Synthesis and biological effects of some kynurenic acid analogs.

The overactivation of excitatory amino acid receptors plays a key role in the pathomechanism of several neurodegenerative disorders and in ischemic and post-ischemic events. Kynurenic acid (KYNA) is an endogenous product of the tryptophan metabolism and, as a broad-spectrum antagonist of excitatory amino acid receptors, may serve as a protective agent in neurological disorders. The use of KYNA is excluded, however, because it hardly crosses the blood-brain barrier. Accordingly, new KYNA analogs which can readily cross this barrier and exert their complex anti-excitatory activity are generally needed. During the past 6 years, we have developed several KYNA derivatives, among others KYNA amides. These new analogs included one, N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride (KYNA-1), that has proved to be neuroprotective in several models. This paper reports on the synthesis of 10 new KYNA amides (KYNA-1-KYNA-10) and on the effectiveness of these molecules as inhibitors of excitatory synaptic transmission in the CA1 region of the hippocampus. The molecular structure and functional effects of KYNA-1 are compared with those of other KYNA amides. Behavioral studies with these KYNA amides demonstrated that they do not exert significant nonspecific general side-effects. KYNA-1 may therefore be considered a promising candidate for clinical studies.

The interferon-induced helicase IFIH1 Ala946Thr polymorphism is associated with type 1 diabetes in both the high-incidence Finnish and the medium-incidence Hungarian populations.

AIMS/HYPOTHESIS: The rs1990760 polymorphism (Ala946Thr) of interferon induced with helicase C domain 1 (IFIH1) has been proposed to associate with type 1 diabetes. In this study, association between IFIH1 Ala946Thr and type 1 diabetes was investigated in two distinct white populations, the Hungarians and Finns. METHODS: The rs1990760 polymorphism was genotyped in 757/509 Hungarian/Finnish childhood-onset cases, 499/250 Hungarian/Finnish control individuals and in 529/924 Hungarian/Finnish nuclear family trios. Disease association was tested using case-control and family-based approaches. A meta-analysis of data from 9,546 cases and 11,000 controls was also performed. RESULTS: In the Hungarian dataset, the A allele was significantly more frequent among cases than among controls (OR 1.29, 95% CI 1.10-1.52; p = 0.002). Combined analysis of Hungarian and Finnish datasets revealed a strong disease association (OR 1.235, 95% CI 1.083-1.408; p = 0.002). Furthermore, the A allele was significantly overtransmitted in both family trio datasets (p = 0.017 in Hungarians; p = 0.007 in Finns). The A allele was increased in Hungarian vs Finnish cases (64.9% vs 60.8% in Finns; p = 0.003). The meta-analysis yielded a significant effect for IFIH1 rs1990760 A allele on type 1 diabetes risk (OR 1.176, 95% CI 1.130-1.225; p = 5.3 x 10(-15)) with significant heterogeneity between effect sizes across the studied populations (p = 0.023). CONCLUSIONS/INTERPRETATION: This study represents the first independent confirmation of the association between type 1 diabetes and the IFIH1 gene in Hungarian and Finnish populations. Summarising the data published so far, a clear association between the Ala946Thr polymorphism and type 1 diabetes was detected, with an apparent difference in the contribution to disease susceptibility in different populations of European ancestry.

In vitro and in vivo multidrug resistance reversal activity by a Betti-base derivative of tylosin.

BACKGROUND: The multidrug resistance (MDR) proteins are present in a majority of human tumours. Their activity is important to understand the chemotherapeutic failure. A search for MDR-reversing compounds was conducted among various Betti-base derivatives of tylosin. METHODS: Here, we evaluate the in vitro and in vivo P-glycoprotein (P-gp)-modulating activity of the most promising compound N-tylosil-1-alpha-amino-(3-bromophenyl)-methyl-2-naphthol (TBN) using human MDR1 gene-transfected and parental L5178 mouse lymphoma cell lines. RESULTS: In vitro experiments showed that TBN dramatically increased the P-gp-mediated cellular uptake of the fluorescent substrate rhodamine 123. Similarly, TBN was found to act as a very potent enhancer of the cytotoxicity of doxorubicin on the resistant cell line. We also provide in vivo evidence using DBA/2 mice in support for an increased tumoural accumulation of doxorubicin, without affecting its tissue distribution, resulting in an enhanced antitumoural effect. CONCLUSION: Our results suggest that TBN is a potent modulator of the P-gp membrane pump and that the compound could be of clinical relevance to improve the efficacy of chemotherapy in MDR cancers.

