Pharmacological aspects of the neuroprotective effects of irreversible MAO-B inhibitors, selegiline and rasagiline, in Parkinson's disease.

The era of MAO-B inhibitors dates back more than 50 years. It began with Kálmán Magyar's outstanding discovery of the selective inhibitor, selegiline. This compound is still regarded as the gold standard of MAO-B inhibition, although newer drugs have also been introduced to the field. It was revealed early on that selective, even irreversible inhibition of MAO-B is free from the severe side effect of the non-selective MAO inhibitors, the potentiation of tyramine, resulting in the so-called 'cheese effect'. Since MAO-B is involved mainly in the degradation of dopamine, the inhibitors lack any antidepressant effect; however, they became first-line medications for the therapy of Parkinson's disease based on their dopamine-sparing activity. Extensive studies with selegiline indicated its complex pharmacological activity profile with MAO-B-independent mechanisms involved. Some of these beneficial effects, such as neuroprotective and antiapoptotic properties, were connected to its propargylamine structure. The second MAO-B inhibitor approved for the treatment of Parkinson's disease, rasagiline also possesses this structural element and shows similar pharmacological characteristics. The preclinical studies performed with selegiline and rasagiline are summarized in this review.

Kynurenic acid and its derivatives are able to modulate the adhesion and locomotion of brain endothelial cells.

The neuroprotective actions of kynurenic acid (KYNA) and its derivatives in several neurodegenerative disorders [characterized by damage to the cerebral endothelium and to the blood-brain barrier (BBB)] are well established. Cell-extracellular matrix (ECM) adhesion is supposedly involved in recovery of impaired cerebral endothelium integrity (endothelial repair). The present work aimed to investigate the effects of KYNA and its synthetic derivatives on cellular behaviour (e.g. adhesion and locomotion) and on morphology of the GP8 rat brain endothelial cell line, modeling the BBB endothelium. The effects of KYNA and its derivatives on cell adhesion were measured using an impedance-based technique, the xCELLigence SP system. Holographic microscopy (Holomonitor™ M4) was used to analyse both chemokinetic responses and morphometry. The GP8 cells proved to be a suitable model cell line for investigating cell adhesion and the locomotion modulator effects of kynurenines. KYNA enhanced cell adhesion and spreading, and also decreased the migration/motility of GP8 cells at physiological concentrations (10-9 and 10-7 mol/L). The derivatives containing an amide side-chain at the C2 position (KYNA-A1 and A2) had lower adhesion inducer effects compared to KYNA. All synthetic analogues (except KYNA-A5) had a time-dependent inhibitory effect on GP8 cell adhesion at a supraphysiological concentration (10-3 mol/L). The immobilization promoting effect of KYNA and the adhesion inducer activity of its derivatives indicate that these compounds could contribute to maintaining or restoring the protective function of brain endothelium; they also suggest that cell-ECM adhesion and related cell responses (e.g. migration/motility) could be potential new targets of KYNA.

Distribution of N-methyl-(14)C-labeled selegiline in the rat.

Tissue distribution of selegiline including N-methyl-(14)C-selegiline was studied with three different techniques. Whole body autoradiography of labeled selegiline in rats completed the former results obtained in mice. Counting radioactivity by liquid scintillation method in various body compartments gave an in-depth numerical estimation of distribution, while RP-HPLC determination of selegiline determined the fate of intact, non-metabolized parent compound. Whole body autoradiography following 15 and 60 min of intraperitoneal application of N-methyl-(14)C-selegiline verified definite and time-dependent blood-brain penetration of selegiline. Quantitative determination of tissue concentrations by liquid scintillation and RP-HPLC methods following 5, 15, 60 and 180 min of intraperitoneal administration of selegiline unanimously verified both blood-brain and blood-testis penetration of the compound through the barrier.

Novel arylalkenylpropargylamines as neuroprotective, potent, and selective monoamine oxidase B inhibitors for the treatment of Parkinson's disease.

