RESUMO
P-glycoprotein is an efflux transporter located in the blood-brain barrier. (R)-[11C]Verapamil is widely used as a PET tracer to investigate its function in patients with epilepsy, Alzheimer's disease, and other neurodegenerative diseases. Currently it is not possible to use this successful tracer in clinics without a cyclotron, because of the short half-life of carbon-11. We developed two new fluorine-18 labeled (R)-verapamil analogs, with the benefit of a longer half-life. The synthesis of (R)-N-[18F]fluoroethylverapamil ([18F]1) and (R)-O-[18F]fluoroethylnorverapamil ([18F]2) has been described. [18F]1 was obtained in reaction of (R)-norverapamil with the volatile [18F]fluoroethyltriflate acquired from bromoethyltosylate and a silver trilate column with a radiochemical yield of 2.7% ± 1.2%. [18F]2 was radiolabeled by direct fluorination of precursor 13 and required final Boc-deprotection with TFA resulting in a radiochemical yield of 17.2% ± 9.9%. Both tracers, [18F]1 and [18F]2, were administered to Wistar rats, and blood plasma and brain samples were analyzed for metabolic stability. Using [18F]1 and [18F]2, PET scans were performed in Wistar rats at baseline and after blocking with tariquidar, showing a 3.6- and 2.4-fold increase in brain uptake in the blocked rats, respectively. In addition, for both [18F]1 and [18F]2, PET scans in Mdr1a/b(-/-), Bcrp1(-/-), and WT mice were acquired, in which [18F]2 showed a more specific brain uptake in Mdr1a/b(-/-) mice and no increased signal in Bcrp1(-/-) mice. [18F]2 was selected as the best performing tracer and should be evaluated further in clinical studies.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/metabolismo , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Fármacos do Sistema Nervoso Central/farmacologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Masculino , Camundongos Knockout , Estrutura Molecular , Quinolinas/farmacologia , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Ratos Wistar , Distribuição Tecidual , Verapamil/síntese química , Verapamil/farmacologia , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
UNLABELLED: Progressive familial intrahepatic cholestasis type 3 is caused by biallelic variations of ABCB4, most often (≥70%) missense. In this study, we examined the effects of 12 missense variations identified in progressive familial intrahepatic cholestasis type 3 patients. We classified these variations on the basis of the defects thus identified and explored potential rescue of trafficking-defective mutants by pharmacological means. Variations were reproduced in the ABCB4 complementary DNA and the mutants, thus obtained, expressed in HepG2 and HEK293 cells. Three mutants were either fully (I541F and L556R) or largely (Q855L) retained in the endoplasmic reticulum, in an immature form. Rescue of the defect, i.e., increase in the mature form at the bile canaliculi, was obtained by cell treatments with cyclosporin A or C and, to a lesser extent, B, D, or H. Five mutations with little or no effect on ABCB4 expression at the bile canaliculi caused a decrease (F357L, T775M, and G954S) or almost absence (S346I and P726L) of phosphatidylcholine secretion. Two mutants (T424A and N510S) were normally processed and expressed at the bile canaliculi, but their stability was reduced. We found no defect of the T175A mutant or of R652G, previously described as a polymorphism. In patients, the most severe phenotypes appreciated by the duration of transplant-free survival were caused by ABCB4 variants that were markedly retained in the endoplasmic reticulum and expressed in a homozygous status. CONCLUSION: ABCB4 variations can be classified as follows: nonsense variations (I) and, on the basis of current findings, missense variations that primarily affect the maturation (II), activity (III), or stability (IV) of the protein or have no detectable effect (V); this classification provides a strong basis for the development of genotype-based therapies.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Colestase Intra-Hepática/genética , Mutação , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Ciclosporina/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Fosfatidilcolinas/metabolismoRESUMO
Progressive familial intrahepatic cholestasis (PFIC) types 1 and 3 are severe cholestatic liver diseases caused by deficiency of ATB8B1 and ABCB4, respectively. Mouse models for PFIC display mild phenotypes compared with human patients, and this can be explained by the difference in bile salt pool composition. Mice, unlike humans, have the ability to detoxify hydrophobic bile salts by cytochrome P450-mediated (re)hydroxylation and thus have a less toxic bile salt pool. We have crossed mouse models for PFIC1 and PFIC3 with Hrn mice that have a reduced capacity to (re)hydroxylate bile salts. Double transgenes were obtained by backcrossing Atp8b1(G308V/G308V) and Abcb4(-/-) mice with Hrn mice that have a liver-specific disruption of the cytochrome P450 reductase gene and therefore have markedly reduced P450 