Convenient Application of a Compact-Type Magnetic Resonance Imager with Blood Pool Contrast Agent Gadolinium-Conjugated Dextran to Assess Vascular Abnormalities in Experimental Animals.

Vascular lesions are symptomatic of lifestyle-related diseases and include blood clots, coarctations, aneurysms, and apoplexy. Furthermore, increased blood vessel permeability is usually observed in tumors. To develop therapeutic drugs treating vascular lesions and tumors, methods with which the vascular abnormalities can be readily assessed in experimental animals are necessary. In this paper, a laboratory-size magnetic resonance imaging (MRI) system with permanent magnets, a compact-type MRI, was used to assess vascular abnormalities. Blood vessels in the head of a mouse were clearly visualized with the compact-type MRI in combination with gadolinium-diethylenetriamine-N,N,N',N″,N″-pentaacetic acid chelate (Gd-DTPA)-linked dextran (Gd-Dex) as blood pool contrast agents. The rat middle cerebral artery was imaged, and artery occlusion was identified. The difference between normal and occluded rats became more apparent upon intravenous injection of sodium nitroprusside, a nitric oxide (NO) donor. The system also visualized poor circulation in a rat saphenous artery by femoral artery occlusion. In a tumor-bearing mouse, a compact-type MRI visualized accumulation of Gd-Dex similar to that of small molecular Gd-DTPA, in the rim of tumor. Gd-Dex accumulation was more consistent than that of Gd-DTPA. Tumor vasculature was characterized by estimating the plasma-to-tumor interstitial tissue transfer constant, Ktrans, of Gd-Dex and fractional plasma volume, Vp, using image data. These results demonstrate the efficacy of a compact-type MRI in combination with Gd-Dex for vascular abnormality assessment in both mice and rats.

Protective effects of 2-aminoethylthiosulfuric acid and structurally analogous organosulfur compounds against ionizing radiation.

High efficacy and minimal toxicity radioprotectors are desirable options for the hazards posed by nuclear medical and energy technologies and the dangers presented by nuclear weapons in an unstable global situation. Although cysteamine is an effective radioprotector, it has considerable toxicity. In this study, the protective effects of the less toxic organosulfur compounds 2-aminoethylthiosulfate (AETS), thiotaurine (TTAU), and hypotaurine (HTAU) against X-ray damage in mice were compared with that of cysteamine. Intraperitoneal injection of either AETS or cysteamine (2.2 mmol/kg body weight) 30 min before X-ray irradiation (7.0 Gy) provided 100% survival for 30 days, limited the decrease in erythrocytes and neutrophils over 9 days, and reduced damage to bone marrow and spleen over 9 days. Neither TTAU nor HTAU provided any protection. In mice, 30 min after AETS administration, non-protein thiol content increased in the spleen, indicating cysteamine generation by AETS hydrolysis, the active protective species of AETS. All examined compounds scavenged •OH under diffusion control in aqueous solution, which is inconsistent with the difference in the protective effects among the compounds. The results indicate that AETS protects animals from ionizing radiation by several mechanisms, including scavenging •OH as cysteamine.

Differences in pharmacokinetic behaviors of two lipophilic 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyl radicals, in vivo probes to assess the redox status in the brain using magnetic resonance techniques.

PURPOSE: The pharmacokinetics of 3-methoxycarbonyl- and 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl radicals (MCP and HMP, respectively), magnetic resonance probes to assess the brain redox status, were examined in healthy mouse brains. METHODS: The time course of the concentration of the radical form of the probe in the brain was examined by signal enhancements on T1 -weighted MR image after an intravenous injection. The distribution of the total probe (sum of radical and reduced forms) was investigated using brain homogenates. RESULTS: MCP distributed to the brain more than HMP. MCP exhibited biphasic decay with fast and slow components, whereas HMP exhibited monophasic decay with a similar rate constant to the slow component of MCP. Similar profiles were observed in various regions of the brain. The total probe for MCP exhibited monophasic decay at a similar rate constant to the slow component of the radical form; however, the initial content of the total probe was similar to its radical form. For HMP, decay of the total probe coincided with that of the radical form. CONCLUSION: The decay of MCP needs to consider the reduction of the probe in and its elimination from the brain, while the decay of HMP may mainly result from its elimination from the brain.

