RESUMO
Sepsis, a life-threatening health issue, lacks effective medicine targeting the septic response. In China, treatment combining the intravenous herbal medicine XueBiJing with conventional procedures reduces the 28-day mortality of critically ill patients by modulating septic response. In this study, we identified the combined active constituents that are responsible for the XueBiJing's anti-sepsis action. Sepsis was induced in rats by cecal ligation and puncture (CLP). The compounds were identified based on their systemic exposure levels and anti-sepsis activities in CLP rats that were given an intravenous bolus dose of XueBiJing. Furthermore, the identified compounds in combination were assessed, by comparing with XueBiJing, for levels of primary therapeutic outcome, pharmacokinetic equivalence, and pharmacokinetic compatibility. We showed that a total of 12 XueBiJing compounds, unchanged or metabolized, circulated with significant systemic exposure in CLP rats that received XueBiJing. Among these compounds, hydroxysafflor yellow A, paeoniflorin, oxypaeoniflorin, albiflorin, senkyunolide I, and tanshinol displayed significant anti-sepsis activities, which involved regulating immune responses, inhibiting excessive inflammation, modulating hemostasis, and improving organ function. A combination of the six compounds, with the same respective doses as in XueBiJing, displayed percentage survival and systemic exposure in CLP rats similar to those by XueBiJing. Both the combination and XueBiJing showed high degrees of pharmacokinetic compatibility regarding interactions among the six active compounds and influences of other circulating XueBiJing compounds. The identification of XueBiJing's pharmacologically significant constituents supports the medicine's anti-sepsis use and provides insights into a polypharmacology-based approach to develop medicines for effective sepsis management.
Assuntos
Medicamentos de Ervas Chinesas , Ratos Sprague-Dawley , Sepse , Animais , Sepse/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/uso terapêutico , Medicamentos de Ervas Chinesas/farmacocinética , Masculino , Ratos , Administração IntravenosaRESUMO
Septic lung damage is associated with endothelial cell and neutrophil activation. This study examines the role of the E3 ubiquitin ligase midline 1 (Mid1) in abdominal sepsis. Mid1 expression was increased in endothelial cells derived from post-capillary venules in septic mice and TNF-α challenge increased Mid1 levels in endothelial cells in vitro. The siRNA-mediated knockdown of Mid1 decreased TNF-α-induced upregulation of ICAM-1 and neutrophil adhesion to endothelial cells. Moreover, Mid1 silencing reduced leukocyte adhesion in post-capillary venules in septic lungs in vivo. The silencing of Mid1 not only decreased Mid1 expression but also attenuated expression of ICAM-1 in lungs from septic mice. Lastly, TNF-α stimulation decreased PP2Ac levels in endothelial cells in vitro, which was reversed in endothelial cells pretreated with siRNA directed against Mid1. Thus, our novel data show that Mid1 is an important regulator of ICAM-1 expression and neutrophil adhesion in vitro and septic lung injury in vivo. A possible target of Mid1 is PP2Ac in endothelial cells. Targeting the Mid1-PP2Ac axis may be a useful way to reduce pathological lung inflammation in abdominal sepsis.
Assuntos
Gastroenteropatias , Molécula 1 de Adesão Intercelular , Sepse , Ubiquitina-Proteína Ligases , Animais , Camundongos , Adesão Celular , Células Endoteliais/metabolismo , Gastroenteropatias/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Pulmão/metabolismo , Neutrófilos/metabolismo , RNA Interferente Pequeno/genética , Sepse/genética , Sepse/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
S100A9, a pro-inflammatory alarmin, is up-regulated in inflamed tissues. However, the role of S100A9 in regulating neutrophil activation, inflammation and lung damage in sepsis is not known. Herein, we hypothesized that blocking S100A9 function may attenuate neutrophil recruitment in septic lung injury. Male C57BL/6 mice were pretreated with the S100A9 inhibitor ABR-238901 (10 mg/kg), prior to cercal ligation and puncture (CLP). Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested for analysis of neutrophil infiltration as well as edema and CXC chemokine production. Blood was collected for analysis of membrane-activated complex-1 (Mac-1) expression on neutrophils as well as CXC chemokines and IL-6 in plasma. Induction of CLP markedly increased plasma levels of S100A9. ABR-238901 decreased CLP-induced neutrophil infiltration and edema formation in the lung. In addition, inhibition of S100A9 decreased the CLP-induced up-regulation of Mac-1 on neutrophils. Administration of ABR-238901 also inhibited the CLP-induced increase of CXCL-1, CXCL-2 and IL-6 in plasma and lungs. Our results suggest that S100A9 promotes neutrophil activation and pulmonary accumulation in sepsis. Targeting S100A9 function decreased formation of CXC chemokines in circulation and lungs and attenuated sepsis-induced lung damage. These novel findings suggest that S100A9 plays an important pro-inflammatory role in sepsis and could be a useful target to protect against the excessive inflammation and lung damage associated with the disease.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Calgranulina B/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Sepse/complicações , Sulfonamidas/uso terapêutico , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/metabolismo , Animais , Quimiocinas CXC/metabolismo , Avaliação Pré-Clínica de Medicamentos , Interleucina-6/metabolismo , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Sepse/imunologia , Sepse/metabolismo , Sulfonamidas/farmacologiaRESUMO
BACKGROUND: Polyphosphates (PolyPs) have been reported to exert pro-inflammatory effects. However, the molecular mechanisms regulating PolyP-provoked tissue accumulation of leukocytes are not known. The aim of the present investigation was to determine the role of specific adhesion molecules in PolyP-mediated leukocyte recruitment. METHODS: PolyPs and TNF-α were intrascrotally administered, and anti-P-selectin, anti-E-selectin, anti-P-selectin glycoprotein ligand-1 (PSGL-1), anti-membrane-activated complex-1 (Mac-1), anti-lymphocyte function antigen-1 (LFA-1), and neutrophil depletion antibodies were injected intravenously or intraperitoneally. Intravital microscopy of the mouse cremaster microcirculation was used to examine leukocyte-endothelium interactions and recruitment in vivo. RESULTS: Intrascrotal injection of PolyPs increased leukocyte accumulation. Depletion of neutrophils abolished PolyP-induced leukocyte-endothelium interactions, indicating that neutrophils were the main leukocyte subtype responding to PolyP challenge. Immunoneutralization of P-selectin and PSGL-1 abolished PolyP-provoked neutrophil rolling, adhesion, and emigration. Moreover, immunoneutralization of Mac-1 and LFA-1 had no impact on neutrophil rolling but markedly reduced neutrophil adhesion and emigration evoked by PolyPs. CONCLUSION: These results suggest that P-selectin and PSGL-1 exert important roles in PolyP-induced inflammatory cell recruitment by mediating neutrophil rolling. In addition, our data show that Mac-1 and LFA-1 are necessary for supporting PolyP-triggered firm adhesion of neutrophils to microvascular endothelium. These novel findings define specific molecules as potential targets for pharmacological intervention in PolyP-dependent inflammatory diseases.
Assuntos
Comunicação Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Microcirculação/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Polifosfatos/farmacologia , Animais , Células Endoteliais/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Antígeno de Macrófago 1/fisiologia , Masculino , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Selectina-P/fisiologiaRESUMO
Methyl (S)-4-(6-amino-9H-purin-9-yl)-2-hydroxybutanoate (DZ2002) is a potent reversible inhibitor of S-adenosyl-L-homocysteine hydrolase (SAHH). Due to its ester structure, DZ2002 is rapidly hydrolyzed in rat blood to 4-(6-amino-9H-purin-9-yl)-2-hydroxybutyric acid (DZA) during and after blood sampling from rats; this hampers accurate determination of the circulating DZ2002 and its acid metabolite DZA in rats. To this end, a method for determining the blood concentrations of DZ2002 and DZA in rats was developed by using methanol to immediately deactivate blood carboxylesterases during sampling. The newly developed bioanalytical assay possessed favorable accuracy and precision with lower limit of quantification of 31â¯nM for DZ2002 and DZA. This validated assay was applied to a rat pharmacokinetic study of DZ2002. After oral administration, DZ2002 was found to be extensively converted into DZA. The level of systemic exposure to DZ2002 was significantly lower than that of DZA. The apparent oral bioavailability of DZ2002 was 90%-159%. The mean terminal half-lives of DZ2002 and DZA were 0.3-0.9 and 1.3-5.1â¯h, respectively. The sample preparation method illustrated here may be adopted for determination of other circulating ester drugs and their acid metabolites in rodents.