To develop novel neuroprotective agents, a library of novel arylalkenylpropargylamines was synthesized and tested for inhibitory activities against monoamine oxidases. From this, a number of highly potent and selective monoamine oxidase B inhibitors were identified. Selected compounds were also tested for neuroprotection in in vitro studies with PC-12 cells treated with 6-OHDA and rotenone, respectively. It was observed that some of the compounds tested yielded a marked increase in survival in PC-12 cells treated with the neurotoxins. This indicates that these propargylamines are able to confer protection against the effects of the toxins and may also be considered as novel disease-modifying anti-Parkinsonian agents, which are much needed for the therapy of Parkinson's disease.

The pharmacokinetic evaluation of selegiline ODT for the treatment of Parkinson's disease.

INTRODUCTION: Parkinson's disease affects a growing number of people. Its major treatment, levodopa therapy, however suffers from response fluctuations in the advanced phase of the disease. Adjunctive therapies have thus gained significant importance. The selective monoamine oxidase B inhibitor, selegiline , is widely used in the treatment of Parkinson's disease. However, selegiline's pharmacokinetics has so far been unfavorable due to its extensive first-pass metabolism to its amphetamine metabolites. A new formulation, an orally disintegrating tablet (ODT), has recently been introduced to overcome these pharmacokinetic problems by avoiding its presystemic metabolism. AREAS COVERED: The authors summarize the pharmacokinetic and clinical efficacy data of selegiline ODT and compare them with the more conventional oral selegiline. This evaluation also presents and compares the drug's safety data. EXPERT OPINION: Selegiline ODT shows a clear pharmacokinetic advantage over the conventional form. It is characterized by improved bioavailability allowing dose reduction and a lower exposure to amphetamine metabolites. This pharmacokinetic improvement, however, is not accompanied with significant clinical advantage, since its efficacy and safety profile are comparable to those of the conventional formulation. This suggests that the metabolites do not participate significantly in the therapeutic or toxic effects of selegiline. On the basis of the available data, the only well-justified advantage of the ODT formulation is its convenient use in parkinsonian patients who have difficulties in swallowing the regular formulation.

The effect of selegiline on total scavenger capacity and liver fat content: a preliminary study in an animal model.

Selegiline is a selective irreversible inhibitor of the B-type of monoamine oxidase (MAO-B). The spectrum of its pharmacological activity is wide, possesses antioxidant, antiapoptotic and neuroprotective properties and, additionally, we found it is effective on the total scavenger capacity (TSC), and the regulation of fat content in rat liver kept on lipid-rich diet. Our aim was to clarify whether the oral treatment with selegiline is protective on oxidative damage of Sprague-Dawley adult rats in vivo. Four groups of rats (five animals in a group) were examined: (1) lipid-rich diet, (2) normal rat food, (3) lipid-rich diet + selegiline and (4) normal rat food + selegiline. Selegiline solution (2.5 µg/ml) was supplied with the drinking water, which was freely available for the animals. Regarding the drinking habit of the rats (20-30 ml/day), the daily dose was roughly equal with that used in the human therapy (5-10 mg/day). TSC was determined both at the beginning (0 day) and at the end of the study (28 days), when the blood samples were taken for chemiluminometric assay. Fat content of the liver was determined in the freshly frozen tissue by Sudan staining. TSC was increased in both the selegiline-treated groups. Selegiline treatment prevented the increase of liver fat in the group fed with lipid-rich diet. Our results led us to the conclusion that prolonged selegiline administration can raise the antioxidant capacity of the animals and prevents the accumulation of fat in their livers.

Basic cell physiological activities (cell adhesion, chemotaxis and proliferation) induced by selegiline and its derivatives in Mono Mac 6 human monocytes.