Synthesis, Aggregation Behavior, and Photodynamic Properties of a Water-Soluble Fulleropyrrolidine Bearing an N-PEG Pyridinium Unit.

Herein, we describe the synthesis of a water-soluble photodynamically active fullerene bearing a polyethylene glycol chain and a hydrophilic cationic group, revealing that the solubility of the above derivative in aqueous medium depends on ultrasonication time, with the particle size of aggregates being correlated with concentration.

Application of a Compact Magnetic Resonance Imaging System with 1.5 T Permanent Magnets to Visualize Release from and the Disintegration of Capsule Formulations in Vitro and in Vivo.

Although magnetic resonance imaging (MRI) has potential in assessments of formulations, few studies have been conducted because of the size and expense of the instrument. In the present study, the processes of in vitro and in vivo release in a gelatin capsule formulation model were visualized using a compact MRI system with 1.5 T permanent magnets, which is more convenient than the superconducting MRI systems typically used for clinical and experimental purposes. A Gd-chelate of diethylenetriamine-N,N,N',N″,N″-pentaacetic acid, a contrast agent that markedly enhances proton signals via close contact with water, was incorporated into capsule formulations as a marker compound. In vitro experiments could clearly demonstrate the preparation-dependent differences in the release/disintegration of the formulations. In some preparations, the penetration of water into the formulation and generation of bubbles in the capsule were also observed prior to the disintegration of the formulation. When capsule formulations were orally administered to rats, the release of the marker into the stomach and its transit to the duodenum were visualized. These results strongly indicate that the compact MRI system is a powerful tool for pharmaceutical studies.

Nitroxyl radicals remarkably enhanced the superoxide anion radical-induced chemiluminescence of Cypridina luciferin analogues.

Measuring the superoxide anion radical (superoxide) with high sensitivity is necessary to clarify the mechanisms of diseases for the development of methods for their prophylaxes, diagnoses, and therapies. The chemiluminescence technique using Cypridina luciferin analogues such as MCLA and CLA is currently the most sensitive method available. Using large concentrations of these reagents, however, leads to increases in background levels due to spontaneous luminescence of the reagent, which is a limitation of this method. This study demonstrated that the superoxide-induced chemiluminescence of MCLA or CLA was markedly enhanced by adding a cyclic nitroxyl radical to the reaction medium. When MCLA was measured spectrophotometrically, the nitroxyl radical was shown to increase the reaction rate of superoxide and MCLA without altering their stoichiometry, whereas consumption of the nitroxyl radical was negligible, as determined by electron paramagnetic resonance (EPR) spectroscopy. These observations indicate that the nitroxyl radical catalytically enhanced the reaction between superoxide and MCLA, resulting in an enhancement in superoxide-dependent MCLA chemiluminescence. This method is applicable to biological systems such as superoxide-generation by neutrophils. The inclusion of the cyclic nitroxyl radical in a sample solution contributed to reductions in the concentration of the chemiluminescence reagent, thereby decreasing background levels. The catalytic mechanism was also discussed.

Simple method for quantification of gadolinium magnetic resonance imaging contrast agents using ESR spectroscopy.

To develop an estimation method of gadolinium magnetic resonance imaging (MRI) contrast agents, the effect of concentration of Gd compounds on the ESR spectrum of nitroxyl radical was examined. A solution of either 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPONE) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) was mixed with a solution of Gd compound and the ESR spectrum was recorded. Increased concentration of gadolinium-diethylenetriamine pentaacetic acid chelate (Gd-DTPA), an MRI contrast agent, increased the peak-to-peak line widths of ESR spectra of the nitroxyl radicals, in accordance with a decrease of their signal heights. A linear relationship was observed between concentration of Gd-DTPA and line width of ESR signal, up to approximately 50 mmol/L Gd-DTPA, with a high correlation coefficient. Response of TEMPONE was 1.4-times higher than that of TEMPOL as evaluated from the slopes of the lines. The response was slightly different among Gd compounds; the slopes of calibration curves for acua[N,N-bis[2-[(carboxymethyl)[(methylcarbamoyl)methyl]amino]ethyl]glycinato(3-)]gadolinium hydrate (Gd-DTPA-BMA) (6.22 µT·L/mmol) and gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid chelate (Gd-DOTA) (6.62 µT·L/mmol) were steeper than the slope for Gd-DTPA (5.45 µT·L/mmol), whereas the slope for gadolinium chloride (4.94 µT·L/mmol) was less steep than that for Gd-DTPA. This method is simple to apply. The results indicate that this method is useful for rough estimation of the concentration of Gd contrast agents if calibration is carried out with each standard compound. It was also found that the plot of the reciprocal square root of signal height against concentrations of contrast agents could be useful for the estimation if a constant volume of sample solution is taken and measured at the same position in the ESR cavity every time.

