RESUMO
Cytochrome P450 (CYP) 2J2 is responsible for the epoxidation of arachidonic acid, producing epoxyeicosatrienoic acids (EETs) that are known to enhance tumorigenesis. CYP2J2 is prominently expressed in the heart and also found in the lungs. Furthermore, the expression level of CYP2J2 in tumour tissues is higher than that in adjacent normal tissues. Non-small cell lung carcinoma is a common cancer, and tyrosine kinase inhibitors (TKIs) are powerful tools for its treatment. This study aimed to elucidate the inhibitory effects of 17 TKIs on CYP2J2 activity using LC-MS/MS.Seventeen TKIs exhibited different inhibitory effects on CYP2J2-catalysed astemizole O-demethylation in recombinant CYP2J2. Pralsetinib and selpercatinib showed strong competitive inhibition, with inhibition constant values of 0.48 and 1.1 µM, respectively. They also inhibited other CYP2J2 activities, including arachidonic acid epoxidation, hydroxyebastine carboxylation, and rivaroxaban hydroxylation.In conclusion, we showed that pralsetinib and selpercatinib strongly inhibit CYP2J2 activity. Inhibition of 14,15-EET production by these TKIs may be a novel mechanism for suppressing tumour growth and proliferation. Additionally, when these TKIs are co-administered with a CYP2J2 substrate, we may consider the possibility of drug-drug interactions via CYP2J2 inhibition.
RESUMO
Single-walled carbon nanotubes (SWCNTs) are made from rolled single graphene sheets with a diameter in the nanometer range and are potential carriers for drug delivery systems. However, their effects on uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A activities remain unclear. The present study aimed to investigate the effect of two kinds of SWCNTs (EC1.5-P- and FH-P-SWCNTs) and other nanocarbons on human UGT1A activity due to the proposed application of SWCNTs in drug and gene delivery. ß-Estradiol 3-glucuronidation, which is catalyzed mainly by UGT1A1, was inhibited by 99 and 76% in the presence of 0.1 mg/mL EC1.5-P- and FH-P-SWCNTs in human liver microsomes, respectively. The observed decrease of free UGT1A1 protein in the enzyme reaction mixture suggests a higher interaction with SWCNTs, and indicates the inhibition of ß-estradiol 3-glucuronidation. Imipramine N-glucuronidation, which is formed mainly by UGT1A4, was also decreased by SWCNTs. Serotonin glucuronidation, which is mainly responsible for UGT1A6, was only influenced by specific nanocarbons in human liver microsomes. The attenuation of free UGT1A6 protein was observed with SWCNTs and carbon black, indicating that UGT1A6 activity was not influenced by the direct interaction of SWCNTs. We also observed a 127% increase by FH-P-SWCNTs for propofol glucuronidation in human liver microsomes, which is catalyzed mainly by UGT1A9. The values of maximum velocity and intrinsic clearance for propofol glucuronidation in the presence of FH-P-SWCNT were 1.8- and 2.0-fold higher than those of the control in human liver microsomes. These results suggest that the effects of SWCNTs on UGT1A are different among isoforms.
Assuntos
Nanotubos de Carbono , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Fígado/metabolismo , Difosfato de UridinaRESUMO
Regorafenib is glucuronidated mainly by uridine 5'-diphosphate glucuronosyltransferase (UGT) 1A9 in humans. UGT1A9 and its orthologues are expressed in the liver, small intestine, and kidney in humans and laboratory animals. The aim of this study was to reveal the species and tissue differences in regorafenib glucuronidation in the liver and extrahepatic tissues of humans and laboratory animals.Regorafenib glucuronidation was fitted to the Michaelis-Menten model in humans, monkeys, and mice using liver, kidney, and small intestine tissue. The hepatic results indicated monophasic kinetics in all species except rats, in which glucuronide could not be detected because rat Ugt1a9 is a pseudogene.The maximum velocity was higher in monkeys (3.41 pmol/min/mg) than in humans (1.21 pmol/min/mg), but was similar between humans and mice (1.11 pmol/min/mg). The maximum velocity in the kidney was higher than that in the liver in both humans and monkeys. Regorafenib glucuronide was not quantified in the kidneys of mice. Small intestinal regorafenib glucuronidation was not detected in any of the species. It is surmised that the degree of regorafenib glucuronidation is dependent on UGT1A9 expression levels.Our study clarified the species and tissue differences in regorafenib glucuronidation in the liver and extrahepatic tissues.
