Effect of P-Glycoprotein on the Rat Intestinal Permeability and Metabolism of the BDDCS Class 1 Drug Verapamil

Abstract

The Biopharmaceutics Drug Disposition Classification System (BDDCS) predicts intestinal transporter effects to be clinically insignificant following oral dosing for highly soluble and highly permeable/metabolized drugs (class 1 drugs). We investigated the effect of inhibiting P-glycoprotein (P-gp) on the in vitro rat intestinal permeability (Papp) and metabolism of the class 1 drug verapamil. Jejunal segments from Sprague-Dawley rats fasted overnight were mounted in Ussing chambers filled with 10 mL of Krebs-Ringer buffer (KRB). For P-gp inhibition studies, GG918 0.5 μM was added to the KRB solution. The experiment started by the addition of verapamil (1 or 10 μM) to either apical or basolateral sides. Samples from verapamil donor and receiver compartments were collected at 30 s and 0.166, 0.5, 1, 1.83 and 3 h after the start of the experiment. Analysis of verapamil and its major metabolite, norverapamil, in the samples and intracellularly at 3 h was performed by HPLC. The same experiment was repeated with norverapamil 10 μM (verapamil metabolite), digoxin 100 nM (positive control for P-gp activity) and atorvastatin 1 and 10 μM (example of a class 2 drug). For 1 μM verapamil, efflux ratio (B to A Papp/A to B Papp) was 4.6 and markedly decreased by GG918 (efflux ratio = 1.1). For 10 μM verapamil efflux ratio was 4.1 (control) vs 1.8 (GG918), comparable to the change seen for digoxin 100 nM with an efflux ratio of 3.6 (control) vs 1.6 (with GG918) and atorvastatin (efflux ratio of 5.2 and 3.0 for atorvastatin 1.0 and 10 μM, respectively, changed to 1.0 and 0.65 with GG918). The changes observed in the norverapamil 10 μM experiment were also significant, where efflux ratio decreased from 13.5 (control) to 1.5 (GG918). The extraction ratio (ER) of 10 μM verapamil to norverapamil decreased from 0.41 after an apical dose to 0.21 after a basolateral dose, but was unaffected by the incubation with GG918. The results suggest that P-gp inhibition has an effect on class 1 drug verapamil and class 2 drug atorvastatin Papp in the rat intestine. Moreover, a stronger P-gp effect on the Papp of the more polar norverapamil metabolite was observed. Papp changes caused by the P-gp inhibitor GG918 do not affect the extent of verapamil metabolism.

Avaliação dos mecanismos envolvidos na permeabilidade de fármacos antirretrovirais por meio do modelo de perfusão <i>in situ </i>em ratos

“…Para os estudos por meio do modelo de perfusão in situ, o uso de animais geneticamente modificados (knockout) permite investigar a interação entre o fármaco e os transportadores específicos, o que possibilita prever os impactos nos processos de absorção oral, além de responder possíveis dúvidas que não são resolvidas por meio dos modelos in vitro e/ou ex vivo. Além disso, a comparação entre animais da linhagem selvagem e os geneticamente modificados permite avaliar os parâmetros farmacocinéticos envolvidos TANG et al, 2013) (ESTUDANTE et al, 2013a).…”

“…Embora as células MDCK-MDR1 permitam estudar com maior clareza a interação fármaco-P-gp, é praticamente impossível concluir a respeito do impacto clínico com base somente nos resultados obtidos neste modelo in vitro. Assim, a associação entre os experimentos em MDCK-MDR1 e o modelo de perfusão in situ permitirá obter conclusões mais significativas em relação aos mecanismos de transporte, uma vez que os segmentos intestinais utilizados expressam os carreadores de maneira biorrelevante ESTUDANTE et al, 2013a;.…”

“…O BCRP (breast cancer resistance protein) e a MRP2 (multidrug resistance associated protein 2) estão localizados na porção apical dos enterócitos e atuam nos processos secretórios de algumas substâncias (AYLIN ARICI et al, 2010;AMIDON, 2009b;ESTUDANTE et al, 2013a;GAO et al, 2014;KIMURA et al, 2007;NIETO;BRIÑÓN, 2011;SCHINKEL;JONKER, 2012;TANG et al, 2013).…”