Peroxidase-like catalytic activity of aqueous- and immobilized-Mn(3+)-octabromo-porphyrins on ion-exchange resin supplied as mimetic of horseradish peroxidase.

In order to explore the capability of metal porphyrins as an alternative of horseradish peroxidase (HRP), HRP-like activity of three manganese-porphyrins (Mn-Ps) and three Mn-octabromo-porphyrins (Mn-OBPs) was examined in both aqueous and immobilized states. It was found that Mn(3+)-octabromotetrakis(1-methyl-pyridinium-4yl)porphine (Mn-OBTMPyP) has an activity of at least 90% of HRP in an aqueous solution. Mn-OBTMPyP exhibited a catalytic activity even in the presence of hydrogen peroxide without suicide reaction. In addition, Mn-OBTMPyP was revealed to function as an alternative to HRP in the quantitative determination of serum uric acid. These results are of great interest because they indicate that metal-octabromo-porphyrins possibly include promising candidates of artificial enzyme capable of substituting for HRP.

5-Iodo-A-85380, a specific ligand for alpha 4 beta 2 nicotinic acetylcholine receptors, prevents glutamate neurotoxicity in rat cortical cultured neurons.

5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-iodo-A-85380, 5IA) has very high affinity and selectivity to nicotinic acetylcholine receptor (nAChR) alpha 4 beta 2 subtype, and a relative safe profile. To assess whether 5IA has neuroprotective properties, we examined the effect of 5IA on glutamate (Glu)-induced neurotoxicity using primary cultures of rat cortical neurons. A 10-min exposure of cultures to Glu followed by 2-h incubation with drug-free medium caused a marked loss of viability, as determined by trypan blue exclusion method. Glu-induced neurotoxicity was prevented by 5IA both in a time- and concentration-dependent manner. 5IA-induced neuroprotection required pretreatment of 5IA prior to Glu exposure with an optimal concentration of 10 nM and an optimal pretreatment time of 2 h. Treatment after Glu exposure could not rescue the cultured cells. The neuroprotective effect of 5IA was antagonized by mecamylamine, a nAChR antagonist, but not by scopolamine, a muscarinic acetylcholine receptor antagonist. Dihydro-beta-erythroidine, an alpha 4 beta 2 nAChR antagonist, completely inhibited 5IA-induced neuroprotection, whereas alpha-bungarotoxin, an alpha 7 nAChR antagonist, had no effect. Furthermore, 5IA did not show neuroprotective effects in the absence of extracellular Ca2+. These results suggest that the neuroprotective effects of 5IA are produced by activation of alpha 4 beta 2 nAChRs followed by the influx of extracellular Ca2+. In conclusion, 5IA is possibly not only useful for the treatment and prevention of glutamate neurotoxicity, but also as an available tool for elucidating the mechanism of neuroprotection associated with alpha 4 beta 2 nAChRs.

Effect of carboxyl-group of D-glutamic acid or gamma-carboxy-D-glutamic acid as N-terminal amino acid of (111)in-diethylenetriaminepentaacetic acid-octreotide on accumulation of radioactivity in kidney.

To establish a strategy for developing (111)In-diethylenetriaminepentaacetic acid ((111)In-DTPA)-octreotide, a diagnostic radiopharmaceutical agent for tumors, with reduced non-specific renal radio-accumulation, the compounds having D-glutamic acid (Glu) or gamma-carboxy-D-glutamic acid (carboxy-Glu) as the N-terminal amino acid were examined for in vivo radio-distribution. Compounds carrying Glu and carboxy-Glu containing one and two negative charges, respectively, showed lower renal radio-accumulation than that carrying D-phenylalanine. It was revealed that the introduction of a negative charge reduces the renal radio-accumulation independently from the number of negative charges. The present result can be a clue for the development of (111)In-DTPA-octreotides with reduced the renal radio-accumulation.

Catalytic activity of silica gels bound manganese(III)-porphyrin on oxidative reaction of adrenaline.