Selegiline (R-deprenyl), a monoamine oxidase-B (MAO-B) inhibitor, has complex pharmacological effect that contributes to treatment of neurodegenerative diseases such as Parkinson's and presumably Alzheimer's disease and might work as an inhibitor of tumor growth. In respect of tumorigenesis and metastasis formation, the controlled modifications of adhesion and migration have high therapeutic significance. In the present study, our purpose was to investigate cell physiological responses (adhesion, chemotaxis and proliferation) induced by selegiline, its metabolites and synthetic derivatives and to find some correlations between the molecular structure and the reported antitumor behavior of the derivatives. Our results demonstrated that both R- and S-deprenyls have the potency to elicit increased adhesion and a chemorepellent activity in monocyte model (Mono Mac 6 cell line derived from monoblastic leukemia); however, only the R-enantiomer proved to be cytotoxic. Among the metabolites R-amphetamine has retained the adhesion inducer and the chemorepellent effect of the parent drug on the most significant level. In contrast, a reversed chemotactic effect and an improved cytotoxic character were detected in the presence of fluoro group (p-fluoro-S-deprenyl). In summary, the adhesion inducer activity, chemorepellent and advantageous cytotoxic effects of selegiline and some derivatives indicate that these drug molecules might have inhibitory effects in metastasis formation in primary tumors.

Improved therapeutic entities derived from known generics as an unexplored source of innovative drug products.

With a New Drug Application (NDA) innovative drug therapies are reaching the market in a specific dosage form for one or more clinically proven indications of which after expiration of the patent or the data exclusivity copies are launched using Abbreviated New Drug Applications (ANDA). Advanced therapies that emerged from launched molecules during their product life-cycle have gained considerable attention as clinical practice provides evidence for additional therapeutic values, patient centric delivery systems show improved therapeutic outcomes or emerging technologies offer efficiency gains in manufacturing or access to emerging markets. The USA and European regulatory framework has set reasonable regulations in place for these "Supergenerics" or "hybrid" applications. While these regulations are relatively recent the pharmaceutical industry is just starting to use this route for their product development and life-cycle management. From a clinical perspective the potential for advanced product development have been demonstrated. Yet, there is still a lag of common understanding between the different stakeholders regarding the development, application process and commercial incentive in developing enhanced therapeutic entities based on existing drug products for the market.

The pharmacology of selegiline.

Selegiline, the R-optical enantiomer of deprenyl (phenyl-isopropyl-methyl-propargylamine), was almost exclusively used MAO-B inhibitor during the past decades to treat Parkinson's disease. Oral treatment prolongs the need of levodopa administration. Selegiline is rapidly metabolized by the microsomal enzymes to amphetamine, methamphetamine, and desmethyl-deprenyl. In addition, the flavin-containing monooxigenase is synthesizing deprenyl-N-oxide. Selegiline in rather low concentrations (10⁻9-10⁻¹³ M), does not influence MAO-B, but it has an antiapoptotic activity in tissue culture. The neuroprotective effect of selegiline has a biphasic character. In higher concentrations than 10⁻7 M increases the rate of apoptosis (proapoptotic activity). The metabolites are also taking part in the complex pharmacological activity of selegiline. The simultaneous presence of the pro- and antiapoptotic effects of selegiline and its metabolites frequently hindered its clinical usage. During the past years rasagiline has been introduced to replace selegiline in clinical application. MAO-B inhibitors beside their effect on the enzyme MAO-B could hold different spectrum of pharmacological activities. Selegiline is administered orally and it possesses an intensive "first pass" metabolism. To circumvent the "first pass" metabolism, parenteral administration of the drug might lead to different distribution and pharmacological activity of selegiline.

Activated MAO-B in the brain of Alzheimer patients, demonstrated by [11C]-L-deprenyl using whole hemisphere autoradiography.