RESUMO
ShenMai, an intravenous injection prepared from steamed Panax ginseng roots (Hongshen) and Ophiopogon japonicus roots (Maidong), is used as an add-on therapy for coronary artery disease and cancer; saponins are its bioactive constituents. Since many saponins inhibit human organic anion-transporting polypeptides (OATP)1B, this investigation determined the inhibition potencies of circulating ShenMai saponins on the transporters and the joint potential of these compounds for ShenMai-drug interaction. Circulating saponins and their pharmacokinetics were characterized in rats receiving a 30-min infusion of ShenMai at 10 mL/kg. Inhibition of human OATP1B1/1B3 and rat Oatp1b2 by the individual saponins was investigated in vitro; the compounds' joint inhibition was also assessed in vitro and the data was processed using the Chou-Talalay method. Plasma protein binding was assessed by equilibrium dialysis. Altogether, 49 saponins in ShenMai were characterized and graded into: 10-100 µmol/day (compound doses from ShenMai; 7 compounds), 1-10 µmol/day (17 compounds), and <1 µmol/day (25 compounds, including Maidong ophiopogonins). After dosing, circulating saponins were protopanaxadiol-type ginsenosides Rb1, Rb2, Rc, Rd, Ra1, Rg3, Ra2, and Ra3, protopanaxatriol-type ginsenosides Rg1, Re, Rg2, and Rf, and ginsenoside Ro. The protopanaxadiol-type ginsenosides exhibited maximum plasma concentrations of 2.1-46.6 µmol/L, plasma unbound fractions of 0.4-1.0% and terminal half-lives of 15.6-28.5 h (ginsenoside Rg3, 1.9 h), while the other ginsenosides exhibited 0.1-7.7 µmol/L, 20.8-99.2%, and 0.2-0.5 h, respectively. The protopanaxadiol-type ginsenosides, ginsenosides without any sugar attachment at C-20 (except ginsenoside Rf), and ginsenoside Ro inhibited OATP1B3 more potently (IC50, 0.2-3.5 µmol/L) than the other ginsenosides (≥22.6 µmol/L). Inhibition of OATP1B1 by ginsenosides was less potent than OATP1B3 inhibition. Ginsenosides Rb1, Rb2, Rc, Rd, Ro, Ra1, Re, and Rg2 likely contribute the major part of OATP1B3-mediated ShenMai-drug interaction potential, in an additive and time-related manner.
Assuntos
Medicamentos de Ervas Chinesas/farmacocinética , Ginsenosídeos/farmacocinética , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/antagonistas & inibidores , Administração Intravenosa , Animais , Combinação de Medicamentos , Interações Medicamentosas , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/metabolismo , Ginsenosídeos/administração & dosagem , Ginsenosídeos/sangue , Ginsenosídeos/metabolismo , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Masculino , Ophiopogon/química , Panax/química , Ligação Proteica , Ratos Sprague-Dawley , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismoRESUMO
BACKGROUND AND PURPOSE: Intravenous glycyrrhizin, having anti-inflammatory and hepatoprotective properties, is incorporated into the management of liver diseases in China. This investigation was designed to elucidate the molecular mechanism underlying hepatobiliary excretion of glycyrrhizin and to investigate its potential for drug-drug interactions on organic anion-transporting polypeptide (OATP)1B. EXPERIMENTAL APPROACH: Human transporters mediating hepatobiliary excretion of glycyrrhizin were characterized at the cellular and vesicular levels and compared with rat hepatic transporters. The role of Oatp1b2 in glycyrrhizin's elimination and pharmacokinetics was evaluated in rats using the inhibitor rifampin. A physiologically based pharmacokinetic (PBPK) model for glycyrrhizin, incorporating transporter-mediated hepatobiliary excretion, was established and applied to predict potential drug-drug interactions related to glycyrrhizin in humans. KEY RESULTS: Hepatobiliary excretion of glycyrrhizin involved human OATP1B1/1B3 (Oatp1b2 in rats)-mediated hepatic uptake from blood and human multidrug resistance-associated protein (MRP)2/breast cancer resistance protein (ABCP)/bile salt export pump (BSEP)/multidrug resistance protein 1 (Mrp2/Abcp/Bsep in rats)-mediated hepatic efflux into bile. In rats, rifampin impaired hepatic uptake of glycyrrhizin significantly increasing its systemic exposure. Glomerular-filtration-based renal excretion of glycyrrhizin was slow due to extensive protein binding in plasma. Quantitative analysis using the PBPK model demonstrated that OATP1B1/1B3 have critical roles in the pharmacokinetics of glycyrrhizin, which is highly likely to be a victim of drug-drug interactions when co-administered with potent dual inhibitors of these transporters. CONCLUSIONS AND IMPLICATIONS: Transporter-mediated hepatobiliary excretion governs glycyrrhizin's elimination and pharmacokinetics. Understanding glycyrrhizin's potential drug-drug interactions on OATP1B1/1B3 should enhance the therapeutic outcome of glycyrrhizin-containing drug combinations on liver diseases.
