Sonodynamically-induced Anticancer Effects of Polyethylene Glycol-Modified Carbon Nano Tubes.

BACKGROUND/AIM: Sonodynamic cancer therapy is based on the preferential uptake and/or retention of a sonosensitizing drug (sonosensitizer) in tumor tissues and the subsequent activation of the drug by ultrasound irradiation. In the present study, we investigated the sonodynamically-induced antitumoral effect with functionalized carbon nanotubes, such as poly-ethylene glycol-modified carbon nanotubes (PEG-modified CNTs). MATERIALS AND METHODS: Antitumor effects were evaluated using histological observation and assessing tumor growth following sonodynamic exposure to PEG-modified CNTs. RESULTS: The combined treatment of 100 µM PEG-modified CNT and ultrasound induced a 2-fold cytotoxicity. Sodium azide, which quenches singlet oxygen, significantly inhibited ultrasonication induced cell damage in the presence of PEG-modified CNTs. This suggests that singlet oxygen produced by the combined use of PEG-modified CNTs and ultrasound is involved in the induction of antitumoral effects. The destruction of tumor tissue was observed with the ultrasonic treatment in combination with PEG-modified CNTs, while neither the treatment with PEG-modified CNTs alone nor ultrasound alone caused any necrosis. CONCLUSION: These results indicate that PEG-modified CNT functions as a sonosensitizer and is effective for sonochemical treatment of solid tumors.

Induction of Apoptosis by Functionalized Fullerene-based Sonodynamic Therapy in HL-60 cells.

Ultrasound has been widely utilized for medical diagnosis and therapy due to its ability to penetrate deep-seated tissue with less attenuation of energy and minimal undesirable side-effects. Functionalized fullerenes, such as polyhydroxy fullerene (PHF), have attracted particular attention due to their water solubility and potential application in tumor imaging and therapy as carbon nanomaterials. The present study investigated sonodynamically-induced apoptosis using PHF. Cell suspensions were treated with 2-MHz continuous ultrasound in the presence of PHF for 3 min and apoptosis was assessed by cell morphology using confocal microscopy, fragmentation of DNA (ladder pattern after agarose-gel electrophoresis) and caspase-3 activation. Cells were ultrasound-irradiated from the bottom of the culture dishes under the following condition: frequency, 2 MHz; output power, 3 W/cm(2) Electron spin resonance was used to measure reactive oxygen species. The number of apoptotic cells after sonodynamic exposure (ultrasound and PHF) was significantly higher than produced from other treatments, such as ultrasound alone and PHF alone. Furthermore, DNA fragmentation, caspase-3 activation and enhanced 2,2,6,6-tetramethyl-4-piperidinyloxy (4oxoTEMPO) formation were observed in the sonodynamically-treated cells. Histidine, a well-known reactive oxygen scavenger, significantly inhibited sonodynamically-induced apoptosis, caspase-3 activation and 4oxoTEMPO formation. Sonodynamic therapy with PHF induced apoptosis that was characterized by a series of typical morphological features, such as shrinkage of the cell and fragmentation into membrane-bound apoptotic bodies, in HL-60 cells. The significant inhibition of sonodynamically-induced apoptosis, caspase-3 activation, and 4oxoTEMPO formation due to histidine and tryptophan suggests that reactive oxygen species, such as singlet oxygen, are involved in the sonodynamic induction of apoptosis. These findings indicate that PHF-mediated sonodynamic therapy can trigger caspase-dependent apoptosis and oxidative injury, thus possibly playing a vital role in apoptotic signaling cascades.

Sonodynamically-induced anticancer effects by functionalized fullerenes.