Generation of radical species from dihydropyrazines having DNA strand-breakage activity and other characteristics.

The various biological activity of dihydropyrazines(DHPs)due to the radical generation potency has been described in previous papers. Detailed data about radical species generating be mentioned here. The electron spin resonance (ESR) spin-trapping technique revealed that DHPs generate free radical species such as ·OH, ·OOH, ·CHR(2) and ·CR(3). Oxygen radicals and two carbon-centered radicals were detected as adducts of the spin traps DMPO and DBNBS, respectively. All the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)- and 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS)-adducts of compounds DHP-1-8 exhibited approximately the same signal patterns, with various levels of intensity depending on the substituent of the dihydropyrazine ring. The ESR signal intensity of DHPs also increased remarkably upon addition of Cu(2+), resulting that the effects of DHPs were enhanced.

Effect of cholesterol on distribution of stable, hydrophobic perchlorotriphenylmethyl triethylester radical incorporated in lecithin liposomal membranes.

Perchlorotriphenylmethyl triethylester radical (PTM-TE) is a hydrophobic, stable radical giving a narrow singlet ESR signal with a small satellite signal for (13)C in organic solvents. In order to use PTM-TE as a label of liposomal membranes, its manner of incorporation into liposomal membranes was studied. Two components, broad and narrow signals, were observed on the ESR spectrum of PTM-TE incorporated into liposomal membranes composed of egg yolk phosphatidylcholine (egg-PC). The broad signal was increased by the presence of cholesterol in the membranes. The spectral anisotropy of the broad signal was very small as analyzed with oriented planar multilamellar membranes. The narrow signal increased with an increase in temperature in the absence of cholesterol, whereas only a small increase in the signal was observed in the presence of cholesterol. The g-value and line width of the narrow signal were very close to those of PTM-TE in mineral oil, whose viscosity is close to the microviscosity in the hydrophobic region of egg-PC membranes. On the other hand, the g-value and line width of the broad signal were close to those of solid PTM-TE. These observations indicate that the broad signal observed in liposomes originates from PTM-TE clusters in the membranes. The clusters were dissolved in egg-PC membranes at a PTM-TE/egg-PC molar ratio of less than 0.017. However, the clusters were hardly dissolved in the presence of cholesterol.

Radical reactions induced by ketoprofen in phospholipid membranes under ultraviolet light irradiation.

2-(3-Benzoylphenyl)propanoic acid (ketoprofen), one of the nonsteroidal anti-inflammatory drugs, causes photocontact dermatitis by ultraviolet (UV) light as a side effect. In this study, we examined radical reactions induced by ketoprofen in the lipid membranes under UV irradiation using egg yolk phosphatidylcholine (egg-PC) liposomal membranes containing 5- or 16-doxyl stearic acid (5- or 16-DSA), which carry nitroxyl radical at the 5- or 16-position of the fatty acid chain, respectively. When the suspension of liposomal membrane was mixed with ketoprofen and irradiated with UV, electron spin resonance signal of 5- and 16-DSA in the membrane decreased. The decay consisted of fast decay and subsequent slow decay. The overall decay for 5-DSA was faster than that for 16-DSA. The rate of slower decay of 16-DSA increased with ketoprofen concentration. The bulk lipid in the membrane affected the rate of slower decay of 5-DSA; the rate increased with the amount of egg-PC and decreased in the rigid membrane composed of dipalmitoylphosphatidylcholine. When spin trapping studies with α-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimetyl-1-pyrroline-N-oxide (DMPO) were performed in ketoprofen solution, C-centered radical adducts of POBN and superoxide anion radical adducts of DMPO were detected after UV irradiation. POBN suppressed the signal decay of 5-DSA in the liposomal membrane, whereas superoxide dismutase accelerated it. These results support that ketoprofen penetrates the lipid membrane and induces a radical reaction near the polar region in the membrane, and that ketoprofen-related C-centered radical is involved in the radical reaction.