Assuntos
Ácido Glicirrízico/farmacologia , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Fígado/efeitos dos fármacos , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Animais , Células Cultivadas , Interações Medicamentosas , Ácido Glicirrízico/farmacocinética , Células HEK293 , Humanos , Funções Verossimilhança , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Ratos , Ratos Sprague-DawleyRESUMO
Anlotinib is a new oral tyrosine kinase inhibitor; this study was designed to characterize its pharmacokinetics and disposition. Anlotinib was evaluated in rats, tumor-bearing mice, and dogs and also assessed in vitro to characterize its pharmacokinetics and disposition and drug interaction potential. Samples were analyzed by liquid chromatography/mass spectrometry. Anlotinib, having good membrane permeability, was rapidly absorbed with oral bioavailability of 28%-58% in rats and 41%-77% in dogs. Terminal half-life of anlotinib in dogs (22.8±11.0 h) was longer than that in rats (5.1±1.6 h). This difference appeared to be mainly associated with an interspecies difference in total plasma clearance (rats, 5.35±1.31 L·h-1·kg-1; dogs, 0.40±0.06 L·h-1/kg-1). Cytochrome P450-mediated metabolism was probably the major elimination pathway. Human CYP3A had the greatest metabolic capability with other human P450s playing minor roles. Anlotinib exhibited large apparent volumes of distribution in rats (27.6±3.1 L/kg) and dogs (6.6±2.5 L/kg) and was highly bound in rat (97%), dog (96%), and human plasma (93%). In human plasma, anlotinib was predominantly bound to albumin and lipoproteins, rather than to α1-acid glycoprotein or γ-globulins. Concentrations of anlotinib in various tissue homogenates of rat and in those of tumor-bearing mouse were significantly higher than the associated plasma concentrations. Anlotinib exhibited limited in vitro potency to inhibit many human P450s, UDP-glucuronosyltransferases, and transporters, except for CYP3A4 and CYP2C9 (in vitro half maximum inhibitory concentrations, <1 µmol/L). Based on early reported human pharmacokinetics, drug interaction indices were 0.16 for CYP3A4 and 0.02 for CYP2C9, suggesting that anlotinib had a low propensity to precipitate drug interactions on these enzymes. Anlotinib exhibits many pharmacokinetic characteristics similar to other tyrosine kinase inhibitors, except for terminal half-life, interactions with drug metabolizing enzymes and transporters, and plasma protein binding.
Assuntos
Indóis/administração & dosagem , Indóis/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Quinolinas/administração & dosagem , Quinolinas/farmacocinética , Administração Oral , Animais , Área Sob a Curva , Disponibilidade Biológica , Células CACO-2 , Cromatografia Líquida , Neoplasias do Colo/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Cães , Interações Medicamentosas , Feminino , Células HEK293 , Meia-Vida , Xenoenxertos , Humanos , Absorção Intestinal , Masculino , Espectrometria de Massas , Taxa de Depuração Metabólica , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Animais , Modelos Biológicos , Transplante de Neoplasias , Ligação Proteica , Ratos Sprague-Dawley , Especificidade da Espécie , Distribuição TecidualRESUMO
Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-ß1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. Stem Cells 2017;35:1719-1732.
Assuntos
Caspases/imunologia , Imunomodulação , Células-Tronco Pluripotentes Induzidas/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Caspases/genética , Diferenciação Celular , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Feminino , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Imunofenotipagem , Células-Tronco Pluripotentes Induzidas/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Ensaio de Cápsula Sub-Renal , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/imunologia , Células Th2/citologia , Células Th2/imunologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Transplante HeterólogoRESUMO
Polyphenols derived from Danshen are responsible for the therapeutic effects of DanHong injection, a two-herb combination of Danshen and Honghua. Whether the pharmacokinetics of Danshen polyphenols is changed by coexisting Honghua constituents remains unknown. A sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed in this study for simultaneous determination of eight Danshen polyphenols (i.e., protocatechuic aldehyde, protocatechuic acid, tanshinol, salvianolic acid D, rosmarinic acid, salvianolic acid A, lithospermic acid, and salvianolic acid B) in rat plasma and applied to a comparative pharmacokinetic study of DanHong injection and Danshen injection. Liquid chromatography conditions, mass spectrometry parameters, and sample preparation were optimized step by step. The calibration curves showed good linearity (r > 0.99) for all the polyphenols. The mean extraction efficiencies ranged from 62.2 to 88.7% with negligible matrix effects. The intrabatch and interbatch precision at all the quality control levels were less than 15% of the nominal concentrations with accuracy of 88.8-114%, except that precision and accuracy at lower limit of quantitation were 3.2-17.3 and 95.7-119%, respectively. Comparative pharmacokinetic study suggested that the coexisting Honghua constituents might have negligible influences on the pharmacokinetics of Danshen polyphenols from DanHong injection. The bioanalytical method could also be applied to pharmacokinetic studies of other Danshen herbal products.