BACKGROUND: Functionalized fullerenes, such as polyhydroxy fullerenes (PHF), have attracted particular attention due to their water solubility and their potential application in tumor imaging and therapy as carbon nanomaterials. In this study, the sonodynamically-induced antitumor effect of PHF was investigated. MATERIALS AND METHODS: Sonodynamically-induced antitumor effects of PHF in combination with ultrasound were investigated using isolated sarcoma 180 cells and solid tumor from colon 26 carcinoma cells. RESULTS: The cell damage induced by sonication was enhanced by two-fold in the presence of 80 µM PHF. Histidine significantly inhibited this enhancement. This inhibitory effect suggests that the sonodynamically-induced antitumor effect was mediated by sonodynamically-generated reactive oxygen species. The combined treatment of ultrasonic exposure with PHF suppressed the growth of implanted colon 26 tumors. The destruction of tumor tissue was observed with the ultrasonic treatment in combination with PHF, while neither the treatment with PHF alone nor that with ultrasound alone caused necrosis. CONCLUSION: These results suggest that PHF is a potential sonosensitizer for sonodynamic treatment of solid tumors.

Effects of acetaldehyde on action potentials and Ca2+ currents in single atrial myocytes from the bullfrog.

AIM: The purpose of the present study was to examine the effects of acetaldehyde on the contractile force and membrane potentials and currents in the bullfrog heart. METHODS: Contractile force was recorded using right atrial tissues, and membrane potentials and currents were measured by using whole cell patch clamp methods in right atrial myocytes. RESULTS: Acetaldehyde at 500 µmol/l and 1 mmol/l increased the contractile force significantly. Acetaldehyde at 300 and 500 µmol/l increased the overshoot and the plateau of electrically induced action potentials in a concentration-dependent and reversible manner, while the resting membrane potential did not change. The duration of the action potential (APD(90)) measured at the 90% repolarization level was shortened. The L-type Ca(2+) current (I(Ca)) increased significantly when 300 and 500 µmol/l were applied. The fast transient inward current, the inward rectifying potassium current and the outward delayed-rectifier potassium current were not changed following acetaldehyde application (500 µmol/l or 1 mmol/l). CONCLUSION: These results suggest that acetaldehyde increased the I(Ca), thereby increased the contractile force, the overshoot and the plateau of action potentials. The shortening of APD(90) may be due to the acceleration of the current decay during the I(Ca) inactivation phase.

Involvement of reactive oxygen species in sonodynamically induced apoptosis using a novel porphyrin derivative.

In this study, we investigated the induction of apoptosis by ultrasound in the presence of the novel porphyrin derivative DCPH-P-Na(I). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of DCPH-P-Na(I), and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Reactive oxygen species were measured by means of ESR and spin trapping technique. Cells treated with 8 µM DCPH-P-Na(I) and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or DCPH-P-Na(I) alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and DCPH-P-Na(I) but not in cells treated with ultrasound or DCPH-P-Na(I) alone. In addition, the combination of DCPH-P-Na(I) and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results indicate that the combination of ultrasound and DCPH-P-Na(I) induced apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

[Mechanism of alcohol consumption-mediated Th2-polarized immune response].

