No abstract

The ineffectiveness and failing of chemotherapeutic treatments are often associated with multidrug resistance (MDR). MDR is primarily linked to the overexpression of ATP-binding cassette (ABC) transporter proteins in cancer cells. ABCG2 (ATP-binding cassette subfamily G member 2, also known as the breast cancer resistance protein (BCRP)) mediates MDR by an increased drug efflux from the cancer cells. Therefore, the inhibition of ABCG2 activity during chemotherapy ought to improve the efficacy of the administered anti-cancer agents by reversing MDR or by enhancing the agents’ pharmacokinetic properties. Significant efforts have been made to develop novel, powerful, selective, and non-toxic inhibitors of BCRP. However, thus far the clinical relevance of BCRP-selective MDR-reversal has been unsuccessful, due to either adverse drug reactions or significant toxicities in vivo. We here report a facile access towards carboranyl quinazoline-based inhibitors of ABCG2. We determined the influence of different methoxy-substitution patterns on the 2-phenylquinazoline scaffold in combination with the beneficial properties of an incorporated inorganic carborane moiety. A series of eight compounds was synthesized and their inhibitory effect on the ABCG2-mediated Hoechst transport was evaluated. Molecular docking studies were performed to better understand the structure-protein interactions of the novel inhibitors, exhibiting putative binding modes within the inner binding site. Further, the most potent, non-toxic compounds were investigated for their potential to reverse ABCG2-mediated mitoxantrone (MXN) resistance. Of these five evaluated compounds, N-(closo-1,7-dicarbadodecaboran(12)-9-yl)-6,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-quinazolin-4-amine (DMQCd) exhibited the strongest inhibitory effect towards ABCG2 in the lower nanomolar ranges. Additionally, DMQCd was able to reverse BCRP-mediated MDR, making it a promising candidate for further research on hybrid inorganic-organic compounds.

A battery of in vitro methods has been developed for the prediction of acute oral toxicity, to reduce the number of animals used for this purpose. However, the results of these tests correlate more closely with lethal serum concentrations than with lethal doses. To address this issue, we have further evaluated the HPCT-1E3 model, which may be better able to emulate toxicokinetic factors that occur in vivo, due to the presence in these hepatocytoma cells of endogenous transmembrane carriers and a basal activity of xenobiotic metabolism. IC50 values produced by using the MTT test after a 48-hour incubation with 20 randomly-selected MEIC substances, correlated better with human oral LD50 values than with LC50 data, supporting this hypothesis. As with other models, the toxicity of receptor-specific rather than cytotoxic substances, for example digoxin, was underpredicted. When digoxin was removed from the correlation analysis, the coefficient of determination (r2) improved to 0.81, and none of remaining chemicals were wrongly predicted by more than one order of magnitude. IC50 values obtained with HepG2 cells under similar conditions (MEIC Test No. 3, 24 hours, MTT) correlated with human LD50 data with a r2 value of 0.55. A direct comparison of HPCT-1E3 and HepG2 cells further suggested that the differences between them may be due to transport processes. In conclusion, the HPCT-1E3 model may be valuable in improving the prediction of lethal doses, rather than lethal serum concentrations.

No abstract

Intrathecal methotrexate (MTX) has been associated with severe neurotoxicity. Because carrier-associated removal of MTX from the cerebrospinal fluid (CSF) into blood remains undefined, we determined the expression and function of MTX transporters in rat choroid plexus (CP). MTX neurotoxicity usually manifests as seizures requiring therapy with antiepileptic drugs (AEDs) such as phenobarbital (PB). Because we have demonstrated that PB reduces activity of MTX influx carrier reduced folate carrier (Rfc1) in liver, we investigated the influence of the AEDs PB, carbamazepine (CBZ), or gabapentin on Rfc1-mediated MTX transport in CP. Reverse transcriptase-polymerase chain reaction and Western blot analysis showed similar expression of the MTX influx carrier Rfc1 and organic anion transporter 3 or efflux transporter multidrug resistance-associated protein 1 (Mrp1) and breast cancer resistance protein (Bcrp) in rat CP tissue and choroidal epithelial Z310 cells. Confocal microscopy revealed subcellular localization of Rfc1 and Bcrp at the apical and of Mrp1 at the basolateral CP membrane. Uptake, efflux, and inhibition studies indicated MTX transport activity of Rfc1, Mrp1, and Bcrp. PB and CBZ but not gabapentin significantly inhibited Rfc1-mediated uptake of MTX in CP cells. Studies on the regulatory mechanism showed that PB significantly inhibited Rfc1 translation but did not alter carrier gene expression. Altogether, removal of intrathecal MTX across the blood-CSF barrier may be achieved through Rfc1-mediated uptake from the CSF followed by MTX extrusion into blood, particularly via Mrp1. Antiepileptic treatment with PB or CBZ causes post-transcriptional down-regulation of Rfc1 activity in CP. This mechanism may result in enhanced MTX toxicity in patients with cancer who are receiving intrathecal MTX chemotherapy by reduced CSF clearance of the drug.