The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors.

BACKGROUND AND PURPOSE: Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. EXPERIMENTAL APPROACH: E1R was tested for sigma receptor binding activity in a [³H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca²âº concentration ([Ca²âº](i)) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. KEY RESULTS: Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca²âº](i) increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. CONCLUSION AND IMPLICATIONS: E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases.

[The influence of mildronate on peripheral neuropathy and some characteristics of glucose and lipid metabolism in rat streptozotocin-induced diabetes mellitus model].

Streptozotocin (STZ) was used to induce the diabetic rat model. STZ rats were treated with mildronate (100 mg/kg daily, per os or intraperitoneally for 6 weeks). Body weight, blood glucose, triglyceride, ketone body concentrations, glycated hemoglobin percent (HbA1c%), glucose tolerance, and the development of neuropathic pain were monitored throughout the experiment. In the STZ + mildronate group, mildronate treatment caused a significant decrease in mean blood glucose (on week 4) and triglyceride concentrations (on weeks 3-6), significantly slowed the increase in HbA1c% (on week 6) and improved glucose tolerance 120 minutes after glucose ingestion during oral glucose tolerance test versus the STZ group. Mildronate completely protected development of STZ-induced neuropathic pain from the first administration week up to end of the experiment. The obtained data indicate clinical usefulness of the drug for the treatment of diabetes mellitus and its complications.

Chronic hepatitis C: quantitative EPR analysis of nitrogen oxide and copper in patients' blood.

Measurements of nitrogen oxide and copper in the blood of 57 patients with chronic viral hepatitis C was carried out before antiviral therapy by electron paramagnetic resonance on a Radiopan EPR spectrometer. The results indicate elevated levels of nitrogen oxide and copper in the blood of these patients in comparison with normal subjects. Comparison of these findings with the results of a previous analysis of redox status of patients with chronic viral hepatitis C indicate that this disease is characterized by a significant pro-oxidant shift in the realization of redox processes and disorders in the metal ligand homeostasis (at least as regards copper).

Paradoxical effects of two oximes on nitric oxide production by purified NO synthases, in cell culture and in animals.

We have studied the impact of two novel compounds TO-85 (2,6-di-(alpha-aziridino-alpha-hydroxyiminomethyl)pyridine and TO-133 (bis-(diaziridinoglyoximato)copper), designed as NO donors, on nitrite production by cell cultures, NO production in rat tissues and their ability to inhibit purified NO synthases (NOS). Both substances induced considerable increase of nitrite production in cell cultures. When NO production was assayed in rat organs by means of ESR using Fe(DETC) as a spin trap the anticipated NO-increasing activity of TO-85 was observed only in kidneys; the NO level increasing almost 10-fold. Treatment of rats with TO-133, decreased the NO concentration in brain cortex, cerebellum and liver. When the drugs were administered to animals with high level of iNOS expression induced by LPS, TO-85 did not significantly modify the LPS-induced NO production; administration of TO-133 caused a significant decrease of NO production in blood, brain cortex and cerebellum. Only high concentrations of TO-85 were capable of inhibiting iNOS (IC50=7 mM), the substance inhibited eNOS at lower concentrations (IC50=250 microM). Inhibitory activities of TO-85 on nNOS were dependent on BH4 concentrations, suggesting eventual competition of TO-85 with BH4 when the substance interacts with nNOS. TO-133 reduced eNOS activity with IC50=200 microM, nNOS activity with IC50=200 microM, iNOS activity was not much affected by this substance. Thus, the two tested compounds manifest opposite effects on NO production by purified enzymes and in cell culture. The pattern of the NO synthesis modification in a living animal appears to be even more complex. Our results stress the importance of direct measurements of NO in the tissues using the ESR method.

[The effect of mildronate and related substances on levels of thyroid hormones and some intermediates of lipid and carbohidrate metabolism in the hyperthyroid and hypothyroid rats].

We have investigated effects of Mildronate, gamma-butyrobetaine (GBB) and their combination ("Neomildronate") on the plasma levels of thyroid gland hormones and some intermediates of basal metabolism (free fatty acids, triglycerides, glucose) in rats with different dysfunctions of thyroid gland, including idiopathic hyperfunction and hypofunction induced by propylthiouracil or L-carnitin administration. Histological investigation of the thyroid gland was also performed. Intraperitoneal injections of Mildronate (150 mg/kg) during 20 days to Wistar male rats with elevated level of thyroid hormones and basal metabolism normalized thyroxine level and parameters of lipid metabolism. Mildronate, GBB and their combination did not affect the natural resurgence of rats with experimental hypofunction induced by propylthiouracil or L-carnitin administration. The possible biochemical role of given treatment in regulation of thyroid gland function is discussed.