Alcohol consumption impairs Th1-mediated cellular immune responses and enhances serum IgE levels. It has been reported that the elevated IgE levels are associated with a Th2 polarization response, but the mechanisms for enhancing Th2 polarization by the ethanol treatment remain to be elucidated. The aim of this review is to present and discuss the mechanism of Th2 polarization response by alcohol. IL-12 production by APCs such as monocytes, macrophages, and dendritic cells (DCs) preferentially leads to Th1 polarization. Acute ethanol consumption results in a significant decrease in IL-12 production in LPS-stimulated DCs and a CD40/CD40L interaction between CD40 on the DCs and CD40 ligand expressed on activated T cells. This suggests that Th2 polarization by ethanol is caused by impaired IL-12 production from APCs. In contrast, the induction of IL-10 by LPS is enhanced by ethanol treatment, suggesting that elevated IL-10 may play a role in ethanol-induced suppression of IL-12. However, ethanol inhibited IL-12 production in LPS-stimulated DCs devoid of IL-10 (IL-10/DC), suggesting that down-regulation of IL-12 by ethanol is independent of the IL-10 levels. Furthermore, several studies report that PGE2, cAMP and linolic acid, and endogenous lipid mediators released in inflammatory conditions, also inhibit IL-12 production. These inhibitory effects are similar to the IL-12 inhibition by ethanol. In addition, increase in the levels of these lipid mediators is induced by ethanol treatment. Alternatively, cytokine signaling studies indicate that IL-12 production by DCs is negatively regulated by PI3K and GSK-3, but positively regulated by p38 MAPK, mTOR, and NF-kappa B. Thus, it seems possible that ethanol may interact on the upstream of IL-12 producing a signal pathway. In fact, ethanol alters the stability of cell membrane, and suppresses clustering of TLR4 and recruitment of signaling molecules into lipid rafts, where it associates with the Ser/Thr kinase and the adaptor proteins, and forms a signaling complex. Down-regulation of lipid raft signaling is results in the impaired IL-12 production leading to the Th1 polarization, and causes CD4+ T cells to differentiation toward the Th2 lineage.

Sonodynamically-induced antitumor effect of mono-l-aspartyl chlorin e6 (NPe6).

BACKGROUND: The sonodynamically-induced in vitro and in vivo antitumor effects of mono-l-aspartyl chlorin e6 (NPe6) was investigated. MATERIALS AND METHODS: Both in vitro and in vivo antitumor effects were tested in combination with ultrasound at 2 MHz. RESULTS: The rate of ultrasonically-induced damage on isolated sarcoma 180 cells in air-saturated suspension was enhanced two-fold with 80 µM NPe6. The co-administration of 25 mg/kg NPe6 followed by ultrasonic exposure at 2 MHz suppressed the growth of implanted colon 26 cell tumors at an intensity at which ultrasound alone showed only a slight antitumor effect. CONCLUSION: These in vitro and in vivo results suggest that NPe6 is a potential sensitizer for sonodynamic tumor treatment. The enhancement of cell damage by NPe6 was significantly inhibited by histidine, which may suggest reactive oxygen species plays a primary role in sonodynamically-induced antitumor effect.

Quantitative analysis of crystalline pharmaceuticals in tablets by pattern-fitting procedure using X-ray diffraction pattern.

A pattern-fitting procedure using an X-ray diffraction pattern was applied to the quantitative analysis of binary system of crystalline pharmaceuticals in tablets. Orthorhombic crystals of isoniazid (INH) and mannitol (MAN) were used for the analysis. Tablets were prepared under various compression pressures using a direct compression method with various compositions of INH and MAN. Assuming that X-ray diffraction pattern of INH-MAN system consists of diffraction intensities from respective crystals, observed diffraction intensities were fitted to analytic expression based on X-ray diffraction theory and separated into two intensities from INH and MAN crystals by a nonlinear least-squares procedure. After separation, the contents of INH were determined by using the optimized normalization constants for INH and MAN. The correction parameter including all the factors that are beyond experimental control was required for quantitative analysis without calibration curve. The pattern-fitting procedure made it possible to determine crystalline phases in the range of 10-90% (w/w) of the INH contents. Further, certain characteristics of the crystals in the tablets, such as the preferred orientation, size of crystallite, and lattice disorder were determined simultaneously. This method can be adopted to analyze compounds whose crystal structures are known. It is a potentially powerful tool for the quantitative phase analysis and characterization of crystals in tablets and powders using X-ray diffraction patterns.

Sonodynamically induced cell damage and membrane lipid peroxidation by novel porphyrin derivative, DCPH-P-Na(I).