Assuntos
Glucuronídeos , Microssomos Hepáticos , Animais , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Cinética , Camundongos , Microssomos Hepáticos/metabolismo , Compostos de Fenilureia , Piridinas , Ratos , Especificidade da Espécie , UDP-Glucuronosiltransferase 1ARESUMO
1. Cytochrome P450 2J2 (CYP2J2) shows high expression in extrahepatic tissues, including the heart and kidney and in tumours. Inhibition of CYP2J2 has attracted attention for cancer treatment because it metabolises arachidonic acid (AA) to epoxyeicosatrienoic acid (EET), which inhibits apoptosis and promotes tumour growth. Multi-kinase inhibitor (MKI) is a molecular-targeted drug with antitumor activities. This study aimed to clarify the inhibitory effects of MKIs on CYP2J2 activity. We also investigated whether MKIs affected CYP2J2-catalysed EET formation from AA.2. Twenty MKIs showed different inhibitory potencies against astemizole O-demethylation in CYP2J2. In particular, apatinib, motesanib, and vatalanib strongly inhibited astemizole O-demethylation. These three MKIs exhibited competitive inhibition with inhibition constant (Ki) values of 9.3, 15.4, and 65.0 nM, respectively. Apatinib, motesanib, and vatalanib also inhibited CYP2J2-catalysed 14,15-EET formation from AA.3. In simulations of docking to CYP2J2, the U energy values of apatinib, motesanib, and vatalanib were low, and measured -84.5, -69.9, and -52.3 kcal/mol, respectively.4. In conclusion, apatinib, motesanib, and vatalanib strongly inhibited CYP2J2 activity, suggesting that the effects of a given CYP2J2 substrate may be altered upon the administration of these MKIs.
Assuntos
Sistema Enzimático do Citocromo P-450RESUMO
Valproic acid (VPA) is well-known as a histone deacetylase (HDAC) inhibitor. It has been reported that HDAC inhibitors enhance basal and aryl hydrocarbon receptor (AhR) ligand-induced aryl hydrocarbon receptor-responsive gene expression. Other studies suggested that HDAC inhibition might significantly activate the NF-E2-related factor-2 (Nrf2). Moreover, VPA activates mitogen-activated protein kinases (MAPKs). MAPK pathways regulate Nrf2 transactivation domain activity. Uridine 5'-diphospho-glucuronosyltransferase (UGT) 1A6 is one of the important isoforms to affect drug pharmacokinetics. UGT1A6 gene is regulated transcriptionally by AhR and Nrf2. The present study aimed to investigate whether UGT1A6 expression was changed by VPA and to elucidate the mechanism of the alteration. Following VPA treatment for 72 h in Caco-2 cells, UGT1A6 mRNA was increased by 7.9-fold. Moreover, UGT1A6 mRNA was increased by other HDAC inhibitors, suggesting that HDAC inhibition caused the UGT1A6 mRNA induction. AhR and Nrf2 proteins in the nucleus of Caco-2 cells were increased by 1.5- and 1.7-fold, respectively, following the VPA treatment. However, VPA treatment did not activate the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways in Caco-2 cells. In conclusion, we observed that VPA induced UGT1A6 mRNA expression via AhR and Nrf2 pathways, but not via the ERK or JNK pathways.
