Research Articles
N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine (CP-100,356) as a “chemical knock-out equivalent” to assess the impact of efflux transporters on oral drug absorption in the rat

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Abstract

The utility of the diaminoquinazoline derivative CP-100,356 as an in vivo probe to selectively assess MDR1/BCRP-mediated drug efflux was examined in the rat. CP-100,356 was devoid of inhibition (IC50 >50 µM) against major human P450 enzymes including P4503A4. In human MDR1-transfected MDCKII cells, CP-100,356 inhibited acetoxymethyl calcein (calcein-AM) uptake (IC50 ∼0.5 ± 0.07 µM) and digoxin transport (IC50 ∼1.2 ± 0.1 µM). Inhibition of prazosin transport (IC50 ∼1.5 ± 0.3 µM) in human BCRP-transfected MDCKII cells by CP-100,356 confirmed the dual MDR1/BCRP inhibitory properties. CP-100,356 was a weak inhibitor of OATP1B1 (IC50 ∼66 ± 1.1 µM) and was devoid of MRP2 inhibition (IC50 >15 µM). In vivo inhibitory effects of CP-100,356 in rats were examined after coadministration with MDR1 substrate fexofenadine and dual MDR1/BCRP substrate prazosin. Coadministration with increasing doses of CP-100,356 resulted in dramatic increases in systemic exposure of fexofenadine (36- and 80-fold increase in Cmax and AUC at a CP-100,356 dose of 24 mg/kg). Significant differences in prazosin pharmacokinetics were also discernible in CP-100,356-pretreated rats as reflected from a 2.6-fold increase in AUC. Coadministration of CP-100,356 and P4503A substrate midazolam did not result in elevations in systemic exposure of midazolam in the rat. The in vivo methodology should have utility in drug discovery in selective and facile assessment of the role of MDR1 and BCRP efflux transporters in oral absorption of new drug candidates. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4914–4927, 2009

Section snippets

INTRODUCTION

During initial stages of hit-to-lead phase in drug discovery, most new chemical entities (NCEs) suffer from poor systemic exposure after oral (p.o.) dosing in animals, necessitating a need for identifying issues limiting oral absorption. Besides first pass metabolism, other factors such aqueous solubility, membrane permeability, and active drug efflux can attenuate oral absorption. With regards to active drug efflux, members of the ATP-binding cassette (ABC) superfamily of efflux transporters

Materials

CP-100,356 (chemical and isomeric purity >99%) was synthesized at Pfizer Global Research and Development (Groton, CT). Detailed synthetic procedure for CP-100,356 and related analogs has been published.31 Solvents used for analysis were of analytical or HPLC grade (Fisher Scientific, Pittsburgh, PA). NADPH, fexofenadine, digoxin, and prazosin were purchased from Sigma–Aldrich Research (St. Louis, MO). Pooled human liver microsomes, probe P450 substrates (phenacetin, diclofenac, S-mephenytoin,

Cytochrome P450 Inhibition by CP-100,356

The ability of CP-100,356 to function as a competitive inhibitor of the five major human P450 enzymes namely P4501A2, 2C9, 2C19, 2D6, and 3A4 was examined in human liver microsomes. Considering that P4503A4 inhibition is dependent on substrate used in the microsomal assay,35 two structurally distinct probe substrates namely testosterone and midazolam were chosen for analysis of P4503A4 inhibition by CP-100,356. Virtually no inhibition (IC50 > 50 µM) of the catalytic activity of the individual

DISCUSSION

An initial literature survey for selecting MDR1 inhibitors in this work was not encouraging considering that many inhibitors possessed additional “off-target” activity including inhibition of P4503A4 and/or other transport proteins like MRP2 and OATP1B1 which are also known to be involved in active efflux or influx.18., 19., 20., 21., 22., 23. Therefore, we focused our attention on the diaminoquinazoline derivative CP-100,356, an optimized lead compound developed in-house as a MDR1 inhibitor.31

Acknowledgements

The authors thank Jonathan Bauman for technical assistance in the pharmacokinetic studies.

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