In the human brain the monoaminooxidase-B enzyme or MAO-B is highly abundant in astrocytes. As astrocyte activity and, consequently, the activity of the MAO-B enzyme, is up-regulated in neuroinflammatory processes, radiolabelled analogues of deprenyl may serve as an imaging biomarker in neuroinflammation and neurodegeneration, including Alzheimer's disease. In the present study [(11)C]-L-deprenyl, the PET radioligand version of L-deprenyl or selegiline®, a selective irreversible MAO-B inhibitor was used in whole hemisphere autoradiographic experiments in human brain sections in order to test the radioligand's binding to the MAO-B enzyme in human brain tissue, with an eye on exploring the radioligand's applicability as a molecular imaging biomarker in human PET studies, with special regard to diagnostic detection of reactive astrogliosis. Whole hemisphere brain sections obtained from Alzheimer patients and from age matched control subjects were examined. In control brains the binding of [(11)C]-L-deprenyl was the highest in the hippocampus, in the basal ganglia, the thalamus, the substantia nigra, the corpus geniculatum laterale, the nucleus accumbens and the periventricular grey matter. In Alzheimer brains significantly higher binding was observed in the temporal lobes and the white matter. Furthermore, in the Alzheimer brains in the hippocampus, temporal lobe and white matter the binding negatively correlated with Braak stages. The highest binding was observed in Braak I-II, whereas it decreased with increasing Braak grades. The increased regional binding in Alzheimer brains coincided with the presence of an increased number of activated astrocytes, as demonstrated by correlative immunohistochemical studies with GFAP in adjacent brain slices. Deprenyl itself as well as the MAO-B antagonist rasagiline did effectively block the binding of the radioligand, whereas the MAO-A antagonist pirlindole did not affect it. Compounds with high affinity for the PBR system did not block the radioligand binding either, providing evidence for the specificity of [(11)C]-L-deprenyl for the MAO-B enzyme. In conclusion, the present observations indicate that [(11)C]-L-deprenyl may be a promising and selective imaging biomarker of increased MAO-B activity in the human brain and can therefore serve as a prospective PET tracer targeting neuroinflammation and neurodegeneration.

Cytoprotective effect of (-)-deprenyl, (-)desmethyl-deprenyl and (-)deprenyl-N-oxide on glutathione depleted A-2058 melanoma cells.

Postulated cytoprotective action of (-)-deprenyl (D), (-)-desmethyl-deprenyl (DD) and (-)-deprenyl-N-oxide (DNO) on L-buthionine-(S,R)-sulfoximine (BSO) toxicity was investigated using in vitro cultures of serum-deprived A-2058 melanoma cells. BSO (10 microM/l) decreased viable cell number and mitotic rate, while increased the apoptotic index. D and both of its metabolites, given together with BSO in the concentration of 50 microM/l, mitigated cell loss and decreased the apoptotic ratio. DD was the most effective compound in decreasing apoptotic activity, while DNO stabilized the cell number on control level and increasing the ratio of mitotic cells above the only serum-deprived control. Surveillance on mitochondrial membrane stability and antioxidant properties may play an important role in these processes.

(-)Deprenyl-N-oxide, a (-)deprenyl metabolite, is cytoprotective after hypoxic injury in PC12 cells, or after transient brain ischemia in gerbils.

BACKGROUND AND AIMS: (-)-Deprenyl (selegiline) possesses cyto-protective effect in a much lower concentration, than it is needed to inhibit MAO-B activity. In permanent MCA occlusion stroke model in rats, the infarct volume and the number of apoptotic neurons in the penumbra region were decreased by low concentration (-)deprenyl treatment. Augmented Bcl-2 protein expression was documented as the responsible factor of this effect. The stabilization of mitochondrial membrane and diminished ROS production are the further possible consequences of (-)deprenyl treatment. It is not clear however that (-)deprenyl, or its metabolites are the acting neuroprotective molecules in the hypoxic/ischemic conditions. We report here the possible cyto-protective effect of deprenyl-N-oxide (DNO), a recently synthesized (-)deprenyl metabolite. METHODS: DNO in a very low dose (10(-5,-8,-12) M) was tested in PC12 cell culture after hypoxia and in gerbils after transient occlusion of bilateral common carotid artery. In PC12 culture the cell death was visualized by PI staining. The level of reactive oxygen species was measured by the Cerium method, and the mitochondrial membrane integrity was labeled by JC1 staining. Apoptotic neurons were counted on formaldehyde fixed gerbil brain slices after TUNEL and caspase-3 immune-staining - NIKON/BIORAD confocal microscopy was used for the quantitative analysis. RESULTS: DNO treatment significantly decreased the frequency of cell death in PC12 cultures after hypoxia, increased the mitochondrial transmembrane potential (DeltaY(m)) and decreased the ROS production. In the CA2 regions of gerbil hippocampus, we found significantly less apoptotic neurons than in the untreated controls. CONCLUSION: Transient hypoxia or ischemia induced cell damage could be diminished by DNO. This (-)deprenyl metabolite is an active cell protective molecule.