The preparation and validation of chitosan tablets that rapidly disperse and disintegrate as an oral adsorbent in the treatment of lifestyle-related diseases.

A carrier and an oral absorbent for the treatment of chronic diseases in the form of a tablet was prepared from granulated chitosan (G-CS) particles. The resulting tablet was highly dispersible and disintegrated rapidly (< 30 s) in aqueous media. The non-granulated chitosan (N-CS) powder partially crystallized (2θ = 12-15° and 20°) during wet granulation to give G-CS crystalline particles. The rate of penetration of water into G-CS aggregates was markedly faster than that for N-CS aggregates, as evidenced by the ease of disintegration of the tablets. The rapid disintegration and dispersion of the tablets in vivo was confirmed by MRI measurements after the oral administration of the both tablets to rats. Some ureic toxins were adsorbed more strongly to G-CS tablets than on N-CS tablets. The results suggest that G-CS tablets have great potential for use as a fast disintegrating carrier and as an oral adsorbent in lifestyle-related diseases.

In vivo ESR imaging of redox status in mice after X-ray irradiation, measured by acyl-protected hydroxylamine probe, ACP.

More detailed investigations on the in vivo redox status are needed to elucidate the mechanisms contributing to damage caused by ionizing radiation. In the present study, the in vivo redox status of mice was examined using in vivo electron spin resonance (ESR) imaging after an intraperitoneal injection of 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) as a probe. ACP is easily hydrolyzed to its hydroxylamine form in the mouse body, and the interconversion between hydroxylamine and the corresponding nitroxyl radical reflects the biological redox status. Liver damage, based on changes in liver weight and plasma aspartate aminotransferase levels, was detected in mice 4 days after X-ray irradiation at 7.5 Gy. ESR imaging showed that the signal intensity of the nitroxyl radical was high at the liver area in both damaged and healthy mice after administration of ACP. Whereas the signal decayed at the liver area for healthy mouse, the decay was negligible in damaged mice. Unlike healthy mouse, signal in the chest for damaged mouse increased with time. The distribution of the sum of hydroxylamine and the nitroxyl radical was similar in damaged and healthy mice. X-ray irradiation slightly lowered the reduction activity of the liver microsomal fraction for the nitroxyl radical. Thiobarbituric acid reactive substances in the liver were higher in damaged mice than in healthy mice; however, no significant differences were noted in reduced glutathione. The present results indicate that the redox status of mice exposed to X-ray irradiation is more oxidative than that in healthy mice.

Relaxation rates for spirocyclohexyl nitroxyl radicals are suitable for interspin distance measurements at temperatures up to about 125 K.

Between 65 and 175 K, nitroxyl radicals with spirocyclohexyl groups at the 2- and 6-positions of the piperidine ring exhibit spin echo dephasing rates that are slower than for nitroxyl radicals with 2,5-gem-dimethyl or 2,6-gem-dimethyl substituents that are currently used as spin labels, and are slow enough to permit DEER measurements at temperatures up to about 125 K for spin labels with analogous ring structures.

Enzymatic reduction-resistant nitroxyl spin probes with spirocyclohexyl rings.

To suppress enzymatic reduction of nitroxyl group of spin probes, this study designed two new nitroxyl probes, 4-hydroxy and 4-oxopiperidine-N-oxyls having 4'-hydroxyspirocyclohexyl groups at the 2- and 6-positions of the piperidine ring (hydroxy-DICPO and oxo-DICPO, respectively). The decay of the EPR signal of these probes in mouse liver homogenates was significantly suppressed compared with that of 4-hydroxy- and 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (hydroxy-TEMPO and oxo-TEMPO, respectively), although hydroxy-DICPO and oxo-DICPO showed no difference in the reactivities with ascorbic acid. While both hydroxy- and oxo-DICPO reacted with hydroxyl radicals, only hydoxy-DICPO lost its EPR signal by the reaction with superoxide anion radical in the presence of cysteine. This feature is similar to that observed for hydroxy- and oxo-TEMPO. These results suggest that the introduction of spirocyclohexyl groups to nitroxyl spin probes is effective for protecting the nitroxyl group against enzymatic reduction without changing the characteristics of the reaction with oxygen radicals.