BACKGROUND: Ultrasonically induced cell damage and active oxygen generation with a novel porphyrin derivative DCPH-P-Na(I), were compared in the same in vitro insonation setup. MATERIALS AND METHODS: Sarcoma 180 cells suspended in air-saturated PBS were exposed to ultrasound at 2 MHz for up to 60 s in the presence and absence of DCPH-P-Na(I). Cell viability was determined with the trypan blue exclusion test. Lipid peroxidation in cell membranes was estimated by measuring the amount of reactive substance produced immediately following the addition of thiobarbituric acid. RESULTS: Significant enhancement of the rates of both ultrasonically induced cell damage and lipid peroxidation was observed in the presence of 2-8 muM DCPH-P-Na(I). Both rates correlated very well. CONCLUSION: The enhancement of both rates with DCPH-P-Na(I) was suppressed by 10 mM histidine. These results suggest that ultrasonically generated active oxygen plays a primary role in the ultrasonically induced cell damage in the presence of DCPH-P-Na(I).

Sonodynamically induced apoptosis and active oxygen generation by gallium-porphyrin complex, ATX-70.

In this study, we investigated the induction of apoptosis by ultrasound in the presence of the photochemically active gallium-porphyrin complex, 7,12-bis(1-decyloxyethyl)-Ga(III)-3,8,13,17-tetramethyl-porphyrin 2,18-dipropionyl diaspartic acid (ATX-70). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of ATX-70, and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Cells treated with 80 µM ATX-70 and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or ATX-70 alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and ATX-70 but not in cells treated with ultrasound or ATX-70 alone. In addition, the combination of ATX-70 and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results indicate that the combination of ultrasound and ATX-70 induces apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests that active species such as singlet oxygen are important in the sonodynamic induction of apoptosis.

Ca2+ extrusion via Na+-Ca2+ exchangers in rat odontoblasts.

INTRODUCTION: Intracellular Ca(2+) is essential to many signal transduction pathways, and its level is tightly regulated by the Ca(2+) extrusion system in the plasma membrane, which includes the Na(+)-Ca(2+) exchanger (NCX). Although expression of NCX1 isoforms has been demonstrated in odontoblasts, the detailed properties of NCX remain to be clarified. In this study, we investigated localization and ion-transporting/pharmacologic properties of NCX isoforms in rat odontoblasts. METHODS: We characterized both the reverse and forward modes of NCX activity in odontoblasts in a dental pulp slice preparation. Ca(2+) influx by reverse NCX activity was measured by fura-2 fluorescence. Ca(2+) efflux by forward NCX activity elicited inward Na(+) current as measured by perforated-patch clamp recording. For immunohistochemical analysis, cryostat sections of incisors were incubated with antibodies against NCX. RESULTS: Immunohistochemical observation revealed localization of NCX1 and NCX3 in the distal membrane of odontoblasts. Inward currents by forward NCX activity showed dependence on external Na(+). Fura-2 fluorescence measurement revealed that Ca(2+) influx by reverse NCX activity depended on extracellular Ca(2+) concentration, and that this influx was blocked by NCX inhibitor KB-R7943 in a concentration-dependent manner. However, Ca(2+) influx by NCX showed a slight sensitivity to SEA0400 (a potent NCX1 inhibitor), indicating that expression potencies in odontoblasts were NCX3 > NCX1. CONCLUSIONS: These results suggest that odontoblasts express NCX1 and NCX3 at the distal membrane, and that these isoforms play an important role in the Ca(2+) extrusion system as well as in the directional Ca(2+) transport pathway from the circulation to the dentin-mineralizing front.

A liquid chromatography/tandem mass spectrometry method for detecting UGT-mediated bioactivation of drugs as their N-acetylcysteine adducts in human liver microsomes.