Reverse Na(+)/Ca(2+)-exchange mediated Ca(2+)-entry and noradrenaline release in Na(+)-loaded peripheral sympathetic nerves.

[(3)H]noradrenaline ([(3)H]NA) released from sympathetic nerves in the isolated main pulmonary artery of the rabbit was measured in response to field stimulation (2Hz, 1ms, 60V for 3min) in the presence of uptake blockers (cocaine, 3 x10(-5)M and corticosterone, 5 x10(-5)M). The [(3)H]NA-release was fully blocked by the combined application of the selective and irreversible 'N-type' voltage-sensitive Ca(2+)-channel (VSCC)-blocker omega-conotoxin (omega-CgTx) GVIA (10(-8)M) and the 'non-selective' VSCC-blocker aminoglycoside antibiotic neomycin (3x10(-3)M). Na(+)-loading (Na(+)-pump inhibition by K(+)-free perfusion) was required to elicit further NA-release after blockade of VSCCs (omega-CgTx GVIA+neomycin). In K(+)-free solution, in the absence of functioning VSCCs (omega-CgTx GVIA+neomycin), the fast Na(+)-channel activator veratridine (10(-5)M) further potentiated the nerve-evoked release of [(3)H]NA. This NA-release was significantly inhibited by KB-R7943, and fully blocked by Ca(o)(2+)-removal. However, Li(+)-substitution was surprisingly ineffective. The non-selective K(+)-channel blocker 4-aminopyridine (4-AP, 10(-4)M) also further potentiated the nerve-evoked release of NA in K(+)-free solution. This potentiated release was concentration-dependently inhibited by KB-R7943, significantly inhibited by Li(+)-substitution and abolished by Ca(o)(2+)-removal. It is concluded that in Na(+)-loaded sympathetic nerves, in which the VSCCs are blocked, the reverse Na(+)/Ca(2+)-exchange-mediated Ca(2+)-entry is responsible for transmitter release on nerve-stimulation. Theoretically we suppose that the fast Na(+)-channel and the exchanger proteins are close to the vesicle docking sites.

[Study on the altered nitric oxide metabolism in experimental diabetes]. / A nitrogen-monoxid metabolizmus változásáinak vizsgálata diabéteszes állatmodellen.

Decreased biological action of nitric oxide (NO) and increased oxidative stress are established to be involved in the development of endothelium dysfunction, early sign of diabetic angiopathy. In the present study, increased nitric oxide synthase (NOS) enzyme activity in the aorta and decreased activity in the kidney tissue of streptozotocin-induced diabetic rats has been found in the early phase of the disease. Augmentation of oxidative transformation of NO in the kidney and heart of the diabetic animals has been demonstrated by the measurement of the stable end-products of NO and other reactive nitrogen species. Insulin treatment was found effective to reduce the intensified oxidative metabolism of NO without increasing its production. Reduced biological effects of NO observed in endothelial dysfunction, is thus probably the consequence of its increased oxidative inactivation.

[Change of the nitric oxide synthase activity after administration of neurotoxic compounds in mice]. / A nitrogen-oxid szintáz enzim aktivitásának megváltozása neurotoxinok adását követoen egérben.

The possible contribution of reactive oxygen and nitrogen species (RONS) to the development of the neurode-generation came up after the investigations with neurotoxic compounds. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH) have detrimental effect on the dopaminergic neurons. The aim of our study was to examine whether altered nitric oxide synthase (NOS) enzyme activity can be involved in the damage induced by these neurotoxins. The other goal of the study was to investigate the applicability of the measurement of the ratio of NADP+/L-citrulline formed by the enzyme to assess the coupled state of the NOS. Elevated NOS activity in mouse striatum and declined enzyme activity in mouse hippocampus have been found after administration of MPTP Decreased NOS activity in mouse striatum and hippocampus was observed after administration of METH. The ratio of the NADP+/L-citrulline produced by the enzyme provides useful information about the coupling state of the NOS. The ratio in the presence of saturating substrate concentration measured in our experiments was comparable with the data found in the literature. Dramatically increased ratio could be observed in case of decreasing substrate concentration indicating the uncoupled function of the enzyme.