[Mildronate in geriatric patients with cardiac failure].

AIM: To study efficacy of mildronate in the treatment of elderly patients with cardiac failure (CF). MATERIAL AND METHODS: A total of 91 patients with chronic CF (NYHA FC I-III) entered the study. The study group of 63 patients received standard therapy for a month and mildronate in a dose 750 mg/day. The control group of 28 patients was treated conventionally. Subjective and objective assessment of the patients' condition, quality of life questionnaire, ECG, 6-min walk test were used for evaluation of the treatment efficacy. RESULTS: In the study group daily frequency of anginal attacks reduced from 1.6 to 0.7 (in the control group from 1.46 to 1.25, p > 0.05), intensity of the attacks--from 1.4 to 0.7 points by 7-score scale (p < 0.05). Crepitation disappeared in 8 (12.8%) patients, edema of the legs--in 3 (4.8%) patients. Systolic blood pressure went down by 8 mmHg, diastolic one--by 4 mmHg. In the control group clinical changes were insignificant. CONCLUSION: Elderly patients tolerated mildronate well. It was a safe drug attenuating CF symptoms, increasing exercise tolerance and improving quality of life.

Plasma NO-binding activity in patients with hematological malignancies: suppressive effect of glucocorticoids.

Plasma NO-binding activity was studied in patients with various forms of hematological malignancies. The method used in the study quantitatively evaluated the plasma capacity to bind NO, which reflects the intensity of NO production and the degree of patient's stress resistance. Plasma NO-binding activity significantly decreases in patients with hematological malignancies. Glucocorticoid treatment promotes the decrease in plasma NO-binding activity, which was dose-dependent.

EPR investigation of in vivo inhibitory effect of guanidine compounds on nitric oxide production in rat tissues.

The aim of the present study was to evaluate in vivo effects on NO production of pharmacologically widely used, commercially available NOS inhibitors, structurally related to guanidine. We compared the NO inhibitory potency and selectivity of L-NAME, aminoguanidine and guanabenz in tissues of normal and LPS-stimulated rats using ex vivo EPR measurements of the NO radical in its complex with dithiocarbamate-Fe(II). The tissues studied were the brain cortex, kidney, liver, heart and testis. Differential inhibitory effects were seen for L-NAME, aminoguanidine and guanabenz when applied during basal or LPS-stimulated conditions. Aminoguanidine exerted inhibition of NO only after stimulation with LPS. Guanabenz had little effect on NO in liver, kidney, testis and heart under normal conditions, while it reduced the basal NO in brain cortex. After stimulation with LPS guanabenz afforded a partial inhibition of the NO formation in all tissues studied. L-NAME was a potent inhibitor of NO synthesis in all tested tissues, both during basal and LPS stimulated conditions. Our results show that compounds containing a guanidine moiety might possess different NOS inhibitory profiles in vivo.

Increased synthesis of nitric oxide in rat brain cortex due to halogenated volatile anesthetics confirmed by EPR spectroscopy.

BACKGROUND: Halogenated volatile anesthetics (HVAs) are considered to be inhibitors of nitric oxide synthase (NOS). On other hand, NO mediates the vasodilation produced by HVAs. Thus, both increase and decrease of NO concentration in brain tissues are possible during anesthesia. Previously, we have observed an increase of NO content in rat brain cortex under halothane anesthesia. The goal of this study was to determine whether the observed phenomenon was general for this anesthetic group, if it was specific for brain cortex, and if the NO increase was due changes in NOS activity. METHODS: NO scavengers were injected to adult rats 30 min prior to anesthesia. Rats were anesthetized by inhalation of an O2 mixture with volatile anesthetics (1.5% for halothane; 1% for isoflurane, 2% for sevoflurane). After 30 min of anesthesia, rats were decapitated and brain cortex, cerebellum, liver, heart, kidneys and testes were dissected, frozen in liquid nitrogen and subjected to EPR spectroscopy. Nitric oxide content was determined quantitatively based on the intensity of the NO-Fe-DETC complex spectrum and its comparison with the calibration curve. RESULTS: In rats anesthetized with HVAs, we observed a greater than twofold increase of NO content in brain cortex as compared to the nonanesthetized animals. No significant changes were detected in other organs. The NOS inhibitor N(omega)-nitro-L-arginine abolished the increase of NO content in brain produced by volatile anesthetics. CONCLUSION: The action of volatile anesthetics is coupled with an increase of NO content in the cortex dependent on NOS activity.

Characterization of a G protein-coupled receptor for nicotinic acid.

