RESUMO
The presence of serum in biological samples often negatively impacts the quality of in vitro assays. However, assays tolerant of serum are useful for assessing the in vivo availability of a small molecule for its target. Electrophysiology assays of ion channels are notoriously sensitive to serum because of their reliance on the interaction of the plasma membrane with a recording electrode. Here we investigate the tolerance of an automated electrophysiology assay for a voltage-gated potassium (K(V)) channel to serum and purified plasma proteins. The delayed rectifier channel, K(V)2.1, stably expressed in Chinese hamster ovary cells produces large, stable currents on the IonWorks Quattro platform (MDS Analytical Technologies, Sunnyvale, CA), making it an ideal test case. K(V)2.1 currents recorded on this platform are highly resistant to serum, allowing recordings in as high as 33% serum. Using a set of compounds related to the K(V) channel blocker, 4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone, we show that shifts in compound potency with whole serum or isolated serum proteins can be reliably measured with this assay. Importantly, this assay is also relatively insensitive to plasma, allowing the creation of a bioassay for inhibitors of K(V)2.1 channel activity. Here we show that such a bioassay can quantify the levels of the gating modifier, guangxitoxin-1E, in plasma samples from mice dosed with the peptide. This study demonstrates the utility of using an automated electrophysiology platform for measuring serum shifts and for bioassays of ion channel modulators.
Assuntos
Proteínas Sanguíneas/metabolismo , Canais de Potássio de Retificação Tardia/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Autoanálise , Células CHO , Cricetinae , Cricetulus , Interpretação Estatística de Dados , Diálise , Eletrofisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Ligação ProteicaRESUMO
Various beta-amino amides containing triazolopiperazine heterocycles have been prepared and evaluated as potent, selective, orally active dipeptidyl peptidase IV (DPP-4) inhibitors. These compounds display excellent oral bioavailability and good overall pharmacokinetic profiles in preclinical species. Moreover, in vivo efficacy in an oral glucose tolerance test in lean mice is demonstrated.
Assuntos
Amidas/química , Amidas/farmacologia , Inibidores da Dipeptidil Peptidase IV , Piperazinas/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Amidas/síntese química , Amidas/farmacocinética , Animais , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Modelos Moleculares , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacocinética , RatosRESUMO
A novel series of beta-amino amides incorporating fused heterocycles, i.e., triazolopiperazines, were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. (2R)-4-Oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine (1) is a potent, orally active DPP-IV inhibitor (IC(50) = 18 nM) with excellent selectivity over other proline-selective peptidases, oral bioavailability in preclinical species, and in vivo efficacy in animal models. MK-0431, the phosphate salt of compound 1, was selected for development as a potential new treatment for type 2 diabetes.