Soluble semicarbazide-sensitive amine oxidase (SSAO) activity is related to oxidative stress and subchronic inflammation in streptozotocin-induced diabetic rats.

Diabetes is known to increase the risk of Alzheimer's disease (AD) and vascular dementia via oxidative stress and inflammation. There are speculations that SSAO activity might be related to the development of AD. Our aim was to investigate whether changes of soluble SSAO activity, oxidative stress and inflammation markers are related to each other in diabetes. Soluble and tissue-bound SSAO activities (from serum and aorta, respectively) were determined in streptozotocin (STZ)-induced diabetic rats without insulin treatment, receiving insulin once, or twice daily compared to control animals. After three weeks of treatment soluble and tissue-bound SSAO activities (seSSAO and aoSSAO, respectively), serum total antioxidant status (TAS), high sensitivity C-reactive protein (hsCRP), fructose amine levels and routine laboratory parameters were determined. SeSSAO activity significantly increased in the diabetic groups without treatment and receiving insulin once daily, and a marked decrease in aoSSAO activity was seen in all diabetic groups. Increased oxidative stress was correlated with hsCRP elevation, while hsCRP and seSSAO activity were also significantly correlated. In all groups seSSAO and aoSSAO activities were in negative correlation with each other. Our results support the view that poor metabolic control leads to increased oxidative stress, which in turn may cause the elevation of hsCRP levels. Soluble SSAO on the one hand acts as an adhesion molecule--thus possibly being a factor responsible for the late complications of diabetes--and on the other hand, it may contribute to oxidative stress. Our parsimonious conclusion is that there is a relation between the risk factors of AD and vascular dementia (diabetes, oxidative stress and chronic inflammation) and SSAO activity, which may originate from the vessel wall.

[Role of R-(-)-deprenyl in adhesion of neuronal and non-neuronal cells]. / Az R-(-)-deprenyl szerepe neuronalis és nem neuronalis sejtek adhéziójában.

Role of R-(-)-deprenyl in adhesion of neuronal and non-neuronal cells. The beneficial effect of the anti-parkinsonian monoamine oxidase-B inhibitor, R-(-)-deprenyl has been shown in a number of different diseases, such as Parkinson's and Alzheimer's disease, atherosclerosis or tumor formation. The role of the cytoskeleton, the main component of cell adhesion, has been suggested in the development of these diseases. Nevertheless, the effect of the drug on cell adhesion has never been examined. In the present study, the authors studied the effect of R-(-)-deprenyl on cell-cell adhesion of neuronal (PC12, rat phaeochromocytoma) and non-neuronal (NIH3T3, NIH3T3/EGFR, NIH3T3/EGFR-e3B1 mouse embryo fibroblasts, and 5180 mouse sarcoma) cells using cell association assay. R-(-)-deprenyl treatment resulted in a cell type- and concentration-dependent increase in cell-cell adhesion of PC12 cells, which contain no monoamine oxidase-B, and we observed the same effect in NIH3T3 cells at concentrations lower than those needed for monoamine oxidase-B inhibition. Interestingly, R-(-)-deprenyl increased cell-cell adhesion of tumor cell lines as well. The effect of R-(-)-deprenyl was not reversible during a 24-hour recovery period. At the same time, the monoamine oxidase-B inactive isomer of the drug, S-(+)-deprenyl had no effect on cell-cell adhesion in PC12 and NIH3T3 cells. In this study, the authors described a new, monoamine oxidase-B independent effect of R-(-)-deprenyl on cell-cell adhesion both in neuronal and non neuronal cells. The authors' results with S-(+)-deprenyl suggest that the sterical structure of the drug is an important factor of the observed effect, which is probably a consequence of an irreversible change in the cells.

Trace analysis of oxidized, nitrated, and chlorinated aromatic amino acids by capillary electrophoresis with electroosmotic flow modification allowing large-volume sample stacking.