Assuntos
Inibidores de Histona Desacetilases , Ácido Valproico , Humanos , Células CACO-2 , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , RNA Mensageiro/metabolismo , Uridina , Ácido Valproico/farmacologiaRESUMO
Despite the prevalence of laboratory animals such as monkeys, rats, and mice in clinical drug trials, we know little regarding the oxidation of regorafenib in these test subjects. This study aimed to elucidate species differences in the kinetics of regorafenib oxidation into two metabolites: regorafenib N-oxide (M-2) and hydroxyregorafenib (M-3).M-2 formation best fitted the Hill equation and showed positive cooperativity in liver and small intestinal microsomes from all species. For all species, M-2 formation had a higher maximum velocity in microsomes from the liver than the small intestines. Maximum velocity was also higher in microsomes from humans and monkeys than those from rats and mice. M-3 formation was well-fitted to the Hill equation and showed positive cooperativity in all microsomes, except those from rat small intestines, where it exhibited biphasic kinetics. At half the maximum velocity, substrate concentration for M-2 and M-3 formation was lower in microsomes from humans than from other species. Moreover, M-2 was the major metabolite in microsomes from humans, monkeys, and mice, whereas M-2 and M-3 were the major metabolites in rat microsomes.M-2 and M-3 formation involving CYP3A4 and CYP3A5 fitted to the Hill equation. However, M-3 formation involving CYP2J2 fitted to the substrate inhibition model.Our study confirmed species differences in regorafenib oxidative metabolism.
Assuntos
Microssomos , Compostos de Fenilureia , Animais , Cinética , Camundongos , Microssomos/metabolismo , Microssomos Hepáticos/metabolismo , Estresse Oxidativo , Compostos de Fenilureia/metabolismo , Piridinas , Ratos , Especificidade da EspécieRESUMO
In arsenic-endemic regions of the world, arsenic exposure correlates with diabetes mellitus. Multiple animal models of inorganic arsenic (iAs, as As3+) exposure have revealed that iAs-induced glucose intolerance manifests as a result of pancreatic ß-cell dysfunction. To define the mechanisms responsible for this ß-cell defect, the MIN6-K8 mouse ß-cell line was exposed to environmentally relevant doses of iAs. Exposure to 0.1-1 µM iAs for 3 days significantly decreased glucose-induced insulin secretion (GIIS). Serotonin and its precursor, 5-hydroxytryptophan (5-HTP), were both decreased. Supplementation with 5-HTP, which loads the system with bioavailable 5-HTP and serotonin, rescued GIIS, suggesting that recovery of this pathway was sufficient to restore function. Exposure to iAs was accompanied by an increase in mRNA expression of UDP-glucuronosyltransferase 1 family, polypeptide a6a (Ugt1a6a), a phase-II detoxification enzyme that facilitates the disposal of cyclic amines, including serotonin, via glucuronidation. Elevated Ugt1a6a and UGT1A6 expression levels were observed in mouse and human islets, respectively, following 3 days of iAs exposure. Consistent with this finding, the enzymatic rate of serotonin glucuronidation was increased in iAs-exposed cells. Knockdown by siRNA of Ugt1a6a during iAs exposure restored GIIS in MIN6-K8 cells. This effect was prevented by blockade of serotonin biosynthesis, suggesting that the observed iAs-induced increase in Ugt1a6a affects GIIS by targeting serotonin or serotonin-related metabolites. Although it is not yet clear exactly which element(s) of the serotonin pathway is/are most responsible for iAs-induced GIIS dysfunction, this study provides evidence that UGT1A6A, acting on the serotonin pathway, regulates GIIS under both normal and pathological conditions.
Assuntos
5-Hidroxitriptofano/efeitos dos fármacos , Arsênio/farmacologia , Diabetes Mellitus/metabolismo , Glucuronosiltransferase/efeitos dos fármacos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Serotonina/metabolismo , 5-Hidroxitriptofano/metabolismo , Adulto , Animais , Linhagem Celular , Feminino , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Mitocôndrias , Consumo de Oxigênio , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismoRESUMO
Thylakoid-rich spinach extract is being used as dietary weight-loss supplements in Japan. A recent rat study has suggested that intake of thylakoid-rich spinach extract with dietary oil inhibits dietary fat absorption via binding to bile acids, which promotes excretion of bile acids in feces. While, we confirmed that a serving size of thylakoid-rich spinach extract contains a large amount of calcium (130 mg/5 g). Therefore, using rats, we evaluated whether one-time ingestion of thylakoid-rich spinach extract affects the gastrointestinal absorption of water-insoluble drugs, such as griseofulvin (GF) and indomethacin (IM), or ciprofloxacin (CPFX) that chelate with polyvalent metal cations. Pretreatment of the rats with thylakoid-rich spinach extract (100 or 300 mg/kg) for 15 min prior to oral administration of GF (50 mg/kg) or IM (10 mg/kg) did not significantly alter the pharmacokinetic properties of either drug. Meanwhile, co-administration of thylakoid-rich spinach extract (500 mg/kg) and CPFX (20 mg/kg) significantly reduced the peak plasma concentration and the area under the plasma concentration-time curve of CPFX to 25 and 40%, respectively in rats. In vitro studies demonstrated that when a mixture of thylakoid-rich spinach extract and CPFX was centrifuged, there was a significant reduction in the supernatant concentration of CPFX relative to the control. When the experiment was repeated in the presence of ethylenediaminetetraacetic acid, the concentration of CPFX was unchanged. These results suggest that the intake of thylakoid-rich spinach extract may reduce the absorption of drugs that form a chelate with polyvalent metal cations, such as CPFX.