To develop a solid catalysis on oxidative reaction of adrenaline (Ad), supports bound metal-tetrakis(4-carboxyphenyl)porphine (M-TCPP) were investigated. Silica gels bound Mn-TCPP were proved to has a superior activity in the oxidation of Ad and be able to serve as a useful oxidase model for Ad.

Peroxidase-like catalytic activity of Mn(3+)-octabromo-tetrakis(4-sulfophenyl)porphine on linoleate hydroperoxide and its analytical application.

To reveal an enzyme-like catalytic activity of metal-octabromo-tetrakis(sulfophenyl)porphines (M-OBPSs), their peroxidease-like catalytic activity on linoleate hydroperoxide (LOOH) were evaluated on the basis of dye-formation in the coloring reaction between N,N-diethylaniline and 4-aminoantipyrine that yields a quinoid-type dye. Among M-OBPSs tested, Mn(3+)-OBPS allowed to produce the largest amount of dye. The optimal conditions of the coloring reaction catalyzed by Mn(3+)-OBPS for the determination of LOOH were determined. A good linear calibration curve was obtained in the concentration range of 0.025-0.4mumole LOOH with good reproducibility (coefficient of variance=1.23%), suggesting that Mn(3+)-OBPS is a good artificial mimesis of the peroxidase for LOOH. In addition, Mn(3+)-OBPS was highly specific for LOOH even in the presence of cumene hydroxyperoxide or hydrogen peroxide. It was revealed that the peroxidase-like activity of Mn(3+)-OBTP is attributable to the redox cycle of Mn(3+)<-->Mn(4+).

Evaluation of the state of active ingredients in pharmaceutical preparations using fourier transform-Raman difference spectra.

To examine the pharmaceutical application of Fourier transform (FT)-Raman spectroscopy, the state of active pharmaceutical ingredients (APIs) in a preparation of several forms was evaluated by investigating the Raman difference spectra between the preparation and excipient. The difference spectra indicated that APIs in alacepril tablets, caffeine sustained-release granules, and quinidine sulfate granules remained unchanged after the manufacturing process. However, the state of sparfloxacin in nanoparticles changed, although it remained unchanged in tablets or powders. These results show that the FT-Raman difference spectrum is expected to be utilized in the field of quality control of crystalline pharmaceutical preparations.

Standard infrared absorption spectrum of betaine and optimal conditions for its measurement.

The infrared absorption (IR) spectrum is often used as a standard reference in identification tests of food additives in Japan. In the case of betaine, many different IR spectra have been reported and, therefore, it is necessary to establish an IR spectrum that is reproducible and reliable enough to be used as a standard for identification. In the present study, suitable conditions to obtain a standard IR spectrum were examined from various viewpoints, including pretreatment, selection of method, and measuring technique. The KBr disk method, which has generally been used to identify betaine, was found to be humidity-dependent, and there was also an interaction between betaine and KBr. A reproducible IR spectrum suitable as a standard could be obtained by drying betaine at 105 degrees C for 3 hours over phosphorus pentoxide, and then measuring the IR spectrum by the liquid paraffin (Nujol) paste method.

HPLC retention behaviors of pi-electron rich compounds on Ni2+- and Cu2+-phthalocyanine derivatives bound to silica gels in polar eluents.

The effect of the central metal of columns packed with silica gels binding Ni(2+)- and Cu(2+)-phthalocyanine derivatives (Ni-and Cu-PCS(D)s) on the retention behavior of poly-aromatic-hydrocarbons (PAHs) thereof in a polar eluent was examined. The retention factors of PAHs on the Ni- and Cu-PCS(D)s in 80% methanol showed a good linear correlation. The Cu-PCS(D) column exhibited the pi-pi interactions for PAHs, while the Ni-PCS(D) column exhibited the pi-d interactions for PAHs in addition to the pi-pi interaction for PAHs. Further, an investigation of the retention behavior of anthracene derivatives having different substituents revealed that the Ni- and Cu-PCS(D) columns could recognize the differences of substituents only in a polar eluent.

