RESUMO
Incorporating bioactive molecules into synthetic ceramic scaffolds is challenging. In this study, to enhance bone regeneration, a magnesium phosphate (MgP) ceramic scaffold was incorporated with a novel indene compound, KR-34893. KR-34893 induced the deposition of minerals and expression of osteoblast marker genes in primary human bone marrow mesenchymal stem cells (BMSCs) and a mouse osteoblastic MC3T3-E1 cell line. Analysis of the mode of action showed that KR-34893 induced the phosphorylation of MAPK/extracellular signal-regulated kinase and extracellular signal-regulated kinase, and subsequently the expression of bone morphogenetic protein 7, accompanied by SMAD1/5/8 phosphorylation. Accordingly, KR-34893 was incorporated into an MgP scaffold prepared by 3D printing at room temperature, followed by cement reaction. KR-34893-incorporated MgP (KR-MgP) induced the expression of osteoblast differentiation marker genes in vitro. In a rat calvaria defect model, KR-MgP scaffolds enhanced bone regeneration and increased bone volume compared with MgP scaffolds, as assessed by micro-computed tomography and histological analyses. In conclusion, we developed a method for producing osteoinductive MgP scaffolds incorporating a bioactive organic compound, without high temperature sintering. The KR-MgP scaffolds enhanced osteoblast activation in vitro and bone regeneration in vivo.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Cerâmica/farmacologia , Compostos de Magnésio/farmacologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Fosfatos/farmacologia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cerâmica/química , Humanos , Técnicas In Vitro , Indenos/química , Indenos/farmacologia , Compostos de Magnésio/química , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Fosfatos/química , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual/métodosRESUMO
Transcriptional coactivator with PDZ-binding motif (TAZ) is considered an attractive target for osteoporosis, obesity, and muscle regeneration. TM-53, a promising TAZ modulator, was recently introduced, and here, we developed a rapid, precise, and reliable analytical method for TM-53 and characterized its pharmacokinetic properties in rat plasma. The hybrid triple quadrupole/linear ion trap coupled to liquid chromatography method was developed and validated to quantify TM-53. Additionally, TM-53 concentrations in plasma were analyzed, and its pharmacokinetic parameters were calculated by non-compartmental analysis. Multiple reaction monitoring at m/z 569.4â207.1 showed the most sensitive signals for TM-53, and the linear scope of the standard curve was between 1.5ng/mL and 500ng/mL. The intra- and inter-day precisions of the quality control samples were <15%, and their accuracies were ranged from 86.2% to 111.0%. Furthermore, the matrix effects, extraction recoveries, and process efficiencies of this analytical method for evaluating TM-53 in rat plasma were 99.1%, 99.9%, and 99.1% respectively. In short- and long-term stability studies, TM-53 showed good stability under frozen conditions, but TM-53 hydrolysis in the plasma matrix was observed following storage at room temperature. This analytical method was successfully applied for pharmacokinetic analysis of TM-53 in rat plasma and demonstrated excellent sensitivity, selectivity, precision, and accuracy. These data indicated that this method can be applied for further preclinical studies of TM-53.
