Expression of the ABC drug transporters Pgp and ABCG2 has been associated with the development of drug resistance to first generation BCR-ABL kinase inhibitors such as Gleevec 12,40-42
.Tasigna, a second generation BCR-ABL kinase inhibitor has been developed as more potent and specific inhibitor that also inhibits the kinase activity of mutant Gleevec-resistant BCR-ABL kinases. Several conflicting reports have been published about the above kinase inhibitors being substrates of ABC drug transporters. While some groups have shown that resistance to Gleevec is due to its low intracellular accumulation 33, 43, 44
, other reports have suggested that the resistance is not dependent on drug efflux by ABC drug transporters 45, 46
. In the case of Tasigna, it has also been shown that Tasigna interacts with ABCG2 with a very high affinity, is a substrate of this drug transporter, and reverses drug resistance mediated by Pgp and ABCG2 17, 33
, while others reported that cellular accumulation of Tasigna in cell lines and primary CD34(+) CML cells is not mediated by active uptake or efflux by major drug transporters and expression of Pgp may not be enough to confer drug resistance to Tasigna 15, 47
. However, the nature of the interaction of Tasigna with Pgp is not very well understood. Moreover, the effectiveness of Tasigna in inhibiting BCR-ABL kinase activity in Pgp- and ABCG2- expressing leukemic cells has also not been studied mechanistically.
The data from this study indicate that Tasigna inhibits Pgp activity by interacting with the substrate binding pocket of Pgp, similar to results obtained previously with ABCG2 ( and 33
). This inhibition of Pgp and ABCG2 function at low micromolar and nanomolar concentrations, respectively, can influence the toxicity or bioavailability of other drugs or metabolites in humans that are substrates of ABC drug transporters. Systemic concentrations of Tasigna after administration of the recommended twice daily dose of 400 mg ranges from 1.7 to 3.6 μM 48
. Therefore, the above experimental concentrations of Tasigna can be achieved systemically in CML patients treated with Tasigna, which may further inhibit Pgp physiologically, representing another cellular target for Tasigna that may arise in a secondary phenotypic outcome. Direct evidence for this is presented in our experiments with rat brain capillaries, where Tasigna inhibits Pgp and ABCG2 function resulting in decreased efflux activity of these transporters at the blood-brain barrier.
A fluorescent derivative of Tasigna was synthesized to study its direct interaction with ABC drug transporters ( and supplementary information
). We showed for the first time that the total intracellular levels of BODIPY® FL Tasigna in Pgp- and ABCG2-expressing cells were lower than in cells that do not express these transporters, indicating that it is actively pumped out of these cells (). This efflux of BODIPY® FL Tasigna was inhibited by specific inhibitors of Pgp and ABCG2 in both in vitro
and ex vivo
assays ( and ). This fluorescent derivative also inhibited the BCR-ABL kinase activity although with a lower efficiency that could be attributed to the structural alteration of the molecule, possibly resulting in decreased affinity to its target ().
H]-labeled Tasigna was also synthesized to test whether it is a transport substrate for Pgp. The polarized LLC-PK1 cells expressing Pgp were used to determine [3
H] Tasigna transport. The data clearly demonstrated a net active transport of [3
H]-Tasigna from the basal to apical side of the polarized LLC-PK1 cells expressing Pgp and Pgp inhibitors significantly blocked the directional flux of radiolabeled Tasigna (). These data validate the results obtained with BODIPY® FL Tasigna. However, Hegedus et al 15
, based on an HPLC-MS assay, reported that there was no significant difference in the amount of intracellular Tasigna in control and Pgp-expressing K562 cells (non polarized cells). At present, we do not know the reason for failure to observe transport in non-polarized cells, but most likely it could be the result of high background due to non-specific binding of Tasigna to cells (also mentioned by these authors).
Our results with both the fluorescent derivative and [3
H]-Tasigna collectively show that this TKI is transported by Pgp and ABCG2. Based on these data, we conclude that Tasigna is a less potent inhibitor of BCR-ABL kinase activity in Pgp- or ABCG2-expressing drug-resistant CML cells compared to sensitive control CML cells because it is transported by Pgp and ABCG2, resulting in reduced intracellular accumulation. This is an important finding given the clinical application of Tasigna, which is administered to drug-resistant CML and ALL patients. This reduced effectiveness becomes more important in a scenario where the expression of ABC transporters is increased in patients who have been treated with a drug that is a transporter substrate and the incidence of secondary resistance increases because the drug becomes ineffective due to active efflux by these transporters. In fact, despite the initial clinical effectiveness of Tasigna in CML resistant patients, lack of response to Tasigna therapy and development of resistance have also been reported 14, 49
A non-toxic concentration of Tasigna in combination with the Pgp substrate doxorubicin was able to inhibit drug resistance mediated by Pgp in MDR1-transfected HEK cells (). These observations could have dual implications in the clinic. Administering Tasigna with drugs that are substrates of Pgp could result in increased systemic concentrations of these drugs. Alternatively, administering Tasigna with drugs that inhibit Pgp function could lead to increased Tasigna toxicity. The FDA summary review for approval of Tasigna reflects these two possibilities, stating that treatment with Tasigna may result in increased toxicity or drug/drug interactions if administered with drugs which are either Pgp substrates or inhibitors (http://www.fda.gov/cder/foi/nda/2007/022068s000_SumR.pdf
). Increased oral bioavailability or antitumor response of anticancer drugs in combination with other TKIs such as Gleevec, Gefitinib or Lapatinib has already been shown by a number of studies 50-53
In conclusion, our findings indicate that interaction of Tasigna with Pgp and ABCG2 may be an important factor in the treatment of CML and AML patients, as efflux of Tasigna by Pgp and ABCG2 may result in resistance to this drug. Moreover, interaction with Pgp and ABCG2 can modify the pharmacokinetic and toxicity profile of Tasigna in vivo. Additional studies to specifically investigate the interaction of Tasigna with ABC transporters may be warranted to determine its efficacy in the treatment of Gleevec-resistant CML and ALL. In addition, BODIPY FL® Tasigna may represent an important tool for researchers to follow the efficacy, pharmacokinetics, and toxicity of Tasigna in preclinical and clinical models. A similar approach may be used to study whether a given TKI is transported by Pgp or ABCG2. It could also be adapted for use in in vivo imaging studies to assess the accumulation of xenobiotics in the brain that are transported by Pgp or ABCG2. To our knowledge, this is the first study that not only demonstrates direct transport of Tasigna by Pgp in transepithelial monolayer assays but also reports synthesis and characterization of a fluorescent derivative of a TKI that is not only active in inhibiting the target kinase but can also be used to study its interaction with other proteins such as ABC drug transporters in cancer cells.