The detection of the reactive metabolites of drugs has recently been gaining increasing importance. In vitro trapping studies using trapping agents such as glutathione are usually conducted for the detection of reactive metabolites, especially those of cytochrome P450-mediated metabolism. In order to detect the UDP-glucuronosyltransferase (UGT)-mediated bioactivation of drugs, an in vitro trapping method using N-acetylcysteine (NAC) as a trapping agent followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed in this study. After the test compounds (diclofenac and ketoprofen) had been incubated in human liver microsomes with uridine diphosphoglucuronic acid (UDPGA) and NAC, the NAC adducts formed through their acyl glucuronides were analyzed using LC/MS/MS with electrospray ionization (ESI). The NAC adduct showed a mass shift of 145 units as compared to its parent, and the characteristic ion fragmentations reflected the parent. This is a concise and high-throughput method for evaluating reactive metabolites by UGT-mediated bioactivation.

Electrophysiological properties of AQP6 in mouse parotid acinar cells.

Salivary gland acinar cells secrete large amounts of water and electrolytes, where aquaporins (AQPs) are thought to be involved in the secretion. In the present study, we investigated expression/localization of AQP6, and the anion transporting properties of AQP6 in mouse parotid acinar cells. RT-PCR, western blotting and immunohistochemical analyses revealed expression of AQP6 in acinar cells, localized in apical membrane. Voltage ramp from -100 mV to +100 mV at a holding potential of -60 mV elicited outwardly-rectifying currents, in the presence of extracellular Cl(-) channel blockers and intracellular solution with 150 mM Cs(+). These outward currents were increased when extracellular Cl(-) was replaced by Br(-), NO(3)(-), I(-), or SCN(-), accompanying a negative shift of reversal potentials. The outward current was enhanced by extracellular Hg(2+). These results were consistent with the biophysical properties of transfected AQP6 oocytes or HEK cells, which indicate that the AQP6 channel is functionally expressed in parotid acinar cells, and suggest that AQP6 contributes to secretion of anions in parotid acinar cells.

Genetic polymorphisms of CYP2A6 affect the in-vivo pharmacokinetics of pilocarpine.

OBJECTIVE: Pilocarpine is metabolized to pilocarpic acid by plasma esterase and to 3-hydroxypilocarpine by CYP2A6. The objective of this study was to identify the determinants of interindividual differences in pilocarpine pharmacokinetics after oral administration and to investigate the possible involvement of genetic polymorphisms of CYP2A6. METHODS: The pharmacokinetic parameters of pilocarpine, pilocarpic acid, and 3-hydroxypilocarpine after oral administration of pilocarpine hydrochloride in 28 Japanese participants were calculated based on the data obtained from two phase-1 clinical studies. Probit analysis was carried out for the pharmacokinetic parameters, and participants were accordingly classified into two groups: poor metabolizers and nonpoor metabolizers of pilocarpine. The poor metabolizers were genotyped for CYP2A6, and the pharmacokinetic parameters in this group were compared with those in the nonpoor metabolizers. RESULTS: Relatively large interindividual differences were observed in the pharmacokinetic parameters of pilocarpine, pilocarpic acid, and 3-hydroxypilocarpine. Probit analysis of the pharmacokinetic parameters revealed that seven of the 28 participants exhibited significantly low plasma concentrations and urinary recovery of 3-hydroxypilocarpine and were classified as poor metabolizers. Genotyping analysis revealed that these poor metabolizers had two inactive CYP2A6 alleles, CYP2A6*4A, CYP2A6*7, CYP2A6*9, or CYP2A6*10. The apparent pilocarpine clearance was significantly lower in the poor metabolizers than in the nonpoor metabolizers (P<0.05). CONCLUSION: We demonstrated that CYP2A6 genotype is a contributor to pilocarpine pharmacokinetics, although the impact of the CYP2A6 polymorphism may be pharmacologically and toxicologically tolerable.

Hypotensive and hypertensive effects of acetaldehyde on blood pressure in rats.