A capillary electrophoresis method has been developed for the simultaneous analysis of the oxidized, nitrated, and chlorinated aromatic amino acids, as well as their parent compounds. These modifications of the aromatic amino acids in proteins or free form are induced by the attack of reactive, mainly free radical species generated during cell stress, and these stable products may serve as biomarkers of cell damage. The analytes tyrosine, phenylalanine, dihydroxyphenylalanine, tryptophan, 3-nitrotyrosine, 3-chlorotyrosine, ortho-tyrosine, meta-tyrosine, 3-hydroxyphenylacetic acid (internal standard 1), and alpha-methyltyrosine (internal standard 2) were separated in their anionic forms in alkaline borate buffer. The polyamine spermine was used as electroosmotic flow (EOF) modifier. Adsorbing to the capillary wall, spermine can either suppress or even reverse the EOF depending on its concentration and the pH. The effects of the pH of the separation buffer, the spermine concentration, the temperature, and the applied field strength on the separation were examined. The modified aromatic amino acids are present in biological fluids in a much lower concentration than their parent compounds, thus high detection sensitivity of the analytical method is required. To achieve good detection sensitivity, field-amplified sample stacking of large injection volumes was applied. Omitting polyamine from the sample buffer allowed local reversal of the EOF, thus removal of the low conductivity sample buffer at the capillary inlet. In this way, 100% of the capillary to the detection window could be filled with the sample, and the detection limits achieved for the modified aromatic amino acids were in the range of 2.5-10 nM.

High sensitivity analysis of nitrite and nitrate in biological samples by capillary zone electrophoresis with transient isotachophoretic sample stacking.

Tissue level of nitrate and nitrite are established indicators of altered nitric oxide metabolism under various pathological conditions. Determination of these anions in biological samples, in the presence of high chloride concentration, using capillary zone electrophoresis suffers from poor detection sensitivity. Separation conditions providing excellent resolution and submicromolar detection sensitivity of nitrate and nitrite have been developed and validated. Simple sample preparation was applied that maintains nitrite stability in tissue extracts and at the same time allows transient isotachophoresis stacking of the analytes. Nitrate and nitrite concentrations in rat brain and liver tissue samples were determined in control and lipopolysaccharide treated animals.

Assessment of the N-oxidation of deprenyl, methamphetamine, and amphetamine enantiomers by chiral capillary electrophoresis: an in vitro metabolism study.

A chiral capillary electrophoresis method using hydroxypropyl-beta-cyclodextrin as chiral selector was developed and validated for the quantification of the N-oxygenated metabolites of deprenyl, methamphetamine, and amphetamine enantiomers, formed in vitro. The influence of various parameters (selector concentration, buffer pH, temperature, polymer additive, etc.) on the simultaneous separation of the optical isomers of the parent drugs and their metabolites has been evaluated. The buffer pH had the greatest impact on the separation selectivity of the N-oxygenated compounds. Linear calibration curves were obtained over the concentration range of 2.5-50 microM for the enantiomers of amphetamine-hydroxylamine, methamphetamine-hydroxylamine, and deprenyl-N-oxide. The inter- and intra-assay precision and accuracy varied by less than 15% for all analytes at concentrations of 5, 10, and 30 microM, and less than 20% at the lower limit of quantitation (2.5 microM). The sample extraction recovery ranged between 109 and 129% at the three concentration levels. The drug enantiomers were incubated with recombinant human flavin-containing monooxygenase enzymes (FMO3 and FMO1), and human liver microsomes, respectively. The enantioselectivity of the substrate preference, as well as the stereoselective formation of the new chiral center upon the oxidation of the prochiral tertiary nitrogen of deprenyl were assessed. FMO1, the extrahepatic form of the enzyme in man, was shown to be more active in the N-oxygenation of both deprenyl and methamphetamine isomers than FMO3. Deprenyl enantiomers and S-methamphetamine were substrates of human recombinant FMO3. Conversion of amphetamine to its hydroxylamine derivative could not be observed on incubation with either FMO1 or FMO3. Formation of the new chiral center on the nitrogen, during N-oxidation of the tertiary amine deprenyl, was found stereoselective. The two FMO isoforms have shown opposite preference in the formation of this chiral center. Methamphetamine-hydroxylamine formed from methamphetamine was further transformed by FMO, amphetamine-hydroxylamine was identified as the product of a demethylation reaction.