Assuntos
Interações Alimento-Droga/fisiologia , Griseofulvina/farmacocinética , Indometacina/farmacocinética , Extratos Vegetais/metabolismo , Spinacia oleracea , Tilacoides/metabolismo , Animais , Relação Dose-Resposta a Droga , Absorção Gastrointestinal/efeitos dos fármacos , Absorção Gastrointestinal/fisiologia , Masculino , Extratos Vegetais/isolamento & purificação , Ratos , Ratos WistarRESUMO
Sulfotransferase (SULT) has been found in the brain; however, the details of its function remain unclear. The present study aimed to elucidate the regional differences in the expression of SULT1 and SULT2 mRNA and SULT activities in the eight functional regions of the rat brain (cerebellum, cortex, hippocampus, medulla oblongata, midbrain, olfactory bulb, striatum, and thalamus). All SULT1 isoforms were detected in the medulla oblongata and thalamus. SULT2A1 mRNA was not observed in any of the eight regions, whereas SULT2B1a and SULT2B1b were found in all regions. The SULT2B1b mRNA expression level in the medulla oblongata was 1.7-fold higher than that in the liver. The sulfonation of p-nitrophenol and pregnenolone was detected in all regions. The kinetics of p-nitrophenol sulfonation in the cerebellum fitted to the substrate inhibition model (Km = 37.6 nM, Vmax = 2.72 pmol/min/mg, Vinh = 1.60 pmol/min/mg, and Ki = 0.87 µM). The pregnenolone sulfonation also exhibited substrate inhibition kinetics (Km = 0.99 µM, Vmax = 1.53 pmol/min/mg, and Ki = 54.67 µM). We clarified that SULT1 and SULT2 were expressed and had metabolizing capacities in the rat brain, suggesting that brain SULTs may be involved in metabolism of endogenous compounds and drugs.
Assuntos
Arilsulfotransferase/metabolismo , Encéfalo/enzimologia , Animais , Arilsulfotransferase/genética , Cinética , Fígado/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
Status epilepticus (SE) involves severe epileptic seizures that cause oxidative stress in the brain. Oxidative stress is known to influence uridine 5'-diposphate-glucuronosyltransferase (UGT) 1A expression. The present study aimed at elucidating the effect of SE on Ugt1a1, Ugt1a6 and Ugt1a7 expression in the rat brain. Kainic acid was used to create an animal model of SE. Sprague-Dawley rats were treated intraperitoneally with 10 mg/kg kainic acid. Ugt1a1 and Ugt1a7 mRNA levels were increased by SE in the cortex and hippocampus (Ugt1a1: 4.0- and 5.3-fold, respectively; Ugt1a7: 2.8- and 2.5-fold, respectively). Moreover, the induction degree of heme oxygenase-1 mRNA, an oxidative stress marker, was high in these regions, suggesting that oxidative stress could be involved in Ugt1a1 and Ugt1a7 induction. Ugt1a6 was elevated by 1.8-fold in the cortex in both SE and non-response (non-epileptic seizure response) rats, implying that Ugt1a6 induction may be independent from SE. An intraperitoneal single administration of 25 mg/kg diazepam (DZP) for the treatment of SE could attenuate heme oxygenase-1 induction in the cortex, whereas Ugt1a1 was decreased in the hippocampus, but not in the cortex, suggesting that there likely exists an alternative mechanism for Ugt1a1 reduction by DZP treatment. Continuous 14-day administration of DZP inhibited Ugt1a1 induction in the cortex, but did not have an effect on Ugt1a7 induction. This study indicated that SE altered the expression of brain Ugt1a1 and Ugt1a7, which could alter glucuronidation in the brain.