The kynurenate analog SZR-72 prevents the nitroglycerol-induced increase of c-fos immunoreactivity in the rat caudal trigeminal nucleus: comparative studies of the effects of SZR-72 and kynurenic acid.

Administration of nitroglycerol in a migraine model results in an increased number of c-fos-expressing secondary sensory neurons in the caudal trigeminal nucleus. Since synapses between first- and second-order trigeminal neurons are mediated by excitatory amino acids, NMDA receptors are inhibited by kynurenic acid, though this crosses the blood-brain barrier only poorly. Systemic treatment of rats with SZR-72, a newly synthetized kynurenic acid analog, diminished the nitroglycerol-induced increase of c-fos immunoreactivity in the brain stem highly significantly, while treatment with kynurenic acid resulted in a significantly smaller decrease, proving that SZR-72 is much more effective than kynurenic acid.

Telomeric repeat amplification, without shortening or lengthening of the telomerase products: a method to analyze the processivity of telomerase enzyme.

The Telomeric Repeat Amplification Protocol (TRAP) and its modified versions (including ours, TP-TRAP) change the size and/or the ratio of the telomerase products in the amplification stage of the assay. Based on our recently published method we developed a new TRAP. This method ensures that the number of telomeric repeats present in the original telomerase products does not change on PCR amplification. The usefulness of the method was proved with amplification of chemically synthesized telomerase products and a newly designed telomerase substrate oligonucleotide. This is the first report in which the PCR products directly reflect the size distribution of telomerase products generated by the enzyme.

A novel non-opioid binding site for endomorphin-1.

Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the µ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the µ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the µ-opioid receptor. Our recent data indicate that a radiolabelled [3H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd = 0.4 nM / Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM / Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems to be present in nervous tissues carrying low density or no µ-opioid receptors, such as rodent cerebellum, or brain of µ-opioid receptor deficient (MOPr-/-) transgenic or 'knock-out' (K.O.) mice. The newly described non-opioid binding component is not coupled to regulatory G-proteins, nor does it affect adenylyl cyclase enzyme activity. Taken together endomorphin-1 carries opioid and, in addition to non-opioid functions that needs to be taken into account when various effects of endomorphin-1 are evaluated in physiological or pathologic conditions.

Activation of telomerase rna gene promoter activity by NF-Y, Sp1, and the retinoblastoma protein and repression by Sp3.

Expression of the human telomerase RNA component gene, hTERC is essential for telomerase activity. The hTERC gene is expressed during embryogenesis and then downregulated during normal development, leaving most adult somatic cells devoid of hTERC expression. During oncogenesis, however, hTERC is re-expressed consequently contributing to the unrestricted proliferative capacity of many human cancers. Thus the identification of the molecular basis for the regulation of the telomerase RNA component gene in normal cells and its deregulation in cancer cells is of immediate interest. We have previously cloned the hTERC promoter and in this study have identified several transcription factors that modulate the expression of hTERC. We demonstrate that NF-Y binding to the CCAAT region of the hTERC promoter is essential for promoter activity. Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. These factors appear to act in a species-specific manner. Whereas Sp1 and Sp3 act on the human, bovine, and mouse TERC promoters, pRb activates only the human and bovine promoter, and NF-Y is only essential for the human TERC gene.

Ipriflavone as an inhibitor of human cytochrome P450 enzymes.