Nicotinic acid is a lipid-lowering agent widely used to treat hypertriglyceridemia and to elevate low high density lipoprotein levels. However, the underlying mechanisms are poorly understood. In this study, G protein activation by nicotinic acid and derivatives was assessed as stimulation of guanosine 5'-(gamma-[(35)S]-thio)triphosphate ([(35)S]GTPgammaS) binding, and [(3)H]nicotinic acid was used for specific labeling of binding sites. Nicotinic acid (EC(50) approximately 1 microM) stimulated [(35)S]GTPgammaS binding in membranes from rat adipocytes and spleen, but not from other tissues. G protein activation in adipocyte membranes in the presence of maximally activating concentrations of the selective A(1) adenosine receptor agonist 2-chloro-N(6)-cyclopentyladenosine and nicotinic acid was almost additive, indicating that G proteins of mostly distinct pools were activated by these agonists. G protein activation by nicotinic acid and related substances in spleen and adipocytes revealed identical pharmacological profiles. [(3)H]Nicotinic acid specifically detected guanine nucleotide-sensitive binding sites of identical pharmacology in adipocyte and spleen membranes. The site of action of nicotinic acid is distinct from other G protein-coupled receptors. These data indicate that nicotinic acid most probably acts on a specific G protein-coupled receptor.

Improved method for EPR detection of DEPMPO-superoxide radicals by liquid nitrogen freezing.

5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) is frequently used as a spin trap for the measurement of superoxide by EPR spectrometry. However, its half life is fairly short in room temperature. We here show that superoxide radicals trapped by DEPMPO can be successfully recorded at -196 degrees C. Moreover, we show that the signal intensity remains unaltered for up to 7 days, when the samples are stored in liquid nitrogen. Our new approach for measurement of superoxide should greatly simplify the studies of this important radical in biological systems.

Synthesis and structure-affinity relationships of 1,3, 5-alkylsubstituted cyclohexylamines binding at NMDA receptor PCP site.

A series of 1,3,5-alkylsubstituted cyclohexylamines 2 were synthesized as ligands for the N-methyl-D-aspartate (NMDA) receptor phencyclidine (PCP) binding site. Pure diastereomers with defined configuration of amino group 2-ax and 2-eq were obtained. The optimal size of 1,3,5-substituents was determined for cyclohexylamines 2 with an equatorial amino group in the lowest energy conformation using Hansch analysis. According to the data, the lipophilic part of cyclohexylamines 2 does not discriminate between hydrophobic regions of the PCP binding site but rather recognizes this site as a whole lipophilic pocket.

Synthesis and antitumor activity of selected 7-alkylidene substituted cephems.

Selected 7-alkylidene substituted cephems were synthesized and subjected to antitumor assay. The effect of substituents was examined to establish structure-activity relationships. It was found that the intensive intracellular generation of nitric oxide induced by tert-butyl 7-alkylidene cephalosporanate sulfones could be also regarded as an additional cytotoxic factor taking place both in vitro and in vivo experiments.

N-Hydroxyguanidine compound 1-(3,4-dimethoxy- 2-chlorobenzylideneamino)-3-hydroxyguanidine inhibits the xanthine oxidase mediated generation of superoxide radical.

We here show that the novel N-hydroxyguanidine derivative PR5 (1-(3, 4-dimethoxy-2-chlorobenzylideneamino)-3-hydroxyguanidine) is acting as an alternative electron acceptor in xanthine oxidase catalyzed oxidation of xanthine. The reduction product is the corresponding guanidine derivative 1-(3, 4-dimethoxy-2-chlorobenzylideneamino)guanidine (PR9). The reaction occurs under both anaerobic and aerobic conditions. Moreover, EPR measurements show that the action of PR5 is associated with the inhibition of superoxide radical formation seen under aerobic conditions. PR5 also supports xanthine oxidase catalyzed anaerobic oxidation of NADH. Kinetic studies indicate that increasing xanthine concentration significantly increases the apparent K(m) of PR5, but it remains unaltered by changing NADH concentration. Moreover, the molybdenum center inhibitor allopurinol inhibits the PR5-sustained oxidation of xanthine and NADH equally well, whereas the flavin adenine dinucleotide site inhibitor diphenyliodonium (DPI) markedly inhibits only the PR5-sustained oxidation of NADH. We suggest that PR5 binds and becomes reduced at the molybdenum center of the xanthine oxidase. We also found that both PR5 and its reduction product PR9 can inhibit the oxygen-sustained xanthine oxidase reaction. The properties of PR5 suggest that it is a member of a novel class of compounds which we have termed xanthine oxidase electron acceptor-inhibitor drugs. The potential use of xanthine oxidase electron acceptor-inhibitors in the prevention of free radical mediated tissue damage in organ ischemia-reperfusion diseases is discussed.