activity. In these mice, a more hydrophobic bile salt pool was instilled by cholic acid supplementation of the diet, and bile formation and liver pathology was studied. As opposed to single transgenes, Atp8b1(G308V/G308V)/Hrn and Abcb4(-/-)/Hrn mice rapidly developed strong cholestasis that was evidenced by increased plasma bilirubin and bile salt levels. The bile salt pool was more toxic in both models; Atp8b1(G308V/G308V)/Hrn mice had a more hydrophobic plasma pool compared with the single transgene, whereas Abcb4(-/-)/Hrn mice had a more hydrophobic biliary pool compared with the single transgene. In line with these findings, liver damage was not aggravated in Atp8b1(G308V/G308V)/Hrn but was more severe in Abcb4(-/-)/Hrn mice. These data indicate that bile salt pool composition is a critical determinant in the initiation and progression of cholestasis and liver pathology in PFIC1 and PFIC3. Most importantly, our data suggest that the hydrophobicity of the plasma bile salt pool is an important determinant of the severity of cholestasis, whereas the hydrophobicity of the biliary bile salt pool is an important determinant of the severity of liver pathology.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Adenosina Trifosfatases/genética , Colestase Intra-Hepática , Modelos Animais de Doenças , Proteínas de Transferência de Fosfolipídeos/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/sangue , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Colestase Intra-Hepática/sangue , Colestase Intra-Hepática/patologia , Sistema Enzimático do Citocromo P-450/deficiência , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
UNLABELLED: Death rates from hepatocellular carcinoma (HCC) are steadily increasing, yet therapeutic options for advanced HCC are limited. We identify a subset of mouse and human HCCs harboring VEGFA genomic amplification, displaying distinct biologic characteristics. Unlike common tumor amplifications, this one seems to work via heterotypic paracrine interactions; stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte growth factor (HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and reduced proliferation, specifically in amplicon-positive mouse HCCs. Sorafenib-the first-line drug in advanced HCC-targets multiple kinases, including VEGFRs, but has only an overall mild beneficial effect. We found that VEGFA amplification specifies mouse and human HCCs that are distinctly sensitive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival of sorafenib-treated patients with VEGFA-amplified HCCs, suggesting that VEGFA amplification is a potential biomarker for HCC response to VEGF-A-blocking drugs. SIGNIFICANCE: Using a mouse model of inflammation-driven cancer, we identified a subclass of HCC carrying VEGFA amplification, which is particularly sensitive to VEGF-A inhibition. We found that a similar amplification in human HCC identifies patients who favorably responded to sorafenib-the first-line treatment of advanced HCC-which has an overall moderate therapeutic efficacy.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Macrófagos/metabolismo , Masculino , Camundongos Knockout , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Sorafenibe , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Animal models are an important tool to understand the complex pathogenesis of inflammatory bowel diseases (IBDs). This study tested the anti-inflammatory potential of a green tea extract rich in polyphenols (GrTP) in the colon of the multidrug resistance targeted mutation (Mdr1a(-/-)) mouse model of IBD. Insights into mechanisms responsible for this reduction in inflammation were gained using transcriptome and proteome analyses. Mice were randomly assigned to an AIN-76A (control) or GrTP-enriched diet. At 21 or 24 weeks of age, a colonic histological injury score was determined for each mouse, colon mRNA transcript levels were assessed using microarrays, and colon protein expression was measured using two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry protein identification. Mean colonic histological injury score of GrTP-fed Mdr1a(-/-) mice was significantly lower compared to those fed the control diet. Microarray and proteomics analyses showed reduced abundance of transcripts and proteins associated with immune and inflammatory response and fibrinogenesis pathways, and increased abundance of those associated with xenobiotic metabolism pathways in response to GrTP, suggesting that its anti-inflammatory activity is mediated by multiple molecular pathways. Peroxisome proliferator-activated receptor-α and signal transducer and activator of transcription 1 appear to be two key molecules which regulate these effects. These results support the view that dietary intake of polyphenols derived from green tea can ameliorate intestinal inflammation in the colon of a mouse model of IBD, and are in agreement with studies suggesting that consumption of green tea may reduce IBD symptoms and therefore play a part in an overall IBD treatment regimen.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Colite/prevenção & controle , Colo/metabolismo , Polifenóis/farmacologia , Animais , Colite/patologia , Colo/efeitos dos fármacos , Colo/patologia , Doenças Inflamatórias Intestinais/prevenção & controle , Masculino , Camundongos , Modelos Animais , PPAR alfa/fisiologia , Proteoma , Fator de Transcrição STAT1/fisiologia , Chá/química , TranscriptomaRESUMO