Intravenous injection of acetaldehyde produced hypotensive actions in pentobarbital-anaesthetised whole rats, but hypertensive actions in pithed rats. The hypotensive effects of acetaldehyde in whole rats were abolished by pre-treatment with yohimbine. In pithed rats, the hypertensive effects of acetaldehyde were significantly attenuated by prazosin and phentolamine, and in rats that had been pre-treated with reserpine. Our results suggest that the hypertensive actions of acetaldehyde in pithed rats are due to the release of catecholamines, which subsequently leads to vasoconstriction. In whole rats the hypotensive actions of acetaldehyde may be due to alpha2-adrenoceptor stimulation in the central nervous or peripheral system.

Prediction of aminoglycoside response against methicillin-resistant Staphylococcus aureus infection in burn patients by artificial neural network modeling.

OBJECTIVE: To predict the response of aminoglycoside antibiotics (arbekacin: ABK) against methicillin-resistant Staphylococcus aureus (MRSA) infection in burn patients after considering the severity of the burn injury by using artificial neural network (ANN). Predictive performance was compared with logistic regression modeling. METHODOLOGY: The physiologic data and some indicators of the severity of the burn injury were collected from 25 burn patients who received ABK against MRSA infection. A three-layered ANN architecture with six neurons in the hidden layer was used to predict the ABK response. The response was monitored using three clinical criteria: number of bacteria, white blood cell count, and C-reactive protein level. Robustness of models was investigated by the leave-one-out cross-validation. RESULTS: The peak plasma level, serum creatinine level, duration of ABK administration, and serum blood sugar level were selected as the linear input parameters to predict the ABK response. The area of the burn after skin grafting was the best parameter for assessing the severity of the burn injury in patients to predict the ABK response in the ANN model. The ANN model with the severity of the burn injury was superior to the logistic regression model in terms of predicting the performance of the ABK response. CONCLUSION: Based on the patients' physiologic data, ANN modeling would be useful for the prediction of the ABK response in burn patients with MRSA infection. Severity of the burn injury was a parameter that was necessary for better prediction.

Effects of thiopental on Ca2+ currents and intracellular Ca2+ transient in single atrial cells from guinea pig.

The effects of thiopental on Ca2+ currents and intracellular Ca2+ transient in single atrial cells from guinea pigs were studied by means of a whole-cell voltage-clamp method and Ca2+-sensitive fluorescent dye. Thiopental inhibited L-type voltage-dependent Ca2+ currents in a concentration-dependent manner (IC50=2.8.10(-5) mol/l). Moreover, the mode of Ca2+ current inhibition by thiopental showed no use dependency. Electrical stimulation-induced intracellular Ca2+ transient was significantly suppressed by 10(-5) mol/l thiopental. However, the caffeine-sensitive Ca2+ releasing pathway from sarcoplasmic reticulum was not affected by thiopental. Our results indicate that thiopental inhibits L-type Ca2+ currents, but not release of Ca2+ from sarcoplasmic reticulum. These results suggest that the negative inotropic action of thiopental is mainly due to inhibition of L-type Ca2+ channels in guinea pig atrial myocytes.

Crystallization kinetics of amorphous griseofulvin by pattern fitting procedure using X-ray diffraction data.

PURPOSE: A pattern fitting procedure using X-ray powder diffraction patterns was applied to study the crystallization kinetics of amorphous griseofulvin. From the optimized parameters obtained by pattern fitting, a change in the quantity and quality of griseofulvin crystals with crystallization was also investigated. MATERIALS AND METHODS: Amorphous griseofulvin was prepared by cooling the melts followed by pulverization. X-ray diffraction patterns of amorphous griseofulvin were repeatedly measured every 20 h. The observed pattern was separated into crystalline diffraction intensity and amorphous scattering intensity by the nonlinear least-squares procedure. RESULTS: The fitting between the observed and simulated diffraction patterns was satisfactorily independent of the degree of crystallinity. Since a good linear relationship was found in a plot of amorphous scattering intensity against crystalline diffraction intensity, the degree of crystallinity can be determined according to Hermans' method. The diffraction peak width increased with higher diffraction angles with crystallization. The crystallization was biphasic: fast and slow crystallization with the growth of low disordered crystals and disordered crystals, respectively. CONCLUSION: The pattern fitting procedure is a powerful tool to analyze the X-ray diffraction patterns of semicrystalline materials. This procedure can simultaneously analyze the degree of crystallinity and crystal disorder in semicrystalline samples during crystallization.

