylamine (BZA) +/- vanadate (V) or insulin. In contrast to insulin, BZA + V treatment did not reduce HbA(1C) levels. However, it reduced the elevated serum SSAO activity, decreased the accumulation of advanced-glycation end products and increased the bioavailability of nitric oxide in diabetic animals, similarly to insulin. BZA alone did not affect any of these parameters.

Influence of acute and chronic administration of benzylamine on glucose tolerance in diabetic and obese mice fed on very high-fat diet.

The combination of vanadate plus benzylamine has been reported to stimulate glucose transport in rodent adipocytes and to mimic other insulin actions in diverse studies. However, benzylamine alone activates glucose uptake in human fat cells and increases glucose tolerance in rabbits. The aim of this work was to unravel the benzylamine antihyperglycemic action and to test whether its chronic oral administration could restore the defective glucose handling of mice rendered slightly obese and diabetic by very high-fat diet (VHFD). When VHFD mice were i.p. injected with benzylamine at 0.7 to 700 micromol/kg before glucose tolerance test, they exhibited reduced hyperglycemic response without alteration of insulin secretion. Whole body glucose turnover, as assessed by the glucose isotopic dilution technique, was unchanged in mice perfused with benzylamine (total dose of 75 micromol/kg). However, their in vivo glycogen synthesis rate was increased. Benzylamine appeared therefore to directly facilitate glucose utilisation in peripheral tissues. When given chronically at 2000 or 4000 micromol/kg/d in drinking water, benzylamine elicited a slight reduction of water consumption but did not change body weight or adiposity and did not modify oxidative stress markers. Benzylamine treatment improved glucose tolerance but failed to normalize the elevated glucose fasting plasma levels of VHFD mice. There was no influence of benzylamine ingestion on lipolytic activity, basal and insulin-stimulated glucose uptake, and on inflammatory adipokine expression in adipocytes. The improvement of glucose tolerance and the lack of adverse effects on adipocyte metabolism, reported here in VHFD mice allow to consider orally given benzylamine as a potential antidiabetic strategy which deserves to be further studied in other diabetic models.

Lipopolysaccharide exposure makes allergic airway inflammation and hyper-responsiveness less responsive to dexamethasone and inhibition of iNOS.

Allergic airway disease can be refractory to anti-inflammatory treatment, whose cause is unclarified. Therefore, in the present experiment, we have tested the hypothesis that co-exposure to lipopolysacharide (Lps) and allergen results in glucocorticoid-resistant eosinophil airway inflammation and hyper-responsiveness (AHR). Ovalbumin (Ova)-sensitized BALB/c mice were primed with 10 microg intranasal Lps 24 h before the start of Ova challenges (20 min on 3 consecutive days). Dexamethasone (5 mg/kg/day) was given on the last 2 days of Ova challenges. AHR, cellular build-up, cytokine and nitrite concentrations of bronchoalveolar lavage fluid (BALF) and lung histology were examined. To assess the role of iNOS-derived NO in airway responsiveness, mice were treated with a selective inhibitor of this enzyme (1400W) 2 h before AHR measurements. More severe eosinophil inflammation and higher nitrite formation were found in Lps-primed than in non-primed allergized mice. After Lps priming, AHR and concentrations of T-helper type 2 cytokines in BALF were decreased, but still remained significantly higher than in controls. Eosinophil inflammation was partially, while nitrite production and AHR were observed to be largely dexamethasone resistant in Lps-primed allergized animals. 1400W effectively and rapidly diminished the AHR in Ova-sensitized and challenged mice, but failed to affect it after Lps priming plus allergization. In conclusion, Lps inhalation may exaggerate eosinophil inflammation and reduce responsiveness to anti-inflammatory treatment in allergic airway disease.

Deprenyl: from chemical synthesis to neuroprotection.

