Ensartinib (X-396) is a promising tyrosine kinase inhibitor currently undergoing advanced clinical evaluation for the treatment of non-small cell lung cancer. In this work, we investigate possible interactions of this promising drug candidate with ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 biotransformation enzymes (CYPs), which play major roles in multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). Accumulation studies showed that ensartinib is a potent inhibitor of ABCB1 and ABCG2 transporters. Additionally, incubation experiments with recombinant CYPs showed that ensartinib significantly inhibits CYP3A4 and CYP2C9. Subsequent molecular docking studies confirmed these findings. Drug combination experiments demonstrated that ensartinib synergistically potentiates the antiproliferative effects of daunorubicin, mitoxantrone, and docetaxel in ABCB1, ABCG2, and CYP3A4-overexpressing cellular models, respectively. Advantageously, ensartinib's antitumor efficiency was not compromised by the presence of MDR-associated ABC transporters, although it acted as a substrate of ABCB1 in Madin-Darby Canine Kidney II (MDCKII) monolayer transport assays. Finally, we demonstrated that ensartinib had no significant effect on the mRNA-level expression of examined transporters and enzymes in physiological and lung tumor cellular models. In conclusion, ensartinib may perpetrate clinically relevant pharmacokinetic DDIs and modulate ABCB1-, ABCG2-, and CYP3A4-mediated MDR. The in vitro findings presented here will provide a valuable foundation for future in vivo investigations.

• Keywords
• ABC transporter, cancer, cytochrome P450, drug-drug interaction, ensartinib, multidrug resistance,
• Publication type
• Journal Article MeSH

Brivanib, a promising tyrosine kinase inhibitor, is currently undergoing advanced stages of clinical evaluation for solid tumor therapy. In this work, we investigated possible interactions of this novel drug candidate with ABC drug efflux transporters and cytochrome P450 (CYP450) drug-metabolizing enzymes that participate in cancer multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). First, in accumulation experiments with various model substrates, we identified brivanib as an inhibitor of the ABCB1, ABCG2, and ABCC1 transporters. However, in subsequent combination studies employing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide proliferation assays in both Madin-Darby canine kidney II (MDCKII) and A431 cellular models, only ABCG2 inhibition was revealed to be able to synergistically potentiate mitoxantrone effects. Advantageous to its possible use as MDR antagonist, brivanib's chemosensitizing properties were not impaired by activity of any of the MDR-associated ABC transporters, as observed in comparative viability assay in the MDCKII cell sublines. In incubation experiments with eight recombinant CYP450s, we found that brivanib potently inhibited CYP2C subfamily members and the CYP2B6 isoform. Finally, in induction studies, we demonstrated that brivanib upregulated ABCB1 and CYP1A2 messenger RNA levels in systemic cell models, although this interaction was not significantly manifested at a functional level. In conclusion, brivanib exhibits potential to cause clinically relevant pharmacokinetic DDIs and act as a modulator of ABCG2-mediated MDR. Our findings might be used as an important background for subsequent in vivo investigations and pave the way for the safe and effective use of brivanib in oncological patients.

• Keywords
• ABC drug efflux transporter, brivanib, cytochrome P450, drug−drug interaction, multidrug resistance,
• MeSH
• ATP Binding Cassette Transporter, Subfamily G, Member 2 antagonists & inhibitors   MeSH
• Alanine analogs & derivatives   pharmacology   MeSH
• Biotransformation drug effects   MeSH
• Cell Line MeSH
• Madin Darby Canine Kidney Cells MeSH
• Drug Resistance, Neoplasm drug effects   MeSH
• Cytochrome P-450 Enzyme Inhibitors pharmacology   MeSH
• Drug Interactions physiology   MeSH
• Humans MeSH
• Drug Resistance, Multiple drug effects   MeSH
• Neoplasm Proteins antagonists & inhibitors   MeSH
• ATP Binding Cassette Transporter, Subfamily B metabolism   MeSH
• Dogs MeSH
• Cytochrome P-450 Enzyme System metabolism   MeSH
• Triazines pharmacology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATP Binding Cassette Transporter, Subfamily G, Member 2 MeSH
• ABCG2 protein, human MeSH Browser
• Alanine MeSH
• brivanib MeSH Browser
• Cytochrome P-450 Enzyme Inhibitors MeSH
• Neoplasm Proteins MeSH
• ATP Binding Cassette Transporter, Subfamily B MeSH
• Cytochrome P-450 Enzyme System MeSH
• Triazines MeSH