Involvement of CYP2A6 in the formation of a novel metabolite, 3-hydroxypilocarpine, from pilocarpine in human liver microsomes.

Pilocarpine is a cholinergic agonist that is metabolized to pilocarpic acid by serum esterase. In this study, we discovered a novel metabolite in human urine after the oral administration of pilocarpine hydrochloride, and we investigated the metabolic enzyme responsible for the metabolite formation. The structure of the metabolite was identified as 3-hydroxypilocarpine by liquid chromatography-tandem mass spectrometry and NMR analyses and by comparing to the authentic metabolite. To clarify the human cytochrome P450 (P450) responsible for the metabolite formation, in vitro experiments using P450 isoform-selective inhibitors, cDNA-expressed human P450s (Supersomes; CYP1A2, -2A6, -2B6, -2C9, -2C19, -2D6, -2E1, and -3A4), and liver microsomes from different donors were conducted. The formation of 3-hydroxypilocarpine in human liver microsomes was strongly inhibited (>90%) by 200 microM coumarin. Other selective inhibitors of CYP1A2 (furafylline and alpha-naphthoflavone), CYP2C9 (sulfaphenazole), CYP2C19 [(S)-mephenytoin], CYP2E1 (4-methylpyrazole), CYP2D6 (quinidine), and CYP3A4 (troleandomycin) had a weak inhibitory effect (<20%) on the formation. The highest formation activity was expressed by recombinant CYP2A6. The K(m) value for recombinant CYP2A6 was 3.1 microM, and this value is comparable with that of human liver microsomes (1.5 microM). The pilocarpine 3-hydroxylation activity was correlated with coumarin 7-hydroxylation activity in 16 human liver microsomes (r = 0.98). These data indicated that CYP2A6 is the main enzyme responsible for the 3-hydroxylation of pilocarpine. In conclusion, we identified a novel metabolite of pilocarpine, 3-hydroxypilocarpine, and we clarified the involvement of CYP2A6 in the formation of this molecule in human liver microsomes.

[Pharmacokinetics and disposition of silodosin (KMD-3213)].

After a single oral dose of silodosin in male rats, male dogs and healthy human male volunteers, C(max) occurred within about 2 h, indicating rapid absorption. The elimination half-life was about 2 h in rat and dog, but 4.7 h (fasted) and 6.0 h (non-fasted) in humans. Absolute bioavailability values in rat, dog and human were about 9, 25 and 32%, respectively. In rat and dog, total blood clearance was almost equivalent to the hepatic blood flow, but that in human was low (20%), demonstrating a large species difference in hepatic clearance. In each species, the apparent volume of distribution exceeded the volume of total body water. After an oral dose of (14)C-silodosin to male rats, radioactivity was rapidly and widely distributed to most tissues. The highest concentrations outside the gastrointestinal tract were found in liver and kidney, with only low concentrations in brain tissues. The in vitro plasma protein binding of silodosin was about 80% in rat and dog, and 95.6% in humans, with alpha(1)-acid glycoprotein (AGP) contributing to the binding profile. Silodosin was found to be a dual substrate for CYP3A4 and p-glycoprotein. In human plasma, two major metabolites generated by UDP-glucuronosyltransferase (UGT; UGT2B7) and alcohol/aldehyde dehydrogenase (ADH/ALDH) were found, but no glucuronide conjugates were detected in rat or dog plasma. After a single oral dose of (14)C-silodosin in rat, dog and human, the urinary excretion of radioactivity was 15-34%, with that of unchanged silodosin being less than 4%. The radioactivity was predominantly excreted via the feces.