A convenient synthesis of 5beta-cholestan-26-oic and 5beta-cholestan-26,27-dioic acids.

A new method for the preparation of 5beta-cholestan-26-oic acids 7 and their analogs is described. The key steps in the synthesis are: iodination of bis- and tris-formyloxy-5beta-cholan-24-ols 3; nucleophilic substitution of iodides 4 with diethyl sodiomalonate; complete alkaline hydrolysis of esters 5; and subsequent decarboxylation of geminal diacids 6 in DMSO.

Cardioprotective effects of N-hydroxyguanidine PR5 in myocardial ischaemia and reperfusion in rats.

1. The potential for the N-hydroxyguanidine compound PR5 (N-(3, 4-dimethoxy-2-chlorobenzylideneamino)-N'-hydroxyguanidine) as a cardioprotective agent in heart ischaemia and reperfusion injury was investigated using rat models. 2. Administration of 1-10 mg kg-1 of PR5 5 min before 10 min of left coronary artery occlusion, followed by 20 min reperfusion, strongly inhibited reperfusion burst of arrhythmias and markedly improved the survival of the animals (e.g. ventricular fibrillation incidence 93 vs 43% (P<0.05); mortality 47 vs 0% (P<0.05), for controls and for 3 mg kg-1 of PR5, respectively). 3. Administration of 3 mg kg-1 of PR5 1 min before reperfusion to rats subjected to 10 min occlusion, 20 min reperfusion was most effective in reducing arrhythmias and decreasing mortality (43 vs 0%, P<0.05), but effects were also seen when PR5 was administered 0, 1 and 5 min after start of reperfusion. 4. Coronary occlusion/reperfusion (10 - 20 min) increased malondialdehyde (MDA) of rat hearts (0.88+/-0.13 for sham vs 1.45+/-0.12 nmol mg-1 protein for ischaemic; P<0.05). In rats where 3 mg kg-1 PR5 were administered 1 min before reperfusion the increase was attenuated (MDA being 1.04+/-0.12; P<0.05 vs ischaemic). 5. PR5 caused a substantial reduction of the infarction size in rats subjected to 180 min left coronary artery occlusion, followed by 120 min of reperfusion; the necrotic zone being 326+/-32 mg for controls vs 137+/-21 mg for animals treated with 3x3 mg kg-1 of PR5 (P<0.01). 6. PR5 reduced the elevation of the ST-segment of the ECGs, as well as caused pronounced attenuation of the rapid blood pressure drop seen at the start of reperfusion following coronary artery occlusion. 7 We conclude that the N-hydroxyguanidine PR5 provides remarkable protection against ischaemia and reperfusion induced myocardial necrosis and life-threatening arrhythmias. These effects of PR5 are discussed in relation to a recently discovered ability of N-hydroxyguanidines to function as electron acceptors at the xanthine oxidase enzyme.

Use of a gel-forming dipeptide derivative as a carrier for antigen presentation.

A dipeptide of the formula Fmoc-Leu-Asp and some other related dipeptides were synthesized in solution by standard methods. When such peptides are dissolved in water at concentrations below 1% at 100 degrees C and cooled below 60 degrees C they form turbid solutions and eventually viscoelastic gels at lower temperatures. Such gels are thermoreversible and can also be disrupted by mechanical agitation. At a concentration of 2 mg/ml the peptide Fmoc-Leu-Asp forms an aqueous gel at 60 degrees C with a shear modulus of 80 Pa measured at a frequency of 1 rad/s. Peptide solutions undergo an abrupt increase in light scattering between 1 and 1.5 mg/ml at both 23 and 60 degrees C. By analogy with previous observations of other systems, this increase appears to be due to the formation of filamentous micelles and the aggregation of filaments into a three-dimensional network. When low molecular weight adamantanamine derivatives, which are inherently non-antigenic antiviral drugs, were incorporated into the Fmoc-Leu-Asp gel and injected into rabbits, high titre specific antibodies were efficiently produced without the need for additional adjuvant. Both the physical properties of the gel and its effect on the antigenicity of low molecular weight compounds suggest a number of practical applications.

A three dimensional model for the interaction of MSH with the melanocortin-1 receptor.

A model for the interaction of the melanocortin-1 (MC1) receptor with MSH peptide has been constructed. The model was built by homology modelling using bacteriorhodopsin as template. A cyclic analogue of MSH could be docked into a binding pocket located between transmembrane (TM) domains 2, 3 and 6 of the receptor. The most significant receptor to ligand interactions occur between D117 in TM3 of receptor with histidine in cyclic MSH-peptide, H260 in TM6 with glutamic in peptide and D121 in TM3 with arginine in peptide. The model finds support from earlier mutagenesis data.