1. Reduction of theophylline metabolism and elimination were observed in a theophylline-treated patient during ipriflavone administration. After withdrawal of ipriflavone, the serum theophylline level decreased to an extent similar to that found before administration of ipriflavone. The effects of ipriflavone and its major metabolites 7-hydroxy-isoflavone and 7-(1-carboxy-ethoxy)-isoflavone on cytochrome P450 activities were studied in vitro in human liver microsomes from three donors. 2. Ipriflavone and 7-hydroxy-isoflavone competitively inhibited phenacetin O-deethylase and tolbutamide hydroxylase activity. The parent compound and its dealkylated metabolite were strong inhibitors exhibiting Ki values around 10-20 microM, while 7-(1-carboxy-ethoxy)-isoflavone had no effect on the cytochrome P450 activities investigated. 7-Hydroxy-isoflavone is the only one that influenced nifedipine oxidase activity. It competitively inhibited this activity with a Ki value of 129.5 microM. 3. The steady state concentrations of ipriflavone and 7-hydroxy-isoflavone in plasma of patients receiving 3 x 200 mg daily doses of ipriflavone for 48 weeks were found to be 0.33 +/- 0.32 microM and 1.44 +/- 0.77 microM, respectively. 4. The results indicate that the decrease in theophylline metabolism observed in a patient treated with ipriflavone may be due to a competitive interaction of ipriflavone or its metabolite, 7-hydroxy-isoflavone with CYP1A2. On the other hand, our in vitro findings predict some more interaction with CYP2C9.

The distribution of orally administered (-)-deprenyl-propynyl-14C and (-)-deprenyl-phenyl-3H in rat brain.

Using alternatively labelled (-)-deprenyl (3H label in the ring and 14C in the propargyl group) the distribution of the compound was studied in 15 brain regions and the plasma of rats over a period of 96 h, after oral administration of 1.5 mg/kg of (-)-deprenyl. The compound is rapidly absorbed (within 15-30 min) from the gastrointestinal tract, as indicated by its high plasma level. It penetrates to the central nervous system, where it reaches a peak level within 45-60 min. During the first 2 h in the plasma the 14C label, whilst in cerebral tissues during the whole period of the experiment the 3H tracer dominates. The difference in the ratio of 3H to 14C radioactivity (compared to the 0 time relation) develops as early as in the first 15 min, which indicates the operation of a rapid "first pass" biotransformation of the compound. Our data represent the tissue molar concentration -time curves of (-)-deprenyl calculated from both the 3H and 14C radiolabels. A ratio of 1 of the concentrations of the two tracers would indicate that the molecule remained unchanged. The changes in the ratio, therefore, suggest the formation of considerable quantities of metabolites (methylamphetamine and amphetamine) and their presence in the brain. The difference between the area under the curves (AUC0-t for 3H and AUC0-t for 14C) represents the amount of metabolites expected to be formed during the experiment. The concentration of the metabolites should be taken into account while evaluating the pharmacological effect of (-)-deprenyl. We proved earlier that a dose of 1.5 mg/kg of (-)-deprenyl completely blocks MAO-B activity in the central nervous system. The fast metabolism of the inhibitor indicates that a minor part of the orally administered (-)-deprenyl is sufficient to produce a high level of selective MAO-B inhibition in the brain.

Synthesis and binding characteristics of [3H] H-Tyr-Ticpsi[CH2-NH] Cha-Phe-OH, a highly specific and stable delta-opioid antagonist.

Substitution of the Phe3 aromatic ring in H-Tyr-Ticpsi[CH2-NH]Phe-Phe-OH with cyclohexylalanine (Cha) has been reported to result in a compound, H-Tyr-Ticpsi[CH2-NH]Cha-Phe-OH (TICP[psi]), showing substantially increased delta-opioid antagonist potency and high delta selectivity. TICP[psi] was radiolabeled by catalytic tritiation of its precursor Tyr(3',5'-I2)1TICP[psi]. Binding characteristics of the new tritiated pseudopeptide were determined using the radioligand binding assay in rat brain membranes. On the basis of the results of saturation binding studies performed at 25 degrees C, an equilibrium dissociation constant (Kd) of 0.35 nM and a receptor density (Bmax) of 112 fmol/mg protein were calculated. This new tritiated ligand exhibits high affinity for delta-opioid receptors, whereas its binding to mu and kappa receptors is weak. A study of [H3]TICP[psi] binding displacement by various receptor-selective opioids showed the following rank order of potency: delta > kappa = mu. These receptor binding characteristics of the ligand, together with its high specific radioactivity (41.3 Ci/mmol) and stability, makes it a useful tool for labeling delta-opioid receptors, both in vitro and in vivo.