Polymeric micelles of zinc protoporphyrin for tumor targeted delivery based on EPR effect and singlet oxygen generation.

Polymeric micelles of zinc protoporphyrin (ZnPP) with water soluble biocompatible and amphiphilic polymer, polyethylene glycol (PEG) demonstrated unique characteristics to target tumor tissues selectively based on the enhanced permeability and retention (EPR) effect. The micellar macromolecular drug of ZnPP (SMA-ZnPP and PEG-ZnPP) previously showed notable anticancer activity as a consequence of selective tumor targeting ability and its potent HO-1 inhibitory potential, resulting in suppressed biliverdin/bilirubin production in tumors thereby leading to oxystress induced tumor cell killing. Furthermore, recent findings also showed that ZnPP efficiently generated reactive singlet oxygen under illumination of visible light, laser, or xenon light source, which could augment its oxystress induced cell killing abilities. In the present paper, we report the synergistic effects of light induced photosensitizing capabilities and HO-1 inhibitory potentials of these unique micelles when tested in vitro and in vivo on tumor models under localized, mild illumination conditions using a tungsten-xenon light source. The results indicate that these water soluble polymeric micelles of ZnPP portend to be promising candidates for targeted chemotherapy as well as photodynamic therapy against superficial tumors as well as solid tumors located at light penetrable depths.

Surface-deacetylated chitin nanofibers reinforced with a sulfobutyl ether ß-cyclodextrin gel loaded with prednisolone as potential therapy for inflammatory bowel disease.

Surface-deacetylated chitin nanofibers (SDACNFs) reinforced with a sulfobutyl ether ß-cyclodextrin (SBE-ß-CD) (NFs-CDs) gel were developed to obtain a controlled release carrier of prednisolone (PD) for the treatment of colitis. PD was released slowly from the gel at both pH 1.2 and 6.8. The in vitro slow release of PD from the NFs-CDs gel was reflected in the in vivo absorption of the drug after oral administration to rats. These results suggest that a simple gel composed of a mixture of SDACNFs and SBE-ß-CD has the potential for use in the controlled release of PD. We also evaluated the therapeutic effects of the NFs-CDs gel containing PD on dextran sulfate sodium (DSS)-induced colitis model mice. The administration of the NFs-CDs gel at intervals of 3days from the beginning of the DSS treatment resulted in a significant improvement, not only in colitis symptoms but also histopathological changes in colon tissue. In addition, the therapeutic effects of the NFs-CDs gel on colitis can be attributed to decreased levels of neutrophil infiltration and the development of oxidative stress. These efficacy profiles of the NFs-CDs gel containing PD suggest that it has the potential for use in the treatment of, not only colitis, but also a variety of other disorders associated with inflammation and oxidative injuries.

In vivo generation of free radicals in the skin of live mice under ultraviolet light, measured by L-band EPR spectroscopy.

Although free radicals may be involved in various types of UV-induced injuries, only a few in vivo studies of the generation of free radicals, including oxygen radicals, during exposure to ultraviolet light (UV) have been reported. In this study, the nitroxyl probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl was intravenously injected into hairless mice, and its decay was monitored in the skin with an in vivo EPR spectrometer equipped with a surface-coil-type resonator. The rate of decay of the EPR signal increased during UV (UVA+B) irradiation. This increase in signal decay was suppressed by preadministration of a spin trap, N-tert-butyl-alpha-phenylnitrone (PBN). PBN did not change the rate of signal decay in nonirradiated mice. The correlation between signal decay rate and physiological parameters such as blood velocity, blood mass, or skin temperature was low. The decay rate responded rapidly and reversibly to starting and stopping the UV illumination. Hydroxyl and peroxyl radicals caused reduction of the probe signal in vitro, and PBN inhibited only the peroxyl radical-induced signal reduction. These observations suggest that peroxyl radicals are generated in the skin of live mice during UVA+B irradiation.

Comparison of stable nitroxide, 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyls, with respect to protection from radiation, prevention of DNA damage, and distribution in mice.