Assuntos
Glucuronosiltransferase/biossíntese , Estado Epiléptico/enzimologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Diazepam/farmacologia , Heme Oxigenase-1/biossíntese , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Ácido Caínico , Masculino , Ratos , Estado Epiléptico/induzido quimicamenteRESUMO
Single-walled carbon nanotubes (SWCNTs) are made from a rolled single sheet of graphene with a diameter in the nanometer range. SWCNTs are potential carriers for drug delivery systems because antibodies or drugs can be loaded on their surface; however, their effect on the activities of cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the effect of two kinds of SWCNTs with different lengths (FH-P- and SO-SWCNTs) on human CYP activity. In addition, other nano-sized carbon materials, such as carbon black, fullerene-C60 , and fullerene-C70 were also evaluated to compare their effects on CYP activities. Ten CYP substrates (phenacetin, coumarin, bupropion, paclitaxel, tolbutamide, S-mephenytoin, dextromethorphan, chlorzoxazone, midazolam, and testosterone) were used. Testosterone 6ß-hydroxylation and midazolam 1'-hydroxylation, which are catalysed by both CYP3A4 and CYP3A5 in liver microsomes, were decreased by 25% and 45%, respectively, in the presence of 0.1 mg/ml SO-SWCNT. Dextromethorphan O-demethylation, which is catalysed mainly by CYP2D6, was decreased by 40% in the presence of SO-SWCNT. Other CYP activities, however, were not attenuated by SO-SWCNT. FH-P-SWCNT, carbon black, fullerene-C60 , and fullerene-C70 at 0.1 mg/ml had no effect on CYP activities. The Ki values for testosterone 6ß-hydroxylation, midazolam 1'-hydroxylation, and dextromethorphan O-demethylation in liver microsomes were 136, 34, and 56 µg/ml, respectively. SO-SWCNT was determined to be a competitive inhibitor of CYP3A4, CYP3A5, and CYP2D6. These results suggest that the effect of SO-SWCNT differs among CYP isoforms, and that the inhibition potency depends on the physicochemical properties of the nanocarbons.
Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Nanotubos de Carbono , Humanos , Isoenzimas/metabolismo , Microssomos Hepáticos/metabolismo , Fuligem/farmacologiaRESUMO
ß-Estradiol is conjugated by uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A to 3-glucuronide in the human liver. UGT1A has been found in the brain; therefore, UGT1A may be involved in ß-estradiol 3-glucuronidation in the brain. In the present study, we aimed to characterize the ß-estradiol 3-glucuronidation reaction in the rat brain. ß-Estradiol 3-glucuronidation was detected in eight rat brain regions (cerebellum, frontal cortex, parietal cortex, piriform cortex, hippocampus, medulla oblongata, striatum, and thalamus). ß-Estradiol 3-glucuronidation in the cerebellum was fitted to the Hill equation (S50=8.0 µM, n=1.1). In inhibition experiments, ß-estradiol 3-glucuronidation was inhibited to 73.6% in the cerebellum by 50 µM bilirubin, whereas it was reduced to 20.5% with 5 µM bilirubin in the liver. Unlike in the liver, Ugt1a1 may not be the main isoform catalyzing this glucuronidation in the brain. Serotonin and acetaminophen at 10 mM inhibited glucuronidation to 1.17 and 25.5%, respectively, in the cerebellum. In induction experiments, the administration of ß-naphthoflavone, carbamazepine, and phenobarbital did not increase ß-estradiol 3-glucuronidation in the brain except for phenobarbital in the striatum. In addition, ß-estradiol 3-glucuronidation was not correlated with serotonin or acetaminophen glucuronidation in the brain, suggesting that Ugt1a6 and Ugt1a7 are not major isoforms of ß-estradiol 3-glucuronidation in the rat brain. In the present study, although we were unable to identify the isoform responsible for ß-estradiol 3-glucuronidation, we confirmed that ß-estradiol could be metabolized to glucuronide in the brain under a different metabolic profile from that in the liver.