Irreversible labelling of the opioid receptors by a melphalan-substituted [Met5]enkephalin-Arg-Phe derivative.

[Met5]enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe) was modified with the methyl esther of melphalan (Mel; 4-bis(2-chloroethyl)amino-L-phenylalanine) and the resulting compounds were studied for their opioid binding properties in guinea pig and rat brain membranes. Three new peptides, with a substitution of a single amino acid, were synthesized (Mel-Gly-Gly-Phe-Met-Arg-Phe, Tyr-Gly-Gly-Mel-Met-Arg-Phe and Tyr-Gly-Gly-Phe-Met-Arg-Mel). In the rat brain, none of these ligands displayed any type specificity, whereas in guinea pig brain membranes the C-terminally modified peptide, Tyr-Gly-Gly-Phe-Met-Arg-Mel ([Mel7]peptide), displayed a kappa-binding profile and was a weak kappa-opioid-receptor agonist in isolated guinea pig ileum. The effect of sodium ions on [Mel7]peptide competition against [3H]naloxone binding indicated a weak agonist nature of the compound. When guinea pig brain membranes were preincubated with 1-10 microM of [Mel7]peptide, an apparently irreversible inhibition of [3H]naloxone ligand binding was observed. These results suggest that the heptapeptide containing melphalan at the C-terminus can be used as a relatively high-affinity irreversible label for the kappa-opioid receptor.

New opioid affinity labels containing maleoyl moiety.

Opioid receptor binding properties and pharmacological profiles of novel peptides containing maleoyl function were determined in order to develop new affinity labels. Based on the enkephalin structure peptide ligands were synthesized and tested. Both in in vitro receptor binding experiments and pharmacological studies, all ligands showed agonist character with relatively high affinity (Ki values in the nanomolar range) and good to moderate selectivity. Replacement of Gly2 in the enkephalin frame with D-Ala led to higher affinities with a small decrease in selectivity. The longer peptide chains resulted in compounds with high percentage (up to 86%) of irreversible binding. The selectivity pattern of the ligands is in good agreement with the data obtained from the pharmacological assays (guinea pig ileum and mouse vas deferens bioassays). The newly synthesized peptides could be used in further studies in order to determine more detailed characteristics of the ligand-receptor interaction.

Morphology and autowave metric on CNN applied to bubble-debris classification.

In this study, we present the initial results of cellular neural network (CNN)-based autowave metric to high-speed pattern recognition of gray-scale images. the application is to a problem involving separation of metallic wear debris particles from air bubbles. This problem arises in an optical-based system for determination of mechanical wear. This paper focuses on distinguishing debris particles suspended in the oil flow from air bubbles and aims to employ CNN technology to create an online fault monitoring system. For the class of engines of interest bubbles occur much more often than debris particles and the goal is to develop a classification system with an extremely low false alarm rate for misclassified bubbles. The designed analogic CNN algorithm detects and classifies single bubbles es and bubble groups using binary morphology and autowave metric. The debris particles are separated based on autowave distances computed between bubble models and the unknown objects. Initial experiments indicate that the proposed algorithm is robust and noise tolerant and when implemented on a CNN universal chip it provides a solution in real time.

The in vitro biosynthesis and stability measurement with agyl-glycuronide isoformes of the main metabolite of ipriflavone.

