Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Péptido 1 Similar al Glucagón/análogos & derivados , Péptido Hidrolasas/metabolismo , Disponibilidad Biológica , Diabetes Mellitus Tipo 2/enzimología , Péptido 1 Similar al Glucagón/farmacocinética , Péptido 1 Similar al Glucagón/farmacología , HumanosRESUMEN
Recombinant peptide technology offers a promising means alternative to chemical synthesis and natural extraction of peptides. The bottleneck in the process of recombinant peptide production is the paucity of efficient purification protocols to eliminate heterogeneity of the desired preparation. Here, we introduce a combination strategy to facilitate purification of recombinant therapeutic peptide via native chemical ligation and chemical cleavage on a solid support. In this study, one promising therapeutic peptide called for type-2 diabetes, GLP-1(7-37), was prepared with high yield and purity without an expensive HPLC purification. Furthermore, this method is also useful for the preparation of isotopically labeled NMR peptide samples. Hopefully, this strategy combining chemical ligation with chemical cleavage on a solid support will ameliorate the production of important recombinant pharmaceutical peptides.
Asunto(s)
Péptido 1 Similar al Glucagón/aislamiento & purificación , Hipoglucemiantes/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Escherichia coli/metabolismo , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/genética , Hipoglucemiantes/química , Microesferas , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Gabapentin is thought to be absorbed from the intestine of humans and animals by a low-capacity solute transporter localized in the upper small intestine. Saturation of this transporter at doses used clinically leads to dose-dependent pharmacokinetics and high interpatient variability, potentially resulting in suboptimal drug exposure in some patients. XP13512 [(+/-)-1-([(alpha-isobutanoyloxyethoxy)carbonyl] aminomethyl)-1-cyclohexane acetic acid] is a novel prodrug of gabapentin designed to be absorbed throughout the intestine by high-capacity nutrient transporters. XP13512 was stable at physiological pH but rapidly converted to gabapentin in intestinal and liver tissue from rats, dogs, monkeys, and humans. XP13512 was not a substrate or inhibitor of major cytochrome P450 isoforms in transfected baculosomes or liver homogenates. The separated isomers of XP13512 showed similar cleavage in human tissues. The prodrug demonstrated active apical to basolateral transport across Caco-2 cell monolayers and pH-dependent passive permeability across artificial membranes. XP13512 inhibited uptake of (14)C-lactate by human embryonic kidney cells expressing monocarboxylate transporter type-1, and direct uptake of prodrug by these cells was confirmed using liquid chromatography-tandem mass spectrometry. XP13512 inhibited uptake of (3)H-biotin into Chinese hamster ovary cells overexpressing human sodium-dependent multivitamin transporter (SMVT). Specific transport by SMVT was confirmed by oocyte electrophysiology studies and direct uptake studies in human embryonic kidney cells after tetracycline-induced expression of SMVT. XP13512 is therefore a substrate for several high-capacity absorption pathways present throughout the intestine. Therefore, administration of the prodrug should result in improved gabapentin bioavailability, dose proportionality, and colonic absorption compared with administration of gabapentin.