Assuntos
Química Encefálica/fisiologia , Estradiol/metabolismo , Glucuronosiltransferase/metabolismo , Acetaminofen/farmacologia , Animais , Bilirrubina/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Glucuronídeos/metabolismo , Isoenzimas/metabolismo , Cinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Microssomos/metabolismo , Ratos , Ratos Sprague-Dawley , Serotonina/farmacologiaRESUMO
Uridine 5'-diphosphate-glucuronosyltransferase (UGT) is expressed in the liver and extrahepatic tissues. One of the major metabolic pathways of ß-estradiol (E2) is glucuronidation at the 17-hydroxy position by UGTs. This study was performed to determine E2 17-glucuronidation kinetics in human and rodent liver, small intestine, and kidney microsomes and to clarify the species and tissue differences. In the human liver and small intestine, Eadie-Hofstee plots exhibited biphasic kinetics, suggesting that E2 17-glucuronide (E17G) formation was catalyzed by more than two UGT isoforms in both tissues. The Km values for E17G formation by the high-affinity enzymes in the human liver and small intestine were 1.79 and 1.12 µM, respectively, and corresponding values for the low-affinity enzymes were 3.72 and 11.36 µM, respectively. Meanwhile, E17G formation in the human kidney was fitted to the Hill equation (S50=1.73 µM, n=1.63), implying that the UGT isoform catalyzing E17G formation in the kidney differed from that in the liver and small intestine. The maximum clearance for E17G formation in the human kidney was higher than the intrinsic clearance in the liver. E17G formation in the rat liver and kidney exhibited biphasic kinetics, whereas that in the small intestine was fitted to the Hill equation. In mice, all 3 tissues exhibited biphasic kinetics. In conclusion, we reported species and tissue differences in E2 17-glucuronidation, which occurred not only in the human liver but also in the extrahepatic tissues particularly the kidney.
Assuntos
Estradiol/metabolismo , Glucuronídeos/metabolismo , Intestino Delgado/metabolismo , Rim/metabolismo , Fígado/metabolismo , Animais , Humanos , Masculino , Camundongos Endogâmicos C57BL , Microssomos/metabolismo , Ratos Sprague-Dawley , Especificidade da EspécieRESUMO
UDP-glucuronosyltransferase (UGT), a phase II drug-metabolizing enzyme, is expressed in the brain and can catalyze glucuronidation of endogenous and exogenous substrates in the brain. Thus, changes in UGT1A expression could affect homeostasis and drug efficacy. Phenobarbital (PB), a typical inducer of drug-metabolizing enzymes, has been reported to induce oxidative stress and epigenetic changes, which could alter UGT expression in the brain. Here, we aimed to clarify the effects of PB on Ugt1a6 and Ugt1a7 gene expression in rat brains. Sprague-Dawley rats were treated intraperitoneally with PB (80 mg/kg), once daily for 7 days. Ugt1a6 and Ugt1a7 mRNA expression levels were increased in the striatum and thalamus (Ugt1a6, 3.0- and 2.9-fold, respectively; Ugt1a7, 2.6- and 2.6-fold, respectively). Acetaminophen glucuronidation was also increased in the medulla oblongata and thalamus by 1.8- and 1.2-fold, respectively. The induction rates within different brain regions were correlated with Ugt1a6 and Ugt1a7 mRNA expression, and the degree of induction also correlated with that of NF-E2-related factor-2 mRNA. Measurement of oxidative stress markers suggested that PB induced oxidative stress in brain regions in which Ugt1a6 and Ugt1a7 mRNAs were increased. Moreover, histone modifications were altered by PB treatment, resulting in increased histone H3 lysine 4 trimethylation in the striatum and thalamus and decreased histone H3 lysine 9 trimethylation in the thalamus. These results suggested that oxidative stress and histone modifications may promote transcriptional activation of Ugt1a6 and Ugt1a7 genes. In summary, Ugt1a6 and Ugt1a7 mRNA levels were increased by PB treatment, which may alter pharmacokinetics in the brain.