UNLABELLED: The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor and member of the immunoglobulin superfamily. RAGE is mainly involved in tissue damage and chronic inflammatory disorders, sustaining the inflammatory response upon engagement with damage-associated molecular pattern molecules (DAMPs) such as S100 proteins and high-mobility group box 1 (HMGB1). Enhanced expression of RAGE and its ligands has been demonstrated in distinct tumors and several studies support its crucial role in tumor progression and metastasis by still unknown mechanisms. Here we show that RAGE supports hepatocellular carcinoma (HCC) formation in the Mdr2(-/-) mouse model, a prototype model of inflammation-driven HCC formation, which mimics the human pathology. Mdr2(-/-) Rage(-/-) (dKO) mice developed smaller and fewer HCCs than Mdr2(-/-) mice. Interestingly, although in preneoplastic Mdr2(-/-) livers RAGE ablation did not affect the onset of inflammation, premalignant dKO livers showed reduced liver damage and fibrosis, in association with decreased oval cell activation. Oval cells expressed high RAGE levels and displayed reduced proliferation upon RAGE silencing. Moreover, stimulation of oval cells with HMGB1 promoted an ERK1/2-Cyclin D1-dependent oval cell proliferation in vitro. Finally, genetic and pharmacologic blockade of RAGE signaling impaired oval cell activation in an independent mouse model of oval cell activation, the choline deficient ethionine-supplemented dietary regime. CONCLUSION: Our data identified a novel function of RAGE in regulating oval cell activation and tumor development in inflammation-associated liver carcinogenesis.
Assuntos
Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Receptores Imunológicos/fisiologia , Células-Tronco/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Animais , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica , Proteína HMGB1/metabolismo , Inflamação/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Knockout , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/biossíntese , Células-Tronco/patologia , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. METHODS: To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. RESULTS: Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. CONCLUSIONS: ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Adenosina Trifosfatases/fisiologia , Canalículos Biliares/citologia , Membrana Celular/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Animais , Ácidos e Sais Biliares/farmacologia , Canalículos Biliares/fisiologia , Células Cultivadas , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Fosfatidilcolinas/metabolismo , Proteínas de Transferência de Fosfolipídeos , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
BACKGROUND/AIMS: Multidrug resistance protein 2 (Abcb4) gene knockout mice (Mdr2(-/-)) lack phosphatidylcholine (PC) excretion into bile and spontaneously develop sclerosing cholangitis, biliary fibrosis and hepatocellular carcinomas. We therefore aimed to test whether formation and hepatic retention of abnormal PC metabolites contribute to the pathogenesis of liver injury in Mdr2(-/-) mice. METHODS: Mdr2(-/-) mice were either fed a diet supplemented with soybean lecithin 2.5% w/w [phosphatidylcholine-enriched diet (PCD), to increase hepatic PC content] or a choline-deficient diet (CDD, to reduce hepatic PC content) for 4 weeks; controls received chow with energy and nutrient content equivalent to PCD and CDD. Serum liver tests, liver histology, markers of fibrosis, cholangiocyte activation, cell proliferation and thin-layer chromatography for phospholipid (PL) composition were carried out. RESULTS: PCD decreased serum alkaline phosphatase and total bilirubin levels compared with controls, while liver histology as well as hepatic hydroxyproline content as markers of liver fibrosis did not differ among groups. Both PCD and CDD decreased hepatocellular proliferation compared with controls. Hepatic, serum and biliary PLs remained unchanged despite dietary manipulations and no potentially toxic PL metabolites were detected. CONCLUSIONS: Mdr2(-/-) mice maintain stable hepatic, serum and biliary PL metabolism in response to dietary PC manipulations. Our findings therefore suggest that liver injury in Mdr2(-/-) mice is not due to formation of toxic PL metabolites.