Colchicine effect on P-glycoprotein expression and activity: in silico and in vitro studies.
Colchicine is a P-glycoprotein (P-gp) substrate that induces its expression, thus increasing the risk for unexpected pharmacokinetic interactions with this drug. Because increased P-gp expression does not always correlate with increased activity of this efflux pump, we evaluated the changes in both P-gp expression and activity induced by colchicine using an in vitro model. Caco-2 cells were incubated with 0.1-100 μM colchicine up to 96 h. Cytotoxicity was evaluated by the MTT and LDH leakage assays, P-gp expression and activity were evaluated by flow cytometry and P-gp ATPase activity was measured in MDR1-Sf9 membrane vesicles. Furthermore, colchicine fitting in P-gp induction and competitive inhibition pharmacophore hypothesis, and docking studies evaluating the interaction between colchicine and P-gp drug binding pocket were tested in silico. Significant cytotoxicity was noted after 48 h. At 24 h a significant increase in P-gp expression was observed, which was not accompanied by an increase in transport activity. Moreover, colchicine significantly increased P-gp ATPase activity, demonstrating to be actively transported by the pump. New pharmacophores were constructed to predict P-gp modulatory activity. Colchicine fitted both the P-gp induction and competitive inhibition models. In silico, colchicine was predicted to bind to the P-gp drug-binding pocket suggesting a competitive mechanism of transport. These results show that colchicine induced P-gp expression in Caco-2 cells but the activity of the protein remained unchanged, highlighting the need to simultaneously evaluate P-gp expression and activity. With the newly constructed pharmacophores, new drugs can be initially screened in silico to predict such potential pharmacokinetic interactions.
REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Electronic address: rsilva@ff.up.pt.
Renata
R
Silva
REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Electronic address: helenacarmo@ff.up.pt.
Helena
H
Carmo
REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Electronic address: vvilasboas@ff.up.pt.
Vânia
V
Vilas-Boas
REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Electronic address: daniel.barbosa@ff.up.pt.
Daniel José
DJ
Barbosa
Centro de Química Medicinal (CEQUIMED-UP), Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Electronic address: apalmeira@ff.up.pt.
Andreia
A
Palmeira
Centro de Química Medicinal (CEQUIMED-UP), Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal; Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. Electronic address: esousa@ff.up.pt.
Emília
E
Sousa
REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Electronic address: felixdc@ff.up.pt.
Félix
F
Carvalho
REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Electronic address: mlbastos@ff.up.pt.
Maria de Lourdes
Mde L
Bastos
REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Electronic address: remiao@ff.up.pt.
Fernando
F
Remião
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects; D018938:Caco-2 Cells; D003078:Colchicine / Q000494:pharmacology*; D003198:Computer Simulation; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008954:Models, Biological
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003078:Colchicine
ATPase activity; Caco-2 cells; Colchicine; In silico; P-glycoprotein induction; P-glycoprotein transport activity