Release of vesicular Zn2+ in a rat transient middle cerebral artery occlusion model.

In the brain, Zn(2+) is stored in synaptic vesicles of a subgroup of glutamatergic nerve terminals. Although it has been reported that this Zn(2+) is released upon the excitation of nerves in vitro, there has been little study of the release of Zn(2+) during ischemia in vivo. Here, using brain microdialysis, the release of vesicular Zn(2+) was investigated in vivo. When the vesicular Zn(2+) was released into the synaptic cleft by a depolarizing stimulation achieved by perfusion with Ringer's solution containing high K(+) (100mM KCl), a significant increase in the extracellular concentration of Zn(2+) could be detected by microdialysis. Then, we investigated the release of vesicular Zn(2+) in a rat transient middle cerebral artery occlusion model using microdialysis. Consequently, the extracellular Zn(2+) level in the cortex increased within 15 min of the start of occlusion and reached a peak at 30 min, which was about twice the basal level. After 30 min, it declined with time returning to the basal level 15 min after reperfusion, which was performed after 60 min of occlusion. The results suggest that vesicular Zn(2+) would be released into the synaptic cleft during brain ischemia in vivo.

In vivo measurement of presynaptic Zn2+ release during forebrain ischemia in rats.

Previous studies have suggested that during forebrain ischemia, considerable Zn2+ is released from synaptic vesicles of gultamatergic neuronal terminals and accumulates in hippocampal CA1 pyramidal neurons, leading to delayed neuronal death. However, since a time lag exists between the accumulation of Zn2+ and the occurrence of ischemia and there are conflicting reports about the amount of Zn2+ released, the level of released Zn2+ during ischemia in vivo is still unclear. In this study, we investigated the temporal change of extracellular Zn2+ in the hippocampal CA1 area using microdialysis and the accumulation of Zn2+ in hippocampal CA1 neurons with TSQ staining in rats with a transient forebrain ischemia. The level of extracellular Zn2+ in the CA1 area increased transiently reaching a peak 15 min after occlusion, then decreased with time, returning to the basal level 15 min after reperfusion. In addition, at this peak, the level of extracellular Zn2+ was about twice the basal level. Assessment of the intracellular Zn2+ in hippocampal neurons with TSQ revealed that Zn2+ accumulate at 24 h, but not 0 and 6 h after ischemia. These results suggest that, although the synaptic vesicular Zn2+ is released into the synaptic cleft during ischemia in vivo, the amount of released Zn2+ might not be so excessive, and it does not accumulate in hippocampal CA1 pyramidal neurons immediately after ischemia.

Protective effect of zinc against ischemic neuronal injury in a middle cerebral artery occlusion model.

In this study, we investigated the effect of vesicular zinc on ischemic neuronal injury. In cultured neurons, addition of a low concentration (under 100 microM) of zinc inhibited both glutamate-induced calcium influx and neuronal death. In contrast, a higher concentration (over 150 microM) of zinc decreased neuronal viability, although calcium influx was inhibited. These results indicate that zinc exhibits biphasic effects depending on its concentration. Furthermore, in cultured neurons, co-addition of glutamate and CaEDTA, which binds extra-cellular zinc, increased glutamate-induced calcium influx and aggravated the neurotoxicity of glutamate. In a rat transient middle cerebral artery occlusion (MCAO) model, the infarction volume, which is related to the neurotoxicity of glutamate, increased rapidly on the intracerebral ventricular injection of CaEDTA 30 min prior to occlusion. These results suggest that zinc released from synaptic vesicles may provide a protective effect against ischemic neuronal injury.

HPLC retention behaviors of poly-aromatic-hydrocarbones on Cu(II)-octabromotetrakis(4-carboxyphenyl)porphine derivatives-immobilized aminopropyl silica gels in polar and non-polar eluents.