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Colestase Intra-Hepática/metabolismo , Deficiência de Colina/metabolismo , Modelos Animais de Doenças , Lecitinas/metabolismo , Fígado/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Fosfatase Alcalina/sangue , Animais , Bile/metabolismo , Bilirrubina/sangue , Proliferação de Células/efeitos dos fármacos , Colestase Intra-Hepática/induzido quimicamente , Colestase Intra-Hepática/patologia , Colesterol/metabolismo , Deficiência de Colina/patologia , Dieta , Resistência a Múltiplos Medicamentos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Lecitinas/administração & dosagem , Fígado/efeitos dos fármacos , Fígado/patologia , Testes de Função Hepática , Masculino , Camundongos , Camundongos Knockout , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Cryptotanshinone (CTS), a major constituent from the roots of Salvia miltiorrhiza (Danshen), is widely used in the treatment of coronary heart disease, stroke and less commonly Alzheimer's disease. Our recent study indicates that CTS is a substrate for P-glycoprotein (PgP/MDR1/ABCB1). This study has investigated the nature of the brain distribution of CTS across the brain-blood barrier (BBB) using several in vitro and in vivo rodent models. A polarized transport of CTS was found in rat primary microvascular endothelial cell (RBMVEC) monolayers, with facilitated efflux from the abluminal side to luminal side. Addition of a PgP (e.g. verapamil and quinidine) or multi-drug resistance protein 1/2 (MRP1/2) inhibitor (e.g. probenecid and MK-571) in both luminal and abluminal sides attenuated the polarized transport. In a bilateral in situ brain perfusion model, the uptake of CTS into the cerebrum increased from 0.52 +/- 0.1% at 1 min to 11.13 +/- 2.36 ml/100 g tissue at 30 min and was significantly greater than that of sucrose. Co-perfusion of a PgP/MDR1 (e.g. verapamil) or MRP1/2 inhibitor (e.g. probenecid) significantly increased the brain distribution of CTS by 35.1-163.6%. The brain levels of CTS were only about 21% of those in plasma, and were significantly increased when coadministered with verapamil or probenecid in rats. The brain levels of CTS in rats subjected to middle cerebral artery occlusion and rats treated with quinolinic acid (a neurotoxin) were about 2- to 2.5-fold higher than the control rats. Moreover, the brain levels in mdr1a(-/-) and mrp1(-/-) mice were 10.9- and 1.5-fold higher than those in the wild-type mice, respectively. Taken collectively, these findings indicate that PgP and Mrp1 limit the brain penetration of CTS in rodents, suggesting a possible role of PgP and MRP1 in limiting the brain penetration of CTS in patients and causing drug resistance to Danshen therapy and interactions with conventional drugs that are substrates of PgP and MRP1. Further studies are needed to explore the role of other drug transporters in restricting the brain penetration of CTS and the clinical relevance.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Medicamentos de Ervas Chinesas/farmacocinética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fármacos Neuroprotetores/farmacocinética , Fenantrenos/farmacocinética , Salvia miltiorrhiza , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Transporte Biológico , Encéfalo/irrigação sanguínea , Permeabilidade Capilar , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/uso terapêutico , Células Endoteliais/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microcirculação/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fármacos Neuroprotetores/isolamento & purificação , Fármacos Neuroprotetores/uso terapêutico , Síndromes Neurotóxicas/metabolismo , Fenantrenos/isolamento & purificação , Fenantrenos/uso terapêutico , Raízes de Plantas , Ácido Quinolínico , Ratos , Ratos Sprague-Dawley , Salvia miltiorrhiza/química , Acidente Vascular Cerebral/tratamento farmacológico , Distribuição Tecidual , Triterpenos/farmacocinéticaRESUMO
Glabridin is a major constituent of the root of Glycyrrhiza glabra, which is commonly used in the treatment of cardiovascular and central nervous system diseases. This study aimed to investigate the role of P-glycoprotein (PgP/MDR1) in the intestinal absorption of glabridin. The systemic bioavailability of glabridin was approximately 7.5% in rats, but increased when combined with verapamil. In single-pass perfused rat ileum with mesenteric vein cannulation, the permeability coefficient of glabridin based on drug disappearance in luminal perfusates (P(lumen)) was approximately 7-fold higher than that based on drug appearance in the blood (P(blood)). Glabridin was mainly metabolized by glucuronidation, and the metabolic capacity of intestine microsomes was 1/15 to 1/20 of that in liver microsomes. Polarized transport of glabridin was found in Caco-2 and MDCKII monolayers. Addition of verapamil in both apical (AP) and basolateral (BL) sides abolished the polarized transport of glabridin across Caco-2 cells. Incubation of verapamil significantly altered the intracellular accumulation and efflux of glabridin in Caco-2 cells. The transport of glabridin in the BL-AP direction was significantly higher in MDCKII cells overexpressing PgP/MDR1 than in the control cells. Glabridin inhibited PgP-mediated transport of digoxin with an IC(50) value of 2.56 microM, but stimulated PgP/MDR1 ATPase activity with a K(m) of 25.1 microM. The plasma AUC(0-24h) of glabridin in mdr1a(-/-) mice was 3.8-fold higher than that in wild-type mice. These findings indicate that glabridin is a substrate for PgP and that both PgP/MDR1-mediated efflux and first-pass metabolism contribute to the low oral bioavailability of glabridin.