During the last decades (-)-deprenyl has become the golden standard of MAO-B inhibitors. It possesses dopamine potentiating and antioxidant properties; however, its effects cannot be explained solely by the enzyme inhibitory action. (-)-Deprenyl prevents the toxicity of certain selective neurotoxins and recently it was demonstrated to increase cell-cell adhesion as well. The complexity of its pharmacological effects reflects the action of both the parent compound and the active metabolites. (-)-Deprenyl and related propargylamines (DRPs) show neuroprotective features in a variety of in vitro and in vivo models that is dependent on the propargyl moiety. The main presumptive targets to date include glyceraldehyde-3-phosphate dehydrogenase, poly(ADP-ribose) polymerase, some kinase cascades, as well as pro- and antiapoptotic proteins, beside the inhibition of MAO-B. The antiapoptotic activity of DRPs converges upon the maintenance of mitochondrial integrity, due to the initiation of a complex transcriptional program, the details of which are yet to be elucidated.

Pharmacological aspects of (-)-deprenyl.

Deprenyl, the selective irreversible inhibitor of monoamine oxidase-B (MAO-B), has been synthesised as a potential antidepressant, however, due to its dopamine potentiating capacity, became a registered drug in the treatment of Parkinson's disease. Deprenyl possesses a wide range of pharmacological activities; some of them are not related to its MAO-B inhibitory potency. Beside its dopamine potentiating effect, it renders protection against a number of dopaminergic, cholinergic and noradrenergic neurotoxins with a complex mechanism of action. By inducing antioxidant enzymes and decreasing the formation of reactive oxygen species, deprenyl is able to combat an oxidative challenge implicated as a common causative factor in neurodegenerative diseases. In a dose substantially lower than required for MAO-B inhibition (10(-9)-10(-13) M), deprenyl interferes with early apoptotic signalling events induced by various kinds of insults in cell cultures of neuroectodermal origin, thus protecting cells from apoptotic death. Deprenyl requires metabolic conversion to a hitherto unidentified metabolite to exert its antiapoptotic effect, which serves to protect the integrity of the mitochondrion by inducing transcriptional and translational changes. Pharmacokinetic and metabolism studies have revealed that deprenyl undergoes intensive first pass metabolism, and its major metabolites also possess pharmacological activities. The ratio of the parent compound and its metabolites reaching the systemic circulation and the brain are highly dependent on the routes of administration. Therefore, in the treatment of neurodegenerative diseases, reconsideration of the dosing schedule, by lowering the dose of deprenyl and choosing the most appropriate route of administration, would diminish undesired adverse effects, with unaltered neuroprotective potency.

Altered nitric oxide production in mouse brain after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridin or methamphetamine.

Several studies have demonstrated the involvement of reactive nitrogen and oxygen species (RNOS) in the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridin (MPTP) and methamphetamine (METH), so the contribution of altered nitric oxide synthase (NOS) enzyme function can be suspected. In this study, about 50% increase in nitric oxide (NO) production in the mouse striatum was found between 4 and 12 h after a single MPTP injection, allowing an increased peroxynitrite (ONOO-) formation in the target brain region. However, METH injection induced a rapid decrease of NO formation both in mouse striatum and hippocampus, reaching its minimum level at 2 h, and restored to the control value after 6 h in the striatum and 12 h in the hippocampus. The uncoupled function of NOS with increased superoxide (O2*-) production after METH injection is suggested.

The influence of metabolism on the MAO-B inhibitory potency of selegiline.

(-)-Deprenyl (selegiline), a propargylamine derivative of methylamphetamine, is a potent, irreversible inhibitor of monoamine-oxidase type B (MAO-B). The MAO-B inhibitory effects of various doses (0.1-0.25-0.5 mg/kg) of (-)-deprenyl in rat brain and liver were compared, using either oral or subcutaneous drug administration. The intensity of the first pass metabolism of (-)-deprenyl was also estimated. The effect of pre-treatment with phenobarbitone (80 mg/kg i.p., daily for three days) or proadifen (SKF-525A, 50 mg/kg i.p., single dose) on the MAO-B inhibitory potency of (-)-deprenyl was also studied. The oral and subcutaneous administration of selegiline induced a significantly different degree of MAO-B enzyme inhibition in the rat brain, but not in the liver. The inhibitory potency of (-)-deprenyl on MAO-B activity was markedly influenced by pre-treatment of rats with an inducer (phenobarbitone), or an inhibitor (SKF-525A) of cytochrome P-450 mono-oxygenases in the liver. Our results suggest, that (-)-deprenyl is metabolised mainly in the liver by microsomal cytochrome P-450 dependent mono-oxygenases, and it has an intensive first-pass metabolism. The parent compound is responsible for the inhibition of MAO-B enzyme activity.