As one of approaches of developing novel HPLC stationary phases, we prepared Cu-octabromotetrakis(4-carboxyphenyl)porphine derivative-immobilized silica gels (Cu-OBTCPP(D)), and evaluated the availability of the resultant Cu-OBTCPP(D) as a stationary phase for separation of poly-aromatic-hydrocarbons (PAHs) and their related compounds. A Cu-OBTCPP(D) column was revealed to have an ability to separate simple PAHs and be useful as a stationary phase in both polar and non-polar eluents. The retention property of the Cu-OBTCPP(D) column was evaluated in various comparative experiments using commercially available columns. In comparison with 2-(1-pyrenyl)ethyl dimetylsilyl silica gel column (PYE column) regarding the retention behavior for PAHs etc., the Cu-OBTCPP(D) column showed stronger interactions involving pi electron in non-polar eluent than PYE column. In comparison with a pentabromobenzyloxy propylsilyl silica gel column (PBB column) regarding the influence of bromination, the Cu-OBTCPP(D) column was affected differently from the PBB column. In comparison with nitrophenylethyl silica gel column (NPE column) regarding the retention behavior for compounds having a dipole in a non-polar eluent, the Cu-OBTCPP(D) column showed electrostatic interactions such as dipole-dipole interaction equivalent to or larger than the NPE column.

Peroxidase-like catalytic activity of Mn- and Fe-tetrakis(4-carboxyphenyl)porphines bound to aminopropyl-glass bead in oxidative reaction of heterocyclic amines.

Fe- or Mn-tetrakis(4-carboxyphenyl)porphine (Fe- and Mn-TCPP) bound to aminopropyl-glass bead (Fe- and Mn-TCPP(g)s) was examined for the peroxidase (POD)-like function in order to develop a solid catalyst which can exhibit POD-like activity without adsorbing heterocyclic amines (HCAs). Mn-TCPP in aqueous solution had only a slight POD-like catalytic activity on HCAs (IQ and MeIQ). As for Fe-TCPP, it was impossible to examine the POD-like activity since it reacted with hydrogen peroxide in a liquid reaction system. However, both Fe- and Mn-TCPP when immobilized on aminopropyl-glass bead via peptide bond (Fe- and Mn-TCPP(g)s), catalyzed the oxidative reaction of mutagenic HCAs with hydrogen peroxide. The catalytic activity of Fe- and Mn-TCPP(g)s was investigated in more detail using as a substrate IQ and MeIQ which were oxidized more rapidly among the tested HCAs. Consequently, the optimal conditions for the oxidative reaction catalyzed by Fe- and Mn-TCPP(g)s were determined. In addition, ESI-mass and absorption spectra of oxidation products of IQ and MeIQ showed that they are dimers. Thus, it was demonstrated that a solid catalyst with POD-like activity can be obtained by immobilizing Fe- and Mn-TCPPs on aminopropyl-glass beads.

Effect of octabromination of a tetrakis(4-carboxyphenyl)porphine derivative bound to silica gels on HPLC retention behaviors of polyaromatic hydrocarbons.

The effect of bromination of Cu-porphyrin-derivative-immobilized silica gels (Cu-TCPP(D)) was examined by comparing the retention behaviors of polyaromatic hydrocarbons (PAHs) on Cu-TCPP(D) and Cu-octabromotetrakis(4-carboxyphenyl)porphine-derivative-immobilized silica gels (Cu-OBTCPP(D)) columns. It was revealed that bromination affects strongly the pi-pi electron interactions caused from hydration energy in a polar eluent (80% methanol) possibly as a result of a destruction of planar structure of porphine-ring by bromination. It was also revealed that bromination enhances pi-d interactions as well as the pi-pi electron interactions in a broad sense (e.g., dispersion forces) in a non-polar eluent (n-hexane). However, the bromination did not exert much influence on electrostatic interactions caused from polarization of mono-halogenated benzenes.