Assuntos
Análise Química do Sangue/métodos , Medicamentos de Ervas Chinesas/farmacocinética , Polifenóis/sangue , Salvia miltiorrhiza/química , Animais , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
Lymphohematopoietic stem cells (L-HSCs) generated from self-somatic cell-derived induced pluripotent stem cells (iPSCs) are a potential source of cells for the treatment of hematological disorders. However, the generation of truly functional L-HSCs from iPSCs has yet to be achieved. Thus, whether iPSCs have the inherent potential to generate a normal differentiated phenotype and functional population of multiple lineages of terminally differentiated lymphocytes needs to be assessed. Here, we used tetraploid embryo complementation to provide a normal environment for the differentiation of hematopoietic cells from iPSCs and embryonic stem cells (ESCs). We then evaluated the characteristics, populations, and functions of lymphocytes derived from iPSCs, ESCs, and naïve isogenic C57BL/6 mice. The results showed that iPSC-derived lymphocytes (iPSLs) expressed normal levels of major histocompatibility complex-I (MHC-I) and exhibited a fully pluripotent capacity to differentiate into CD4(+) T, CD8(+) T, regulatory T, B, and natural killer cells. Following in vitro stimulation with either concanavalin A or an alloantigen, iPSLs exhibited the same capacities for proliferation and cytokine secretion as ESC-derived or isogenic lymphocytes. Furthermore, iPSC-derived bone marrow cells could differentiate into multiple lymphocyte lineages that reconstituted the lymphocyte population in syngeneic lethally irradiated recipient animals. Our results demonstrated that iPSCs have the inherent potential to differentiate into multiple lineages of functional lymphocytes without bias, and further support the practical application of iPSC-based treatments to hematological disorders.
Assuntos
Linhagem da Célula , Células-Tronco Pluripotentes Induzidas/citologia , Linfócitos/citologia , Linfopoese , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
AIM: Tanshinol is an important catechol in the antianginal herb Salvia miltiorrhiza roots (Danshen). This study aimed to characterize tanshinol methylation. METHODS: Metabolites of tanshinol were analyzed by liquid chromatography/mass spectrometry. Metabolism was assessed in vitro with rat and human enzymes. The major metabolites were synthesized for studying their interactions with drug metabolizing enzymes and transporters and their vasodilatory properties. Dose-related tanshinol methylation and its influences on tanshinol pharmacokinetics were also studied in rats. RESULTS: Methylation, preferentially in the 3-hydroxyl group, was the major metabolic pathway of tanshinol. In rats, tanshinol also underwent considerable 3-O-sulfation, which appeared to be poor in human liver. These metabolites were mainly eliminated via renal excretion, which involved tubular secretion mainly by organic anion transporter (OAT) 1. The methylated metabolites had no vasodilatory activity. Entacapone-impaired methylation did not considerably increase systemic exposure to tanshinol in rats. The saturation of tanshinol methylation in rat liver could be predicted from the Michaelis constant of tanshinol for catechol-O-methyltransferase (COMT). Tanshinol had low affinity for human COMT and OATs; its methylated metabolites also had low affinity for the transporters. Tanshinol and its major human metabolite (3-O-methyltanshinol) exhibited negligible inhibitory activities against human cytochrome P450 enzymes, organic anion transporting polypeptides 1B1/1B3, multidrug resistance protein 1, multidrug resistance-associated protein 2, and breast cancer resistance protein. CONCLUSION: Tanshinol is mainly metabolized via methylation. Tanshinol and its major human metabolite have low potential for pharmacokinetic interactions with synthetic antianginal agents. This study will help define the risk of hyperhomocysteinemia related to tanshinol methylation.
Assuntos
Ácidos Cafeicos/farmacocinética , Fármacos Cardiovasculares/farmacocinética , Medicamentos de Ervas Chinesas/farmacocinética , Fígado/enzimologia , Salvia miltiorrhiza/química , Administração Oral , Animais , Biotransformação , Ácidos Cafeicos/administração & dosagem , Ácidos Cafeicos/isolamento & purificação , Ácidos Cafeicos/toxicidade , Fármacos Cardiovasculares/administração & dosagem , Fármacos Cardiovasculares/isolamento & purificação , Fármacos Cardiovasculares/toxicidade , Catecol O-Metiltransferase/metabolismo , Cromatografia Líquida , Sistema Enzimático do Citocromo P-450/metabolismo , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/toxicidade , Interações Ervas-Drogas , Humanos , Injeções Intravenosas , Túbulos Renais/metabolismo , Masculino , Espectrometria de Massas , Proteínas de Membrana Transportadoras/metabolismo , Metilação , Microssomos Hepáticos/enzimologia , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Fitoterapia , Raízes de Plantas , Plantas Medicinais , Ratos Sprague-Dawley , Eliminação Renal , Sulfatos/metabolismoRESUMO