Protective effect of 7-nitroindazole against DSP-4 induced noradrenaline depletion in mouse hippocampus.

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) is a selective noradrenaline (NA) uptake blocker, capable of inducing a long-lasting depletion of NA in some noradrenergic axon terminals originating from the locus coeruleus in rodents. Pretreatment with 7-nitroindazole, a fairly selective inhibitor of neuronal nitric oxide synthase in vivo, partially prevented DSP-4 induced NA depletion in mouse hippocampus measured seven days after the neurotoxic insult. Administration of L-arginine, the substrate of nitric oxide synthase, altered neither the NA depletion induced by DSP-4, nor the protective effect of 7-nitroindazole. Inhibition of nitric oxide synthesis by N(G)-nitro-L-arginine methyl ester did not attenuate the NA depleting effect of DSP-4. Thus, the contribution of neuronal nitric oxide synthase inhibition to the protective effect of 7-nitroindazole needs further studies. As 7-nitroindazole did not block NA uptake, this cannot play a part in the protective effect. The possible contribution of monoamine oxidase B enzyme inhibition by 7-nitroindazole to the protective effect is also discussed.

The effect of low oral doses of (-)-deprenyl and its metabolites on DSP-4 toxicity.

Treatment with a single oral dose of (-)-deprenyl (selegiline) before DSP-4 administration could dose-dependently decrease the noradrenaline (NA) depleting effect of the toxin in mouse hippocampus. The maximum protective effect was achieved at as low oral dose as 0.25 mg/kg. Pre-treatment with the same doses of the main metabolites of (-)-deprenyl; (-)-amphetamine and (-)-methylamphetamine provided a weaker attenuation of DSP-4 induced NA depletion, than the parent compound. The selective noradrenergic toxin DSP-4, which depletes NA in nerve terminals originating from the locus coeruleus, is presumably metabolised by CYP-450 enzymes. Continuous administration of low, by themselves non-toxic doses of DSP-4 resulted in the cumulation of its NA depleting effect.

Metabolic transformation of deprenyl enantiomers in rats.

The optical isomers of deprenyl have different pharmacological activities and potency. Selegiline, an (-)-isomer, is the more potent monoamine oxidase B enzyme inhibitor, and this substance is used in the therapy of Parkinson's disease. The neuroprotective and neuronal rescue effects of deprenyl, as well as the psychostimulant action of its metabolites, methamphetamine and amphetamine are also different for the enantiomers. In this study the biotransformation of deprenyl enantiomers was compared. Stereoselective dealkylation of both optical isomers was found as major metabolic alteration. The difference in the ratio of the formed metabolites suggests the existence of preferred metabolic pathways for the enantiomers.

Equilibrium binding model of bile salt-mediated chiral micellar electrokinetic capillary chromatography.

Optimum concentration of bile salts in chiral separations depends on both the aggregation properties of the surfactant and the stability of the analyte-micelle complexes. An equilibrium model is proposed in which these two effects are treated separately. First the aggregation constants should be determined under the experimental conditions of the chiral MEKC analysis. With these data, the equilibrium concentrations of bile salt aggregates can be calculated at any total surfactant concentration. Using the Offord equation to approximate the mobilities of the enantiomer-bile salt complexes, a model function has been derived to fit the experimental mobilities. The method yields the binding constants of the enantiomers to each aggregate present. Those species are assumed to be important in the chiral recognition process, which have significantly different stability constants for the enantiomers. The method is demonstrated by the chiral separation of R- and S-1,1'-binaphthyl-2,2'-diyl hydrogen phosphate with sodium taurodeoxycholate. Based on the calculated binding constants, tetrameric aggregates are assumed to be the discriminating species, while no significant difference in enantiomer binding to dimers was found.

