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1.
Bioorg Med Chem ; 17(19): 6926-36, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19747833

RESUMO

Signal transducers and activators of transcription 6 (STAT6) is an important transcription factor in interleukin (IL)-4 signaling pathway and a key regulator of the type 2 helper T (Th2) cell immune response. Therefore, STAT6 may be an excellent therapeutic target for allergic conditions, including asthma and atopic diseases. Previously, we reported 4-aminopyrimidine-5-carboxamide derivatives as STAT6 inhibitors. To search for novel STAT6 inhibitors, we synthesized fused bicyclic pyrimidine derivatives and identified a 7H-pyrrolo[2,3-d]pyrimidine derivative as a STAT6 inhibitor. Optimization of the pyrrolopyrimidine derivatives led to identification of 2-[4-(4-{[7-(3,5-difluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]amino}phenyl)piperazin-1-yl]acetamide (24, AS1810722) which showed potent STAT6 inhibition and a good CYP3A4 inhibition profile. Compound 24 also inhibited in vitro Th2 differentiation without affecting type 1 helper T (Th1) cell differentiation and eosinophil infiltration in an antigen-induced mouse asthmatic model after oral administration.


Assuntos
Pirimidinas/síntese química , Pirróis/síntese química , Fator de Transcrição STAT6/antagonistas & inibidores , Administração Oral , Animais , Asma/tratamento farmacológico , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Eosinófilos/efeitos dos fármacos , Humanos , Imunidade , Camundongos , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirróis/farmacologia , Pirróis/uso terapêutico , Relação Estrutura-Atividade , Células Th2/efeitos dos fármacos
2.
J Pharm Sci ; 108(3): 1085-1089, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30339864

RESUMO

Lilly Laboratories cell porcine kidney 1 (LLC-PK1) cells transfected with human P-glycoprotein (LLC-PK1-P-gp) are widely used in transport assays to identify drug candidates that function as substrates of this efflux transporter. Endogenous transporters expressed in LLC-PK1 cells may complicate the interpretation of findings from P-gp-mediated transport assays. We investigated the impact of porcine breast cancer resistance protein (Bcrp) in P-gp-mediated transport assays in LLC-PK1 cells. Porcine Bcrp mRNA was detected in both LLC-PK1 wildtype (WT) and LLC-PK1-P-gp cells by quantitative RT-PCR. To investigate the activity and impact of porcine Bcrp, we conducted transport assays using 6 typical BCRP substrates in LLC-PK1 cells. Efflux ratios (ER) of the 6 BCRP substrates in LLC-PK1 WT cells were >2, and were reduced in the presence of the BCRP inhibitor Ko143. The efflux activities of the 6 BCRP substrates were confirmed using MDCKII cells transfected with human BCRP. Net ERs of prazosin and fluvastatin, dual substrates of P-gp and BCRP, determined by dividing ERs in LLC-PK1-P-gp cells by those in LLC-PK1 WT cells, were <2, but increased to >2 in the presence of Ko143. These results indicated that endogenous Bcrp in LLC-PK1 cells was involved in the transport of BCRP substrates and may interfere with the identification of P-gp substrates.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Dicetopiperazinas/farmacologia , Fluvastatina/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Células LLC-PK1 , Prazosina/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Suínos , Transfecção
3.
Assay Drug Dev Technol ; 13(2): 79-87, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25785771

RESUMO

Transport assays using P-gp-expressing cell lines are commonly used to identify P-gp substrates and inhibitors in drug discovery. The P-gp cell-based assay is performed manually in 12- or 24-well plates and requires improvement for high-throughput screening. In this study, we established an efficient semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-gp. The protocol was optimized with a microplate washer for exchanging media and buffer to enhance throughput. P-gp substrates and inhibitors, and the paracellular marker Dextran Texas Red® were used to validate the 96-well transport assay. Cell monolayer integrity after washing by a microplate washer was confirmed by measuring paracellular permeability of Dextran Texas Red. Permeability and net flux ratio of the P-gp substrates and the inhibitory potency of the P-gp inhibitors were comparable in 24- and 96-well plates. The regression value of net flux ratio of P-gp substrates was high between the two formats (r²=0.99). The optimized 96-well transport assay using the microplate washer was found to be an efficient high-throughput screening tool that provided the same quality data as the 24-well plate for the identification of P-gp substrates and inhibitors in drug discovery.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Bioensaio/instrumentação , Avaliação Pré-Clínica de Medicamentos/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Rim/efeitos dos fármacos , Rim/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular , Desenho de Fármacos , Desenho de Equipamento , Análise de Falha de Equipamento , Citometria de Fluxo/instrumentação , Rim/citologia , Robótica/instrumentação , Suínos , Transfecção
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