Assuntos
Encéfalo/metabolismo , Glucuronosiltransferase/metabolismo , Fenobarbital/farmacologia , Acetaminofen/farmacologia , Animais , Histonas/metabolismo , Masculino , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ativação Transcricional/efeitos dos fármacosRESUMO
Uridine 5'-diphosphate-glucuronosyltransferase (UGT) catalyzes a major phase II reaction in a drug-metabolizing enzyme system. Although the UGT1A subfamily is expressed mainly in the liver, it is also expressed in the brain. The purpose of the present study was to elucidate the effect of ß-naphthoflavone (BNF), one of the major inducers of drug-metabolizing enzymes, on Ugt1a6 and Ugt1a7 mRNA expression and their glucuronidation in the rat brain. Eight-week-old male Sprague-Dawley rats were treated intraperitoneally with BNF (80 mg/kg), once daily for 7 d. Ugt1a6 and Ugt1a7 mRNA expression increased in the cerebellum and hippocampus (Ugt1a6: 2.1- and 2.3-fold, respectively; Ugt1a7: 1.7- and 2.8-fold, respectively); acetaminophen glucuronidation also increased in the same regions by 4.1- and 2.7-fold, respectively. BNF induced Ugt1a6 and Ugt1a7 mRNA expression and their glucuronidation, and the degree of induction differed among 9 regions. BNF also upregulated CYP1A1, CYP1A2, and CYP1B1 mRNAs in the rat brain. Since the aryl hydrocarbon receptor signaling pathway was activated by BNF, it is indicated that Ugt1a6 and Ugt1a7 were induced via AhR in the rat brain. This study clarified that Ugt1a6 and Ugt1a7 mRNA expression and their enzyme activities were altered by BNF, suggesting that these changes may lead to alteration in the pharmacokinetics of UGT substrate in rat brain.
Assuntos
Encéfalo/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/metabolismo , beta-Naftoflavona/farmacologia , Acetaminofen/metabolismo , Animais , Encéfalo/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/genética , Masculino , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
1. Serotonin is a UGT1A6 substrate that is mainly found in the extrahepatic tissues where some UGT1As are expressed. The aim of the present study was to characterize serotonin glucuronidation in various tissues of humans and rodents. 2. Serotonin glucuronidation in the human liver and kidney fitted to the Michaelis-Menten model, and the Km values were similar to that of recombinant UGT1A6. However, serotonin glucuronidation in the human intestine fitted to the Hill equation, indicating that it is likely catalyzed not only by UGT1A6, but also by another UGT1A isoform. Serotonin glucuronidation in the rat liver, intestine and kidney fitted well to the Michaelis-Menten model and exhibited monophasic kinetics in the kidney, but biphasic kinetics in the liver and intestine. Furthermore, serotonin glucuronidation in the rat brain fitted best to the Hill equation. Serotonin glucuronidation in the mouse tissues fitted to the Michaelis-Menten model and exhibited monophasic kinetics in the liver and intestine microsomes, but biphasic kinetics in the kidney and brain microsomes. 3. In conclusion, we clarified that tissue and species differences exist in serotonin glucuronidation. It is necessary to take these potential differences into account when considering the pharmacodynamics and pharmacokinetics of serotonin.