Tanshinol has desirable antianginal and pharmacokinetic properties and is a key compound of Salvia miltiorrhiza roots (Danshen). It is extensively cleared by renal excretion. This study was designed to elucidate the mechanism underlying renal tubular secretion of tanshinol and to compare different ways to manipulate systemic exposure to the compound. Cellular uptake of tanshinol was mediated by human organic anion transporter 1 (OAT1) (Km, 121 µM), OAT2 (859 µM), OAT3 (1888 µM), and OAT4 (1880 µM) and rat Oat1 (117 µM), Oat2 (1207 µM), and Oat3 (1498 µM). Other renal transporters (human organic anion-transporting polypeptide 4C1 [OATP4C1], organic cation transporter 2 [OCT2], carnitine/organic cation transporter 1 [OCTN1], multidrug and toxin extrusion protein 1 [MATE1], MATE2-K, multidrug resistance-associated protein 2 [MRP2], MRP4, and breast cancer resistance protein [BCRP], and rat Oct1, Oct2, Octn1, Octn2, Mate1, Mrp2, Mrp4, and Bcrp) showed either ambiguous ability to transport tanshinol or no transport activity. Rats may be a useful model, to investigate the contribution of the renal transporters on the systemic and renal exposure to tanshinol. Probenecid-induced impairment of tubular secretion resulted in a 3- to 5-fold increase in the rat plasma area under the plasma concentration-time curve from 0 to infinity (AUC0-∞) of tanshinol. Tanshinol exhibited linear plasma pharmacokinetic properties over a large intravenous dose range (2-200 mg/kg) in rats. The dosage adjustment could result in increases in the plasma AUC0-∞ of tanshinol of about 100-fold. Tanshinol exhibited very little dose-related nephrotoxicity. In summary, renal tubular secretion of tanshinol consists of uptake from blood, primarily by OAT1/Oat1, and the subsequent luminal efflux into urine mainly by passive diffusion. Dosage adjustment appears to be an efficient and safe way to manipulate systemic exposure to tanshinol. Tanshinol shows low propensity to cause renal transporter-mediated herb-drug interactions.
Assuntos
Ácidos Cafeicos/metabolismo , Interações Ervas-Drogas/fisiologia , Túbulos Renais/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Linhagem Celular , Alimentos , Células HEK293 , Humanos , Túbulos Renais/efeitos dos fármacos , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Probenecid/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND AND PURPOSE: Ginsenosides are bioactive saponins derived from Panax notoginseng roots (Sanqi) and ginseng. Here, the molecular mechanisms governing differential pharmacokinetics of 20(S)-protopanaxatriol-type ginsenoside Rg1 , ginsenoside Re and notoginsenoside R1 and 20(S)-protopanaxadiol-type ginsenosides Rb1, Rc and Rd were elucidated. EXPERIMENTAL APPROACH: Interactions of ginsenosides with human and rat hepatobiliary transporters were characterized at the cellular and vesicular levels. A rifampin-based inhibition study in rats evaluated the in vivo role of organic anion-transporting polypeptide (Oatp)1b2. Plasma protein binding was assessed by equilibrium dialysis. Drug-drug interaction indices were calculated to estimate potential for clinically relevant ginsenoside-mediated interactions due to inhibition of human OATP1Bs. KEY RESULTS: All the ginsenosides were bound to human OATP1B3 and rat Oatp1b2 but only the 20(S)-protopanaxatriol-type ginsenosides were transported. Human multidrug resistance-associated protein (MRP)2/breast cancer resistance protein (BCRP)/bile salt export pump (BSEP)/multidrug resistance protein-1 and rat Mrp2/Bcrp/Bsep also mediated the transport of the 20(S)-protopanaxatriol-type ginsenosides. Glomerular-filtration-based renal excretion of the 20(S)-protopanaxatriol-type ginsenosides was greater than that of the 20(S)-protopanaxadiol-type counterparts due to differences in plasma protein binding. Rifampin-impaired hepatobiliary excretion of the 20(S)-protopanaxatriol-type ginsenosides was effectively compensated by the renal excretion in rats. The 20(S)-protopanaxadiol-type ginsenosides were potent inhibitors of OATP1B3. CONCLUSION AND IMPLICATIONS: Differences in hepatobiliary and in renal excretory clearances caused markedly different systemic exposure and different elimination kinetics between the two types of ginsenosides. Caution should be exercised with the long-circulating 20(S)-protopanaxadiol-type ginsenosides as they could induce hepatobiliary herb-drug interactions, particularly when patients receive long-term therapies with high-dose i.v. Sanqi or ginseng extracts.