We compared three 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyls (PROXYLs): carbamoyl-, methoxycarbonyl-, and hydroxymethyl-PROXYL (CM-, MC-, and HM-PROXYL, respectively) with respect to radioprotection, prevention of DNA damage, and in vivo distribution in mice. The PROXYLs provided protection to C3H mice against lethal X-irradiation (8 Gy) with the following order of magnitude, HM- > CM- approximately MC-PROXYL. In contrast, radioprotection at the cellular level assessed by the colony formation of leukemia cell line L5178Y showed no difference among them. The degree of protection from X ray-induced oxidation of DNA bases measured by the formation of 8-hydroxydeoxyguanosine in salmon DNA and the cleavage of DNA measured by electrophoresis of plasmid pBR322 DNA did not differ among the PROXYLs. Redox potentials were also similar for each. However, the blood concentration of the PROXYLs injected ip into the mice showed different maximum concentrations (HM- > CM- approximately MC-PROXYL), although all reached a maximum at around 5-10 min and gradually decreased thereafter. Their concentration in bone marrow showed a similar pattern, suggesting that the difference in in vivo radioprotection among the three PROXYLs is due to the difference in their distribution to bone marrow. In general, the radioprotection provided by stable nitroxides is affected not only by redox potential and reactivity in vitro but also by pharmacokinetics.

Reaction of para-hydroxybenzoic acid esters with singlet oxygen in the presence of glutathione produces glutathione conjugates of hydroquinone, potent inducers of oxidative stress.

The determination and toxicological characterization of products of the reaction between p-hydroxybenzoic acid esters (parabens) and singlet oxygen ((1)O(2)) are very important because of the frequent use of parabens in cosmetics and possible generation of (1)O(2) in the skin. We observed (1)O(2)-dependent production of mono-, di-, and tri-substituted glutathione (GSH) conjugates of hydroquinone (HQ) during visible light-irradiation of a mixture of methyl or ethyl paraben and GSH in the presence of rose bengal (RB). 1,4-Benzoquinone (BQ) and HQ were produced during the irradiation in the absence of GSH. While a mixture of BQ and GSH produced only mono-substituted conjugate, irradiation of the mixture with RB produced mono-, di-, and tri-substituted conjugates. These observations indicate that (1)O(2) is involved both in the production of BQ and HQ from parabens and in the formation of multi-substituted GSH conjugates from mono-substituted conjugate. Tri-substituted conjugate generated larger amounts of hydrogen peroxide in an aqueous solution than mono-substituted conjugates or HQ did. Detection of semiquinone radical suggests that the autoxidation of conjugates is related to the generation of hydrogen peroxide. The results obtained in this study indicate that parabens may induce oxidative stress in the skin after conversion to GSH conjugates of HQ by reacting with (1)O(2) and GSH.

Pharmacokinetics of lipophilically different 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyl radicals frequently used as redox probes in in vivo magnetic resonance studies.

3-Carboxy-, 3-carbamoyl-, 3-hydroxymethyl, and 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl radicals (CxP, CmP, HMP, and MCP, respectively) have been widely used as redox probes in in vivo magnetic resonance studies. Knowledge of the pharmacokinetics of these probes is essential for redox analyses. The apparent partition coefficient (Kp) of these probes at neutral pH was in the order of MCP>HMP>CmP>CxP. After these probes had been injected intravenously, their blood levels decayed in a bi-phasic manner, namely, fast decay followed by slow decay. The order of the area under the curve (AUC) was CxP¼HMP>MCP≥CmP, which roughly coincided with that of Kp in the opposite direction, except for CmP. Decay in the slow phase largely affected the AUC of these probes. The reduction of these probes contributed to their decay in the slow phase. A two-compartment model analysis of blood levels, cyclic voltammetry, and magnetic resonance imaging provided the following pharmacokinetic information. The distribution of the probes between the central and peripheral compartments rapidly reached an equilibrium. In addition to lipophilicity, reduction potential may also be involved in the rate of in vivo reduction of the probes. Hydrophilic probes, such as CxP and CmP, were predominantly excreted in the urine. MCP was distributed to the peripheral tissues and then rapidly reduced. HMP was unique due to its moderate lipophilicity and slower reduction. Among the probes examined, the liver and kidney appear to be included in the central compartment in the two-compartment model analysis. MCP and HMP were rapidly distributed to the brain.