Stephanie van Hoppe

23 ABCG2 & ABCB1 transport afatinib and restrict its oral availability and brain accumulation I N T R ODU C T I ON ATP-binding cassette (ABC) transporters form a superfamily of transmembrane transport proteins. Two members, ABCB1 (MDR1, P-glycoprotein or P-gp) and ABCG2 (Breast Cancer Resistance Protein, BCRP), are especially important in pharmacokinetics [1, 2]. Both affect the disposition of a wide variety of endogenous and exogenous compounds, including many anticancer drugs. They are expressed at pharmacologically important sites such as the apical membranes of enterocytes, hepatocytes and renal tubular epithelial cells, where they can limit gastrointestinal absorption or mediate direct intestinal, hepatic, or renal excretion of their substrates [1, 2]. Furthermore, ABCB1 and ABCG2 are expressed on apical membranes of barriers protecting sanctuary tissues such as the blood-brain, blood-placenta and blood-testis barriers, where substrates are pumped directly out of the epithelial or endothelial cells into the blood. Consequently, several chemotherapeutic agents that are ABCB1 and/or ABCG2 substrates have restricted brain accumulation [1-4]. Improving brain accumulation of drugs is of high interest in the clinic due to the fact that current therapies are often inefficient in eradicating brain tumors or brain metastases situated in whole or in part behind an intact blood-brain barrier (BBB) [3, 4]. Afatinib (Gilotrif/Giotrif, BIBW2992) is an orally administered tyrosine kinase inhibitor (TKI) for the treatment of patients with distinct types of metastatic non-small cell lung carcinoma (NSCLC), whose tumor genes have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations [5]. Approved by the FDA in July 2013, afatinib is a highly selective, irreversible inhibitor of the EGFR and of the human epidermal growth-factor receptor (HER)-2 [6]. It is barely metabolized, with a total recovery percentage of 89.5% of unchanged drug in the urine and feces over 72 h after dosing in humans [7]. Both EGFR and HER-2 are members of the receptor tyrosine kinase (RTK) superfamily [8] and overexpression of both receptors is often found in human cancers such as gliomas, carcinomas of the breast, ovaries, bladder, and lung, including NSCLC. The overexpression, due to gene amplification, is often associated with higher EGFR pathway signaling activity, increased proliferation of cancer cells and reduced apoptosis [9]. It has further been shown preliminarily in cell lines, that afatinib appears effective against a subset of these various cancers overexpressing EGFR and HER-2 [10-12], making it an attractive candidate for further clinical research. Several published studies indicate that afatinib can interact with ABCB1 and ABCG2 [13-15]. It has for instance been shown that afatinib affects ABCB1 and ABCG2 in several cancer cell lines by blocking substrate transport and/or down-regulating mRNA and protein expression of the transporters [13, 15]. However, these same studies reported conflicting data on the ability of afatinib to inhibit ABCB1. FDA and EMA registration