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Medicamentos de Ervas Chinesas/farmacocinética , Flavonoides/farmacocinética , Glycyrrhiza , Absorção Intestinal , Fenóis/farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Administração Oral , Animais , Disponibilidade Biológica , Transporte Biológico Ativo , Células CACO-2 , Permeabilidade da Membrana Celular , Digoxina/metabolismo , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/isolamento & purificação , Flavonoides/administração & dosagem , Flavonoides/sangue , Flavonoides/isolamento & purificação , Glucuronídeos/metabolismo , Glycyrrhiza/química , Humanos , Técnicas In Vitro , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Isoflavonas , Camundongos , Camundongos Knockout , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Estrutura Molecular , Fenóis/administração & dosagem , Fenóis/sangue , Fenóis/isolamento & purificação , Raízes de Plantas , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transfecção , Uridina Difosfato Ácido Glucurônico/metabolismo , Verapamil/farmacologiaRESUMO
BACKGROUND: Intestinal mucus not only facilitates substrate absorption, but also forms a hydrophobic, phosphatidylcholine (PC) enriched, barrier against luminal gut contents. METHODS: For evaluation of the origin of PC in intestinal mucus, we first analyzed the mucus PC in mice with absent biliary phospholipid secretion (mdr2 (-/-) mice) using electrospray ionization (ESI) tandem mass spectroscopy (MS/MS). Second, in situ perfused rat jejunum, ileum and colon were analyzed after i.v. bolus injections of 155 pmol [(3)H]-PC. Additional in vitro experiments were performed with isolated mucosal cells after incubation with the PC precursor [(3)H]-choline. RESULTS: In mdr2 (-/-) mice and control animals no significant quantitative difference in mucus PC was found, indicating that mucus PC is of intestinal and not biliary origin. In situ perfusion studies detected intestinal secretion of [(3)H]-PC, which was stimulated in presence of 2 mM taurocholate (TC). Secretion rates of [(3)H]-PC were highest in ileum (9.0+/-0.8 fmol h(-1)xcm(-1)), lower in jejunum (4.3+/-0.5) and minimal in colon (0.8+/-0.2). It compares to an intestinal secretion of native PC originating to 64% from bile, 9% from jejunum, 28% from ileum, and 1% from colon. Complementary in vitro studies showed 30-min secretion rates for [(3)H]-PC to be highest in enterocytes from ileum (26.5+/-5.3% of intracellular [(3)H]-PC) and jejunum (19.8+/-2.9%), and significantly lower in colonocytes (8.4+/-1.3%). CONCLUSION: PC in the intestinal mucus originates from secretion by ileal and jejunal enterocytes.
Assuntos
Íleo/metabolismo , Fosfatidilcolinas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Colo/metabolismo , Células Epiteliais/metabolismo , Jejuno/metabolismo , Masculino , Camundongos , Muco/metabolismo , Ratos , Trítio/metabolismoRESUMO
Previous studies have demonstrated that phenobarbital treatment impairs the biliary excretion of acetaminophen glucuronide (AG), although the transport system(s) responsible for AG excretion into bile has not been identified. Initial studies in rat canalicular liver plasma membrane vesicles indicated that AG uptake was stimulated modestly by ATP, but not by membrane potential, HCO(3)(-), or pH gradients. To examine the role of the ATP-dependent canalicular transporter multidrug resistance-associated protein 2 (Mrp2)/canalicular multispecific organic anion transporter (cMOAT) in the biliary excretion of AG, the hepatobiliary disposition of acetaminophen, AG, and acetaminophen sulfate (AS) was examined in isolated perfused livers from control and TR(-) (Mrp2-deficient) Wistar rats. Mean bile flow in TR(-) livers was approximately 0.3 microl/min/g of liver ( approximately 4-fold lower than control). AG biliary excretion was decreased (>300-fold) to negligible levels in TR(-) rat livers, indicating that AG is an Mrp2 substrate. Similarly, AS biliary excretion in TR(-) livers was decreased ( approximately 5-fold); however, concentrations were still measurable, suggesting that multiple mechanisms, including Mrp2-mediated active transport, may be involved in AS biliary excretion. AG and AS perfusate concentrations were significantly higher in livers from TR(-) compared with control rats. Pharmacokinetic modeling of the data revealed that the rate constant for basolateral egress of AG increased significantly from 0.028 to 0.206 min(-1), consistent with up-regulation of a basolateral organic anion transporter in Mrp2-deficient rat livers. In conclusion, these data indicate that AG biliary excretion is mediated by Mrp2, and clearly demonstrate that substrate disposition may be influenced by alterations in complementary transport systems in transport-deficient animals.