Peroxidase-like catalytic activity of anion-exchange resins modified with metal-porphyrins in oxidative reaction of hetrocyclic amines with hydrogen peroxide.

To elucidate the peroxidase (POD)-like catalytic activity of anion-exchange resins modified with metal-tetrakis(sulfophenyl)porphine (M-TSPP(r)s), an oxidative reaction of seven mutagenic heterocyclic amines (HCAs) with hydrogen peroxide, which reaction is catalyzed by horse radish POD, was investigated in the presence of M-TSPP(r)s. Among six M-TSPP(r)s tested, Mn- and Fe-TSPP(r)s were found to have a relatively strong POD-like activity for HCAs, in particular for a typical HCA, 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ). The optimal condition for the POD-like activity was selected using Fe- and Mn-TSPP(r)s. For evaluation of an oxidation product of IQ produced in the presence of Fe-TSPP(r), the absorption, NMR and FAB-mass spectra thereof were compared with those of an oxidation product of IQ produced by horse radish POD or a chemical oxidizing agent, sodium hypochlorite. When Fe-TSPP(r) was present as a catalysts, IQ was converted into the dimmer (hydorazone type) which has no mutagenic activity in umu-test. It was revealed that Fe- and Mn-TSPP(r)s exhibit a POD-like catalytic activity in oxidative reaction of HCAs with hydrogen peroxide.

Application of liquid chromatography-tandem mass spectrometry for the determination of opioidmimetics in the brain dialysates from rats treated with opioidmimetics intraperitoneally.

We have determined three opioidmimetics (compounds I-III) in the rat brain dialysates after intraperitoneal (i.p.) administration of compounds I-III using a liquid chromatography/mass spectrometry with tandem mass spectrometry (LC-MS/MS). The dialysate samples with methanol were directly analyzed by online column-switching liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 421 of m/z 657 for compound I, m/z 421 of m/z 643 for compound II, and m/z 407 of m/z 629 for compound III) on LC-MS/MS with electrospray ionization (ESI), opioidmimetics in rat brain dialysates were determined. Calibration curves of the method showed a good linearity in the range of 10-100 ng/ml for each compound. The limit of determination was estimated to be ca. 1 ng/ml for compounds II and III, and ca. 5 ng/ml for compound I, respectively. The precision of analysis showed coefficients of variation ranging from 4.7 to 10.4% at compound III concentration (10-100 ng/ml) in Ringer's solution. As a result, the procedure proved to be very suitable for routine analysis. The method was applied to the analysis of three opioidmimetics in the brain dialysate samples from rats treated with these compounds.

Binding of 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium ion (HPP+), a metabolite of haloperidol, to synthetic melanin: implications for the dopaminergic neurotoxicity of HPP+.

The toxicity of 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium ion (HPP+), a metabolite of haloperidol, toward dopaminergic neurons was investigated. When HPP+ (approximately 100 microM) was added to primary cultures prepared from rat embryonic mesencephalon for 1 h, the survivability of dopaminergic neurons decreased significantly, and this effect was not inhibited by the dopamine transporter (DAT) inhibitor GBR 12909. In addition, HPP+ bound to neuromelanin, which is abundant in dopaminergic neurons. A binding analysis using the Scatchard method showed that there are two classes of binding sites: high affinity sites with a dissociation constant K(d1) of 20.2 nM, and low affinity sites with a K(d2) of 4.0 microM. HPP+ was released easily from synthetic melanin using phosphate buffer (pH 7.0), suggesting that this binding is reversible. The results suggest that the toxicity of HPP+ in dopaminergic neurons is due not to DAT-mediated uptake, but to the binding to neuromelanin.

In vivo imaging of brain dopaminergic neurotransmission system in small animals with high-resolution single photon emission computed tomography.