The formation and stability of 1-beta-glucuronide conjugate of the main metabolite of ipriflavone [7-(1-carboxy-ethoxy)-isoflavone] (CI)--were studied by using liver microsomes, hepatocytes, and isolated perfused liver of untreated and 3-methylcholanthrene (MC) treated dog and rat, and human liver microsomes. MC treatment enhanced the rate of conjugation twice as much as that of the control in the microsomes of both dogs and rats. Conjugation of CI by microsomes results in two metabolites, both sensitive to pH and temperature. Other two glucuronide forms appeared in experiments with hepatocytes and perfused liver. Mass spectrometry supported. The conclusion, assumption that both metabolites produced by microsomes are glucuronide conjugate isoforms of CI, and that they could be distinguished according to the intensity of peaks on FAB-MIKE spectra. The beta-glucuronidase enzyme hydrolysed only the 1-beta-glucuronide isomer, the other, migrated form remained unchanged. D-saccharic-acid-1,4-lactone, a specific inhibitor of beta-glucuronidase enzyme, decreased the rate of enzymatic cleavage. Standard curves of CI were prepared by HPLC, and 1-beta-CI-glucuronide was quantified according to the amount of CI formed by hydrolysis. The stability of conjugates greatly depends on pH and temperature, and the rate of degradation and isomerization is sensitive to the value of both. Lowering the pH from 7.4 to 5.0 and the temperature from 37 degrees C to 18 degrees C increased the stability of glucuronides. Increasing the pH to 12.0 results in very rapid acyl migration and hydrolysis.

The fate of drotaverine-acephyllinate in rat and man. I. Absorption, distribution and excretion in the rat.

Two different labelled forms were used for the pharmacokinetic investigations: the carbon 1 in the isoquinoline ring (Drotaverine-14C-Acephyllinate) and the carboxyl group of theophylline-7-acetic acid (Drotaverine-Acephylline-14C-ate). Drotaverine-14C-Acephyllinate was rapidly absorbed from duodenal and ileal segments. Biliary excretion was substantial after oral administration and radioactivity was excreted mostly in the feces. Absorption of Drotavenine-Acephylline-14-C-ate from the gastrointestinal tract was very poor and radioactivity was therefore excreted for the most part in the feces. The results of the study were confirmed by whole body autoradiography.

The fate of drotaverine-acephyllinate in rat and man. II. Human pharmacokinetics of drotaverine-14C-acephyllinate.

Pharmacokinetics of Drotaverine-Acephyllinate, Chinoin was investigated in seven male volunteers using 14C labelled drug. Drotaverine-Acephyllinate was administered at a 100 mg single oral dose. Measurements of total radioactivity showed that the drug was absorbed completely and was eliminated by renal and biliary routes. Within 72 hours 39.9 +/- 9.9% and 47.1 +/- 4.9% of the dose were recovered in the urine and faeces respectively. Experimental results were interpreted on the basis of a complex linear compartment model. The structural identifiability of the model was proved by computer analysis, and the pharmacokinetic parameters were determined.

[Pharmacokinetics and metabolism of selegiline]. / A Selegilin farmakokinetikája és metabolizmusa.

As it has been shown is animal experiments, selegiline administered orally is absorbed rapidly. The compound penetrates the blood-brain barrier and the concentration of selegiline and/or its metabolites is high in the brain. Selegiline is eliminated primarily by renal excretion (73%) and 14% of the dose is eliminated with faeces. The main metabolic pathway is N-dealkylation. N-demethyl-selegiline, amphetamine and methamphetamine are the main metabolites. p-Hydroxylated derivatives of amphetamine and methamphetamine could also be identified in rat urine. (-)-Selegiline gives rise to (-) metabolites and does not undergo racemization. Selegiline is bound to macromolecules extensively. The metabolites have lower affinity to plasma proteins than the parent drug do. Selegiline and its metabolites do not accumulate in the organism not even during prolonged administration.

[Positron emission tomography of C-11-labelled selegiline]. / Vizsgálatok pozitron emissziós tomográfia és 11C jelzett selegilin alkalmazásával.

The combination of C-11-labelled Selegiline with PET gives the possibility of a non-invasive method for the determination of the distribution, activity and turnover of MAO-B enzyme and all the enzyme-related changes in the brain as well as for the early detection of Parkinson's disease.

Syntheses, transformations and pharmaceutical applications of kynurenic acid derivatives.

The syntheses and transformations of 4-hydroxyquinoline-2-carboxylic acid, kynurenic acid, are reviewed, and special attention is paid to the pharmacological activities and pharmaceutical applications of its derivatives.