RESUMEN
Collagen tripeptide (CTP) is a collagen-derived compound containing a high concentration of tripeptides with a Gly-X-Y sequence. In this study, the concentrations and metabolites of CTP were monitored in rat plasma after its administration. We performed a quantitative analysis using high-performance liquid chromatography tandem mass spectrometry according to the isotopic dilution method with stable isotopes. We confirmed that the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp were transported into the plasma. Dipeptides, which are generated by degradation of the N- or C-terminus of the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp, were also present in plasma. The plasma kinetics for peroral and intraperitoneal administration was similar. In addition, tripeptides and dipeptides were detected in no-administration rat blood. The pharmacokinetics were monitored in rats perorally administered with Gly-[(3)H]Pro-Hyp. Furthermore, CTP was incorporated into tissues including skin, bone, and joint tissue. Thus, administering collagen as tripeptides enables efficient absorption of tripeptides and dipeptides.
Asunto(s)
Absorción Fisicoquímica , Colágeno/química , Oligopéptidos/administración & dosificación , Oligopéptidos/sangre , Administración Oral , Animales , Cromatografía Líquida de Alta Presión , Inyecciones Intraperitoneales , Cinética , Masculino , Espectrometría de Masas , Oligopéptidos/metabolismo , Oligopéptidos/farmacocinética , Ratas , Ratas WistarRESUMEN
The domains of bispecific diabodies (BsDbs) can be ordered in four different ways; however, the influence of domain order on the cytotoxicity of BsDbs that retarget immune cells against tumor cells had not been addressed. We previously reported the marked antitumor effects of a humanized BsDb that targets epidermal growth factor receptor and CD3 (hEx3-Db). Here, we rearranged the domains of hEx3-Db to examine the influence of domain order on the function of BsDbs. We successfully prepared homogenous dimers of hEx3-Db in all four domain configurations. Interestingly, all three rearranged hEx3s inhibited cancer growth more effectively than did the original hEx3-Db, in which both components were in variable heavy domain (VH)-variable light domain (VL) order (redesignated as hEx3-HL), and the highest effects were observed with hEx3-LH (hEx3-Db with both components in VL-VH order). In addition, hEx3-LH had comparable in vitro growth inhibitory effects to those of the tandem single-chain variable fragment (scFv) format of hEx3-Db (hEx3-tandem scFv (taFv)), which we previously showed to have greater cytotoxicity than does hEx3-HL. Flow cytometry suggested that the enhanced cytotoxicity of hEx3-LH is attributable to structural superiority for cross-linking, similar to that of hEx3-taFv. Furthermore, hEx3-LH inhibited cancer growth in mice more effectively than did hEx3-taFv; this difference may be due to differences in antibody stability. Our results show that merely rearranging the domain order of BsDbs can enhance their effects beyond those with structural format conversion.
Asunto(s)
Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Complejo CD3/inmunología , Receptores ErbB/inmunología , Neoplasias/terapia , Animales , Anticuerpos Biespecíficos/inmunología , Antineoplásicos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , Neoplasias/inmunología , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
α-L-Arabinofuranosidase from the hyperthermophilic bacterium Thermotoga maritima (Tm-AFase) is an extremely thermophilic enzyme belonging to glycoside hydrolase family 51. It can catalyze the transglycosylation of a novel glycosyl donor, 4,6-dimethoxy-1,3,5-triazin-2-yl (DMT)-ß-D-xylopyranoside. In this study we determined the crystal structures of Tm-AFase in substrate-free and complex forms with arabinose and xylose at 1.8-2.3 Å resolution to determine the architecture of the substrate binding pocket. Subsite -1 of Tm-AFase is similar to that of α-L-arabinofuranosidase from Geobacillus stearothermophilus, but the substrate binding pocket of Tm-AFase is narrower and more hydrophobic. Possible substrate binding modes were investigated by automated docking analysis.