Assuntos
Ginsenosídeos/farmacocinética , Interações Ervas-Drogas , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Feminino , Células HEK293 , Humanos , Rim/metabolismo , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Ratos Sprague-Dawley , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
Adverse effects of nickel ions being released into the living organism have resulted in development of high nitrogen nickel-free austenitic stainless steels for medical applications. Nitrogen not only replaces nickel for austenitic structure stability but also improves steel properties. The cell cytocompatibility, blood compatibility and cell response of high nitrogen nickel-free austenitic stainless steel were studied in vitro. The mechanical properties and microstructure of this stainless steel were compared to the currently used 316L stainless steel. It was shown that the new steel material had comparable basic mechanical properties to 316L stainless steel and preserved the single austenite organization. The cell toxicity test showed no significant toxic side effects for MC3T3-E1 cells compared to nitinol alloy. Cell adhesion testing showed that the number of MC3T3-E1 cells was more than that on nitinol alloy and the cells grew in good condition. The hemolysis rate was lower than the national standard of 5% without influence on platelets. The total intracellular protein content and ALP activity and quantification of mineralization showed good cell response. We conclude that the high nitrogen nickel-free austenitic stainless steel is a promising new biomedical material for coronary stent development.
Assuntos
Materiais Biocompatíveis , Ligas Dentárias/química , Nitrogênio/química , Aço Inoxidável , Células 3T3 , Fosfatase Alcalina/metabolismo , Animais , Plaquetas/citologia , Adesão Celular , Hemólise , Técnicas In Vitro , Camundongos , NíquelRESUMO
AIM: To investigate the pharmacokinetics and disposition of simmitecan (L-P) that was a water-soluble ester prodrug of chimmitecan (L-2-Z) with potent anti-tumor activities in different experimental animals, and to assess its drug-drug interaction potential. METHODS: SD rats were injected with a single iv bolus doses of L-P (3.75, 7.5 and 15 mg/kg). The pharmacokinetics, tissue distribution, excretion and metabolism of L-P and its active metabolite L-2-Z were studied through quantitative measurements and metabolite profiling with LC/MS. The binding of L-P and L-2-Z to rat plasma proteins was examined using an ultrafiltration method. Systemic exposures of beagle dogs to L-P as well as drug distribution in tumors of the nude mice xenograft model of human hepatic cancer SMMC-7721 cells were also examined. The metabolism of L-P by liver mcirosomal carboxylesterase in vitro was investigated in different species. The effects of L-P and L-2-Z on cytochrome P450 enzymes were examined using commercial screening kits. RESULTS: The in vivo biotransformation of L-P to L-2-Z showed a significant species difference, with a mean elimination half-life t1/2 of approximately 1.4 h in rats and 1.9 h in dogs. The systemic exposure levels of L-P and L-2-Z were increased in a dose-dependent manner. In rats, approximately 66% of L-P and 79% of L-2-Z were bound to plasma proteins. In rats and the nude mice bearing human hepatic cancers, most organ tissues had significantly higher concentrations of L-P than the corresponding plasma levels. In the tumor tissues, the L-P levels were comparable to those of plasma, whereas the L-2-Z levels were lower than the L-P levels. In rats, L-P was eliminated mainly via biliary excretion, but metabolism played an important role in elimination of the intact L-P. Finally, L-P and L-2-Z exerted moderate inhibition on the activity of CYP3A4 in vitro. CONCLUSION: L-P and L-2-Z have relatively short elimination half-lives and L-P is mainly eliminated via biliary excretion. The species difference in the conversion of L-P to L-2-Z and potential drug-drug interactions due to inhibition of CYP3A4 should be considered in further studies.
Assuntos
Antineoplásicos/farmacocinética , Camptotecina/análogos & derivados , Inibidores Enzimáticos/farmacocinética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Camptotecina/administração & dosagem , Camptotecina/farmacocinética , Camptotecina/farmacologia , Carboxilesterase/metabolismo , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Cães , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Feminino , Meia-Vida , Humanos , Injeções Intravenosas , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Nus , Microssomos Hepáticos/metabolismo , Pró-Fármacos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Simmitecan (L-P) is an anticancer ester prodrug, which involves activation to chimmitecan (L-2-Z). In the current study, a liquid chromatography/tandem mass spectrometry-based method was developed for simultaneous determination of L-P and L-2-Z in various plasma samples. Because L-P is rapidly converted to L-2-Z by blood carboxylesterase during and after sampling, which hampers accurate determination of L-P and L-2-Z in the biological samples, different carboxylesterase inhibitors were tested. As a result, bis(4-nitrophenyl)phosphate gave the best results with respect to inhibitory capability, hemolysis, and matrix effects and was used to deactivate blood carboxylesterases when sampling. The plasma samples were precipitated with acetonitrile and the resulting supernatants were separated using a pulse gradient method on a C18 column. Irinotecan and camptothecin were used as internal standards for quantification of L-P and L-2-Z, respectively. Protonated L-P, L-2-Z and their internal standards were generated by electrospray ionization and their precursor-product ion pairs (m/z 599â124, 405â361, 587â195, and 349â305, respectively) were used for measurement. The newly developed bioanalytical assay processed favorable accuracy and precision with lower limits of quantification of 2.1 nM for L-P and 3.4 nM for L-2-Z, and was successfully applied to pharmacokinetic studies in tumor-bearing nude mice, rats, and dogs. There are substantial species differences in the ester activity. The experimental strategies illustrated in our report may be adopted for measurement of other prodrugs (including irinotecan) or drugs subject to ester hydrolysis, as well as their metabolites, in biological matrices.