High-resolution single photon emission computed tomography (SPECT) provides a unique capability to image the biodistribution of radiolabeled molecules in small laboratory animals. Thus, we applied the high-resolution SPECT to in vivo imaging of the brain dopaminergic neurotransmission system in common marmosets using two radiolabeled ligands, [123I]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane (beta-CIT) as a dopamine transporter (DAT) ligand and [123I]iodobenzamide (IBZM) as a dopamine D2 receptor (D2R) ligand. Specific images of the striatum, a region with a high density of dopaminergic synapses, were obtained at 240 min and 60 min after injection of [123I]beta-CIT and [123I]IBZM, respectively. Furthermore, a significantly low accumulation of [123I]beta-CIT in the striatum was observed in MPTP-treated animals compared with results for a control group, and a similar accumulation in the control group was observed with the pretreatment of deprenyl in the MPTP-treated animals. However, the striatal accumulation of [123I]IBZM showed no changes among the control, MPTP-treated, and deprenyl-MPTP-treated groups. These SPECT imaging results agreed well with those of DA concentration and motor behavior. Since MPTP destroys nigrostriatal dopamine nerves and produces irreversible neurodegeneration associated with Parkinsonian syndrome, SPECT imaging data in this study demonstrated that deprenyl shows its neuroprotective effect on Parkinsonism by protecting against the destruction of presynaptic dopamine neurons.

Brain extraction of 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium ion (HPP+), a neurotoxic metabolite of haloperidol: studies using [3H]HPP+.

Tritium-labeled 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium ion (HPP+) was synthesized enzymatically from [3H]haloperidol using rat liver microsomal preparations, and using prepared [3H]HPP+, the passage of HPP+ into the brain was investigated. Consequently, HPP+ showed a moderate brain uptake index, indicating that it is able to permeate the blood-brain barrier. Furthermore, HPP+ was detected in murine brains after being intravenously injected. These results suggested that HPP+, produced mainly in the liver, is taken up into the brain and induces damage to brain dopaminergic neurons.

Evaluation of radioiodinated 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine as a ligand for SPECT investigations of brain nicotinic acetylcholine receptors.

5-Iodo-3-(2(S)-azetidinylmethoxy)pyridine (5IA), an A-85380 analog iodinated at the 5-position of the pyridine ring, was evaluated as a radiopharmaceutical for investigating brain nicotinic acethylcholine receptors (nAChRs) by single photon emission computed tomography (SPECT). [123/125I]5IA was synthesized by the iododestannylation reaction under no-carrier-added conditions and purified by high-performance liquid chromatography (HPLC) with high radiochemical yield (50%), high radiochemical purity (> 98%), and high specific radioactivity (> 55 GBq/micromol). The binding affinity of 5IA for brain nAChRs was measured in terms of displacement of [3H]cytisine and [125I]5IA from binding sites in rat cortical membranes. The binding data revealed that the affinity of 5IA was the same as that of A-85380 and more than seven fold higher than that of (-)-nicotine, and that 5IA bound selectively to the alpha4beta2 nAChR subtype. Biodistribution studies in rats indicated that the brain uptake of [125I]51A was rapid and profound. Regional cerebral distribution studies in rats demonstrated that the accumulation of [125I]5IA was consistent with the density of high affinity nAChRs with highest uptake observed in the nAChR-rich thalamus, moderate uptake in the cortex and lowest uptake in the cerebellum. Administration of the nAChR agonists (-)-cytisine and (-)-nicotine reduced the uptake of [125I]5IA in all regions studied with most pronounced reduction in the thalamus, and resulted in similar levels of radioactivity throughout the brain. [125I]5IA binding sites were shown to be saturable with unlabeled 5IA. Behavioral studies in mice demonstrated that 5IA did not show signs of behavioral toxicity. Furthermore, SPECT studies with [123I]5IA in the common marmoset demonstrated appropriate brain uptake and regional localization for a high-affinity nAChR imaging radiopharmaceutical. These results suggested that [123I]5IA is a promising radiopharmaceutical for SPECT studies of central nAChRs in human subjects.