RESUMO
UDP-glucuronosyltransferase (UGT) is an enzyme that catalyses a major phase II reaction in drug metabolism. Glucuronidation occurs mainly in the liver, but UGTs are also expressed in extrahepatic tissues, where they play an important role in local metabolism. UGT1A isoforms catalyse the glucuronidation of several drugs, neurotransmitters and neurosteroids that exert pharmacological and physiological effects on the brain. The aim of the current study was to determine UGT1A mRNA expression levels and glucuronidation activities in different regions of the rat brain (namely the cerebellum, frontal cortex, parietal cortex, piriform cortex, hippocampus, medulla oblongata, olfactory bulb, striatum and thalamus). It was found that all UGT1A isoforms were expressed in all the nine regions, but their expression levels differed between the regions. The difference between the regions of the brain where the mRNA levels were highest and those where they were lowest ranged between 2.1- to 7.8-fold. Glucuronidation activities were measured using the UGT substrates such as mycophenolic acid, p-nitrophenol and umbelliferone. Glucuronidation activity was detected in all nine regions of the brain. Activity levels differed between the regions, and were highest in the cerebellum, medulla oblongata and olfactory bulb. Differences in glucuronidation activity between regions with the highest rates and those with the lowest rates ranged from 5.3- to 10.1-fold. These results will contribute to our current understanding of the physiological and pharmacokinetic roles of drug-metabolizing enzymes in the brain. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Encéfalo/metabolismo , Glucuronosiltransferase/genética , Animais , Glucuronídeos/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Ácido Micofenólico/metabolismo , Nitrofenóis/metabolismo , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Umbeliferonas/metabolismoRESUMO
Uridine 5'-diphosphate (UDP)-glucuronosyltransferase 1A (UGT1A), which catalyzes major phase II reactions, is regulated by endogenous and exogenous factors via nuclear receptors such as the aryl hydrocarbon receptor (AhR). Glucocorticoid, one of the adrenocortical hormones, regulates AhR and UGT1A expression. We examined the effects of adrenalectomy on the expression and induction of UGT1A via AhR in the rat liver and small intestine. Rats were adrenalectomized bilaterally (ADX) or sham-operated (SHAM) and received intraperitoneal treatment with ß-naphthoflavone (BNF) for 4 d. Hepatic UGT1A6 and UGT1A7 mRNA levels were altered by ADX (0.1-fold and 1.6-fold, respectively). BNF treatment increased UGT1A6 and UGT1A7 mRNA expression and the intrinsic clearance of acetaminophen (APAP) glucuronidation, which is primarily catalyzed by UGT1A6 and UGT1A7, in both SHAM and ADX rats. Therefore, ADX rats maintained a functional AhR signaling pathway in the presence of BNF, expressed UGT1A6 and UGT1A7 mRNA, and showed APAP glucuronidation, namely induction by BNF via AhR was not abolished. Our results indicate that adrenal-dependent factors such as glucocorticoids are partially involved in the basal regulation of UGT1A6 and UGT1A7 transcription.
Assuntos
Adrenalectomia , Glucuronosiltransferase/biossíntese , Acetaminofen/metabolismo , Animais , Citocromo P-450 CYP1A1/metabolismo , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Microssomos/metabolismo , Ratos , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , beta-Naftoflavona/farmacologiaRESUMO
A fentanyl patch is widely used for the treatment of cancer pain. Its few adverse effects include constipation and drowsiness. The absorption volume of transdermally applied fentanyl may differ according to its site of application and variability in patch adhesion. Since fentanyl is predominantly metabolized by the drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 in the liver, its concentration may vary in cases of physiologically reduced CYP3A4 activity in the liver (liver disease and aging) or on co-administration of drugs. The clinical significance of measuring plasma concentration of fentanyl is high, but conventional methods require complicated processes such as solid-phase extraction and liquid-liquid extraction before the sample is injected into an HPLC system. In this study, a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for determining plasma fentanyl concentrations by deproteinization with acetonitrile. A recovery test was conducted using an absolute calibration curve to confirm the method's linearity and inter- and intra-day reproducibility. The required plasma volume for detection was reduced from 1 mL in the conventional method to 20 µL in the present study, and a good calibration curve was obtained in the concentration range from 0.05 to 5 ng/mL. These findings suggest that the method for sample preparation and quantification developed in this study are appropriate for measuring fentanyl concentration in human plasma in clinical settings.
Assuntos
Analgésicos Opioides/sangue , Cromatografia Líquida/métodos , Fentanila/sangue , Neoplasias/fisiopatologia , Dor Intratável/tratamento farmacológico , Espectrometria de Massas em Tandem/métodos , Idoso , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos WistarRESUMO
Onetaxotere®(OTAX)injection, which is a docetaxel(DOC)injection formulation, cannot be administered to those patients with alcohol intolerance or hypersensitivity, because it contains ethanol as a dissolving agent. To broaden treatment options for those patients, we tried to eliminate ethanol from OTAX injection. Under sterile conditions, dealcoholization was carried out using nitrogen gas in a hot water bath at 50°C. By this method, the ethanol included in OTAX injection was almost completely removed and DOC in the formulation was stable for 28 days. When the dealcoholized or untreated OTAX injection was intravenously injected in rats, no significant differences in the pharmacokinetic parameters of DOC were observed between those with dealcoholized and untreated OTAX injections. It is expected that dealcoholized OTAX may be useful in patients with alcohol-related difficulties.