Analysis of deprenyl metabolites in the rat brain using HPLC-ES-MS.

Methylamphetamine and amphetamine, the two major metabolites of deprenyl in the rat brain were analyzed using HPLC method combined with electrospray-mass spectrometer. (-)-Deprenyl and (+)-deprenyl were orally administered to rats either in a single dose of 10 mg/kg, or three times a week for three weeks. The metabolites were determined in four different parts of the rat brain, such as in the frontal cortex, corpus striatum, hippocampus, and hypophysis. The ratio of methylamphetamine to amphetamine was also compared after (-)-deprenyl and (+)-deprenyl treatments.

Biotransformation of deprenyl enantiomers.

Rats were treated with either (-)- or (+)-deprenyl, and the metabolites extracted from the plasma, liver, kidney and heart homogenates were studied by chiral capillary electrophoresis (CE). Stereoselective dealkylation of both optical isomers with the formation of desmethyldeprenyl (DD), methamphetamine (MA), and amphetamine (A) was found. (-)-MA appears to be the main metabolite of (-)-D, and (+)-A for (+)-D. This suggests that the enantiomers undergo a different dealkylation process.

Trace analysis of proteins by capillary zone electrophoresis with on-column transient isotachophoretic preconcentration.

The qualitative and quantitative aspects of transient isotachophoretic (ITP) sample preconcentration in the capillary zone electrophoretic analysis of protein samples have been demonstrated. By the proper selection of components of the background electrolyte and/or additives to the sample solution, two basic electrolyte arrangements have been employed. In the first, a typical isotachophoretic electrolyte system consisting of a leading and terminating electrolyte was used, and after focusing and preconcentration, the terminating electrolyte was replaced by the leading electrolyte, with the separation being continued in the zone electrophoretic mode. In the second, only one background electrolyte was used, containing a co-ion with low electrophoretic mobility, and the sample was supplemented with a salt of a highly mobile co-ion. In this case transient isotachophoretic migration of the sample ions took place at the beginning of the migration and gradually changed to the zone electrophoretic mode. Sample mixtures containing basic (positively charged) or acidic (negatively charged) proteins were examined using surface-coated fused-silica capillaries. For acidic proteins, bare silica was also tested. The isotachophoretic sample stacking permitted injection and preconcentration of sample volumes two to three orders of magnitude higher than usual in capillary zone electrophoresis. For example, up to 1 microL was injected into a 75 microns ID capillary. This approach afforded quantitative analysis of protein samples in the concentration range of 10(-7)-10(-8) M, with detection limits of approximately 10(-9) M. Furthermore, with constant sample volume injected, good reproducibility of migration times was obtained. Finally, the determination of trace components in the presence of a major sample component using transient ITP preconcentration has been demonstrated.

Properties of the melanin binding of p-bromo-methylamphetamine (V-111).

The aim of the present study was to obtain detailed information on the binding properties of p-bromomethylamphetamine (V-111) to melanins under in vitro and in vivo conditions. The results obtained by the methods of both equilibrium and dynamic dialysis revealed that the binding of V-111 to bovine eye melanin was reversible, at least for the part of the binding sites, whereas the dissociation of V-111-melanin adduct was slow. The binding capacity of two different classes of binding sites of bovine eye melanin and dissociation constants of the drug-melanin complexes have been determined. The in vivo melanin binding of V-111-1-14C was studied by the method of whole body autoradiography. Extensive accumulation and retention was observed in the eyes of the pigmented mice while in the albino animal uptake was low in the corresponding tissues. In conclusion, V-111 may most probably be accumulated and retained for long periods in the pigmented cells of intact animals. Our data also imply that melanin in the pigmented cells serves as a depot, which gradually releases p-bromo-methylamphetamine resulting in a prolonged local effect of this compound.