Bioluminescent imaging of drug efflux at the blood–brain barrier mediated by the transporter ABCG2

Abstract

ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood-brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that D-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter. P rovision of nutrients and maintenance of chemical homeostasis in the brain is performed by the endothelial cells of brain capillaries within a neurovascular unit termed the bloodbrain barrier (BBB) (1). In contrast to endothelial cells of capillaries elsewhere in the body, those in the brain are joined by tight junctions forming a physiologic barrier. Drug delivery to the brain depends on physicochemical characteristics such as lipophilicity, molecular weight, and ionic state. For many compounds, brain entry is lower than other tissues/organs because of the presence of ATP-binding cassette (ABC) efflux transporters at the apical surface of endothelial cells at the BBB (2, 3). These transporters maintain chemical homeostasis in the brain, and prevent toxins from interfering with neural processes by regulating the compounds that can enter the brain.ABC transporters contribute to the clinical challenge of drug delivery to the brain, and it has been estimated that only 2% of drug discovery compounds can cross the BBB to reach therapeutic targets (4). ABCG2 (also known as breast cancer resistance protein) and ABCB1 (also called P-glycoprotein) are the two most highly expressed efflux transporters at the BBB (5). Altered expression of ABC transporters at the BBB has been associated with a range of pathophysiological conditions (2, 6). ABC efflux transporters at the BBB also play a major role in limiting effective concentrations of chemotherapeutic agents to treat primary and metastatic tumors in the brain (7). ABCG2 has been shown to work in tandem with ABCB1 at the BBB (8, 9). However, its individual contribution is not understood.Molecular imaging allows the measurement of the individual contribution and function of transporters in vivo (10). Efflux of a substrate by transporters at the BBB is reflected by...

In Vivo Imaging of Human MDR1 Transcription in the Brain and Spine of MDR1-Luciferase Reporter Mice

“…The brain penetration of 14 C-D-luciferin in mice has been previously shown to be very low (Berger et al, 2008), which is consistent with the finding that luciferin is a Bcrp substrate (Zhang et al, 2007). Indeed, treatment with Bcrp inhibitors enhanced D-luciferin brain penetration of a low dose of D-luciferin (18 mg/kg) (Bakhsheshian et al, 2013), suggesting BBB Bcrp can limit brain availability of D-luciferin. At the D-luciferin concentrations used in most studies (and this one) (i.p.…”

“…3). Since D-luciferin is a substrate of Bcrp (Zhang et al, 2007), a transporter that can limit brain availability of D-luciferin at the BBB (Bakhsheshian et al, 2013), we treated mice with an oral dose of elacridar (100 mg/kg) for 4 hours in order to maximize the brain-to-plasma concentration (Sane et al, 2012). Elacridar increased the total MDR1-luc bioluminescent signal up to 2-fold in vehicle-and drug-treated mice (Fig.…”

The Inhibitor Ko143 Is Not Specific for ABCG2

“…In transgenic mice expressing the luciferase gene, activity was measured using the ABCG2 substrate D-luciferin before and after Ko143 administration (Bakhsheshian et al, 2013b). Because bioluminescence is not an option in the CNS of humans, a specific ABCG2 substrate that can be efficiently radiolabeled is needed instead.…”

“…1A) has been used over the past decade to examine the interaction between ABCG2 and pharmaceutical drugs in vitro and in vivo (Allen et al, 2002). For example, Ko143 was found to elevate mouse brain levels of the ABCG2 substrate D-luciferin in a bioluminescence-based imaging strategy (Bakhsheshian et al, 2013b). Given the frequent use of Ko143, characterization of Ko143 in terms of specificity in both human and mouse cell lines is of particular significance given the known species differences of ABC transporters.…”

Molecular Imaging of Membrane Transporters’ Activity in Cancer: a Picture is Worth a Thousand Tubes

“…The same group also demonstrated that D-luciferin cellular accumulation is facilitated by an uptake mechanism, likely OCTN1 (107). The work of Bakhsheshian et al (108) showed that D-luciferin entry into the brain is restricted by BCRP and that this process correlates with BCRP expression and function. This study provided the groundwork for a novel method to examine transporter function at the blood-brain barrier.…”