Assuntos
Antineoplásicos/sangue , Antineoplásicos/metabolismo , Camptotecina/análogos & derivados , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Cromatografia Líquida de Alta Pressão/métodos , Inibidores Enzimáticos/sangue , Pró-Fármacos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Camptotecina/sangue , Hidrolases de Éster Carboxílico/sangue , Cães , Inibidores Enzimáticos/metabolismo , Masculino , Camundongos , Camundongos Nus , Pró-Fármacos/metabolismo , Ratos , Ratos Sprague-Dawley , Sensibilidade e EspecificidadeRESUMO
In this communication, we report the development of a new ultra-performance liquid chromatographic/tandem mass spectrometry (UPLC-MS-MS) assay for measurement of amrubicin (an anthracycline anti-cancer agent) and its active metabolite, amrubicinol, in plasma. The enhanced electrospray ionization signal intensity of the analytes achieved by modifying the mobile phase with formic acid was associated with improvement in the lower limit of quantification. These favorable effects were electrolyte concentration-dependent. In order to maximize assay throughput, we used methanol protein precipitation to prepare the plasma samples, and simplified sample preparation by injecting 40 microL of the supernatant containing methanol at 87.5% (v/v) directly onto the UPLC column without any intermediary solvent evaporation step. The large-volume injection of highly organic supernatant sample increased matrix and elutropic effects, but these drawbacks were respectively overcome by using a 5mM formic acid-modified mobile phase and a new pulse gradient method. To our knowledge, this is the first report successfully using large-volume injection of strong organic samples with UPLC-MS-MS bioanalysis. The pulse gradient elution also resulted in band compression and enhanced the robustness of the chromatography. The promising new approach illustrated herein is extremely straightforward to optimize, and may be used for UPLC-MS-MS bioanalytical assay of other compounds.
Assuntos
Antraciclinas/sangue , Antineoplásicos/sangue , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Humanos , Sensibilidade e EspecificidadeRESUMO
Flavonoids are important naturally occurring polyphenols with antioxidant properties. In this study, we report the development of a liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method capable of simultaneously quantifying multiple active licorice flavonoids (including liquiritin apioside, liquiritin, liquiritigenin, isoliquiritin apioside, isoliquiritin, and isoliquiritigenin) in plasma. Electrospray ionization was used to efficiently generate precursor deprotonated molecules of all the analytes and the [M-H]- ions were used to produce characteristic product ions for MS/MS analysis. We found that inclusion of a very low concentration of HCOONH4 (0.01 per thousand) in the LC mobile phase dramatically improved the detection limit for the tested flavonoids and decreased the interference by matrix effects, which have been referred to as "LC-electrolyte effects." Liquid-liquid extraction with ethyl acetate was effective for isolation of all the analytes and resulted in the lowest matrix effects of several tested sample cleanup methods. This bioanalytical method showed good linearity between 0.32 ng/mL and 1 microg/mL analyte in 50-microL plasma samples. The accuracy and precision at different analyte concentrations varied from 85 to 110% and from 0.8 to 8.8%, respectively. Finally, we demonstrated the applicability of this method in a pilot pharmacokinetic study of rats receiving an oral dose of Xiaochaihu-tang, an important Chinese herbal remedy for chronic hepatitis. The use of a low concentration of HCOONH4 in the LC mobile phase could be used to improve LC-mass spectroscopy- or LC-MS/MS-based methods.
Assuntos
Flavonoides/sangue , Glycyrrhiza/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Administração Oral , Animais , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/farmacocinética , Ratos , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
A series of triaminotriazine derivatives (compounds 5a-f, 6a-x, and 7a-g) was designed, synthesized, and evaluated for their inhibition activities to colorectal cancer (CRC) cell lines (HCT-116 and HT-29). Most of the synthesized compounds demonstrated moderate anti-proliferatory effects on both HCT-116 and HT-29 cell lines at the concentration of 10 microM. The inhibitory activities against HCT-116 and HT-29 cell lines were discussed to develop the structure-activity relationships of this new series. Compounds 6l and 6o exhibited prominent inhibition activities toward HCT-116, with IC50s of 0.76 and 0.92 microM, respectively. The in vivo antitumor studies and pharmacokinetics of compound 6l showed that it might be a promising new hit for further development of antitumor agents.