Cyclin-dependent kinase inhibitors (CDKi) have high potential applicability in anticancer therapy, but various aspects of their pharmacokinetics, especially their interactions with drug efflux transporters, have not yet been evaluated in detail. Thus, we investigated interactions of five CDKi (purvalanol A, olomoucine II, roscovitine, flavopiridol and SNS-032) with the ABCB1 transporter. Four of the compounds inhibited efflux of two ABCB1 substrates, Hoechst 33342 and daunorubicin, in MDCKII-ABCB1 cells: Olomoucine II most strongly, followed by roscovitine, purvalanol A, and flavopiridol. SNS-032 inhibited ABCB1-mediated efflux of Hoechst 33342 but not daunorubicin. In addition, purvalanol A, SNS-032 and flavopiridol lowered the stimulated ATPase activity in ABCB1 membrane preparations, while olomoucine II and roscovitine not only inhibited the stimulated ATPase but also significantly activated the basal ABCB1 ATPase, suggesting that these two CDKi are ABCB1 substrates. We further revealed that the strongest ABCB1 inhibitors (purvalanol A, olomoucine II and roscovitine) synergistically potentiate the antiproliferative effect of daunorubicin, a commonly used anticancer drug and ABCB1 substrate, in MDCKII-ABCB1 cells as well as in human carcinoma HCT-8 and HepG2 cells. We suggest that this pronounced synergism is at least partly caused by (i) CDKi-mediated inhibition of ABCB1 transporter leading to increased intracellular retention of daunorubicin and (ii) native cytotoxic activity of the CDKi. Our results indicate that co-administration of the tested CDKi with anticancer drugs that are ABCB1 substrates may allow significant dose reduction in the treatment of ABCB1-expressing tumors.

• MeSH
• Adenosine Triphosphatases metabolism   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Cell Line MeSH
• Cyclin-Dependent Kinases antagonists & inhibitors   MeSH
• Daunorubicin pharmacology   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• ATP Binding Cassette Transporter, Subfamily B antagonists & inhibitors   genetics   MeSH
• Cell Proliferation drug effects   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Purines pharmacology   MeSH
• Gene Expression Regulation drug effects   MeSH
• Roscovitine MeSH
• Drug Synergism MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 6-((3-chloro)anilino)-2-(isopropyl-2-hydroxyethylamino)-9-isopropylpurine MeSH Browser
• ABCB1 protein, human MeSH Browser
• Adenosine Triphosphatases MeSH
• Cyclin-Dependent Kinases MeSH
• Daunorubicin MeSH
• Protein Kinase Inhibitors MeSH
• RNA, Messenger MeSH
• olomoucine II MeSH Browser
• ATP Binding Cassette Transporter, Subfamily B MeSH
• Antineoplastic Agents MeSH
• Purines MeSH
• Roscovitine MeSH

Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.

• MeSH
• ATP Binding Cassette Transporter, Subfamily G, Member 2 MeSH
• ATP-Binding Cassette Transporters metabolism   MeSH
• Biological Transport MeSH
• Cell Line MeSH
• Drug Resistance, Neoplasm drug effects   MeSH
• Neoplasm Proteins metabolism   MeSH
• ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism   MeSH
• ATP Binding Cassette Transporter, Subfamily B MeSH
• Dogs MeSH
• Purines pharmacokinetics   MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 6-((3-chloro)anilino)-2-(isopropyl-2-hydroxyethylamino)-9-isopropylpurine MeSH Browser
• ATP Binding Cassette Transporter, Subfamily G, Member 2 MeSH
• ATP-Binding Cassette Transporters MeSH
• ABCB1 protein, human MeSH Browser
• ABCG2 protein, human MeSH Browser
• Neoplasm Proteins MeSH
• olomoucine II MeSH Browser
• ATP Binding Cassette Transporter, Subfamily B, Member 1 MeSH
• ATP Binding Cassette Transporter, Subfamily B MeSH
• Purines MeSH