Assuntos
Benzimidazóis/farmacologia , Benzimidazóis/farmacocinética , Domínios PDZ/efeitos dos fármacos , Tetrazóis/farmacologia , Tetrazóis/farmacocinética , Ativação Transcricional/efeitos dos fármacos , Animais , Benzimidazóis/sangue , Cromatografia Líquida/métodos , Masculino , Plasma/química , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem/métodos , Tetrazóis/sangueRESUMO
This study evaluated the pharmacokinetics of the novel TAZ modulator TM-25659 in rats following intravenous and oral administration at dose ranges of 0.5-5 mg/kg and 2-10 mg/kg, respectively. Plasma protein binding, plasma stability, liver microsomal stability, CYP inhibition, and transport in Caco-2 cells were also evaluated. After intravenous injection, systemic clearance, steady-state volumes of distribution, and half-life were dose-independent, with values ranging from 0.434-0.890 mL · h(-1) · kg(-1), 2.02-4.22 mL/kg, and 4.60-7.40 h, respectively. Mean absolute oral bioavailability was 50.9% and was not dose dependent. Recovery of TM-25659 was 43.6% in bile and <1% in urine. In pharmacokinetic modeling studies, the three-compartment (3C) model was appropriate for understanding these parameters in rats. TM-25659 was stable in plasma. Plasma protein binding was approximately 99.2%, and was concentration-independent. TM-25659 showed high permeation of Caco-2 cells and did not appear to inhibit CYP450. TM-25659 was metabolized in phase I and II steps in rat liver microsomes. In conclusion, the pharmacokinetics of TM-25659 was characterized for intravenous and oral administration at doses of 0.5-5 and 2-10 mg/kg, respectively. TM-25659 was eliminated primarily by hepatic metabolism and urinary excretion.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Tetrazóis/farmacocinética , Administração Oral , Algoritmos , Animais , Proteínas Sanguíneas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/sangue , Células CACO-2 , Inibidores das Enzimas do Citocromo P-450 , Interações Medicamentosas , Humanos , Injeções Intravenosas , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Cinética , Masculino , Taxa de Depuração Metabólica , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Ratos , Ratos Sprague-Dawley , Tetrazóis/administração & dosagem , Tetrazóis/sangue , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
TM-25659 compound, a novel TAZ modulator, is developed for the control of bone loss and obesity. TAZ is known to bind to a variety of transcription factors to control cell differentiation and organ development. A selective and sensitive method was developed for the determination of TM-25659 concentrations in rat plasma. The drug was measured by liquid chromatography-tandem mass spectrometry after liquid-liquid extraction with ethyl acetate. TM-25659 and the internal standard imipramine were separated on a Hypersil GOLD C18 column with a mixture of acetonitrile-ammonium formate (10 mM) (90:10, v/v) as the mobile phase. The ions m/z 501.2â207.2 for TM-25659 and m/z 281.0â86.0 for imipramine in multiple reaction monitoring mode were used for the quantitation. The calibration range was 0.1-100 µg/ml with a correlation coefficient greater than 0.99. The lower limit of quantitation of TM-25659 in rat plasma was 0.1 µg/ml. The percent recoveries of TM-25659 and imipramine were 98.6% and 95.7% from rat plasma, respectively. The intra- and inter-batch precisions were 3.17-15.95% and the relative error was 0.38-10.82%. The developed assay was successfully applied to a pharmacokinetic study of TM-25659 administered intravenously (10 mg/kg) to rats.
Assuntos
Fármacos Antiobesidade/sangue , Fármacos Antiobesidade/farmacocinética , Conservadores da Densidade Óssea/sangue , Conservadores da Densidade Óssea/farmacocinética , Compostos Bicíclicos Heterocíclicos com Pontes/sangue , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Cromatografia Líquida , Espectrometria de Massas em Tandem , Tetrazóis/sangue , Tetrazóis/farmacocinética , Fatores de Transcrição/metabolismo , Acetatos/química , Acetonitrilas/química , Aciltransferases , Animais , Fármacos Antiobesidade/administração & dosagem , Conservadores da Densidade Óssea/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Calibragem , Cromatografia Líquida/normas , Estabilidade de Medicamentos , Formiatos/química , Imipramina/química , Injeções Intravenosas , Limite de Detecção , Masculino , Ratos , Ratos Sprague-Dawley , Padrões de Referência , Reprodutibilidade dos Testes , Solventes/química , Espectrometria de Massas em Tandem/normas , Tetrazóis/administração & dosagemRESUMO
This paper describes the design, syntheses, and biological evaluations of novel ATP-sensitive potassium channel (K(ATP)) openers, benzopyranyl indoline and indole derivatives. Among those, two enantiomers of indoline-2-carboxylic ethyl esters (14, 18) showed the best cardioprotective activities both in vitro and in vivo, while their vasorelaxation potencies were very low (concentration for 50% inhibition of vasorelaxation >30 microM). The cardioprotective effect of 14 was completely reversed by 5-hydroxydecanoate, a selective mitochondrial K(ATP) blocker, indicating its provable protective mechanism through the mitochondrial K(ATP) opening. In addition, we performed conformational analyses using 2D-NMR, X-ray crystallography and molecular modeling to study the structure-activity relationships in this series of compounds.