Bortezomib, carfilzomib and ixazomib do not mediate relevant transporter-based drug-drug interactions.

In order to optimize the clinical application of an increasing number of proteasome inhibitors, investigations into the differences between their respective pharmacodynamic and pharmacokinetic profiles, including their ability to act as a perpetrator in drug-drug interactions, are warranted. Therefore, in the present in vitro study, it was investigated whether bortezomib, carfilzomib and ixazomib are able to alter the expression, and/or the activity, of specific drug transporters generally relevant for pharmacokinetic drug-drug interactions. Through induction experiments, the current study demonstrated that the aforementioned three proteasome inhibitors do not induce mRNA expression of the transporter genes ATP binding cassette (ABC)B1, C1, C2 and G2 in the LS180 cell line, which was used as a model for systemic induction. By contrast, in certain myeloma cell lines, ixazomib provoked minor alterations in individual transporter gene expression. None of the proteasome inhibitors tested relevantly inhibited drug transporters within the range of physiological plasma concentrations. Taken together, transporter-based drug-drug interactions are unlikely to be a primary concern in the clinical application of the tested compounds.

Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays.

Firefly luciferase-based reporter gene assays are the most commonly used assays to investigate the transcriptional regulation of gene expression. However, direct interaction of tested compounds with the firefly luciferase leading to altered enzymatic activity may lead to misinterpretation of experimental data. When investigating the proteasome inhibitors bortezomib, carfilzomib, and ixazomib, we observed increased luminescence for bortezomib and ixazomib, but not for carfilzomib, in a pregnane-X-receptor (PXR) reporter gene assay, which was inconsistent with the mRNA expression levels of the main PXR target gene CYP3A4. To further scrutinize this phenomenon, we performed experiments with constitutively expressed firefly luciferase and demonstrated that the increase in cellular firefly luciferase activity is independent from PXR activation or CYP3A4 promoter. Using cell-free assays with recombinant firefly luciferase enzyme, we made the counterintuitive observation that firefly luciferase activity is inhibited by bortezomib and ixazomib in a reversible and competitive manner. This inhibition stabilizes the firefly luciferase enzyme against proteolytic degradation (e.g., toward trypsin), thereby increasing its half-life with subsequent enhancement of total cellular luminescence that eventually mimicked PXR-driven luciferase induction. These data show that particular compounds can strikingly interfere with firefly luciferase and once more illustrate the importance of careful interpretation of data obtained from luciferase-based assays.

Cellular uptake kinetics of bortezomib in relation to efficacy in myeloma cells and the influence of drug transporters.

PURPOSE: Despite overall successful application to multiple myeloma patients, clinical efficacy of the proteasome inhibitor bortezomib is typically challenged by primary and secondary resistance of unknown origin. So far, the potential impact of intracellular concentrations on drug efficacy of bortezomib and the influence of drug transporters are unknown. METHODS: We determined cellular bortezomib kinetics in nine myeloma cell lines using ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry. The potential influence of drug transporters on the uptake kinetics observed in these cell lines was investigated by testing substrate characteristics of bortezomib for several transporters in over-expressing model cells. Additionally, transporter mRNA expression was quantified in myeloma cell lines by real-time polymerase chain reaction (RT-PCR). RESULTS: All myeloma cells revealed an extensive intracellular bortezomib accumulation (47.5-183 ng/ml) exceeding extracellular concentrations (0.04-0.17 ng/ml) by more than factor 1,000. Only organic anion-transporting polypeptide 1B1 facilitated the uptake in over-expressing cells, however, to a negligible extent (factor 1.36). Bortezomib efflux via P-glycoprotein was confirmed by demonstrating reduced sensitivity (IC50 11.6 vs. 2.8 ng/ml) and intracellular concentrations (-56.1%) in over-expressing cells compared to controls. RT-PCR revealed a varying but overall weak transporter expression in the studied myeloma cells without any correlation to intracellular concentrations. Although principally valid as demonstrated in the P-glycoprotein over-expressing cell model, there was no significant correlation between intracellular concentrations and bortezomib efficacy in myeloma cell lines. CONCLUSION: Differences in intracellular concentrations in myeloma cell lines neither result from variable transporter expression nor represent the main factor determining bortezomib efficacy in vitro.

Stability of the proteasome inhibitor bortezomib in cell based assays determined by ultra-high performance liquid chromatography coupled to tandem mass spectrometry.

Bortezomib represents the first clinically approved proteasome inhibitor for multiple myeloma. Research conducted on its intracellular kinetics in target cells and on possibly related mechanisms of resistance is sparse so far. We therefore developed and validated a highly sensitive ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS) method for bortezomib quantification within cultured myeloma cells and media. Fast gradient UPLC based on a BEH C18 column (1.7 µm particle size) with aqueous formic acid and acetonitrile as mobile phase. Selective extraction procedures using protein precipitation extraction (PPE) and liquid-liquid extraction (LLE) were established and compared. Extracted bortezomib was quantified by positive electrospray tandem mass spectrometry using deuterated D8-bortezomib as internal standard. The calibrated ranges were 0.5-2500 pg per sample. For LLE, overall accuracies varied between 99.2% and 112% (medium) and 89.9% and 111% (cells), while overall precision ranged from 1.13% to 13.0% (medium) and 2.80% to 12.7% (cells), respectively. Recovery rates (cells/medium) were >77%/>65% for LLE and >89%/63% for PPE. Matrix effects were generally lower for LLE compared to PPE. Regardless of the extraction method, retrievable amounts of bortezomib were considerably reduced after 24h of incubation (0.2, 1, 5, and 25 nM). Revealing greater dependence on the extent of acidification, retrieval of bortezomib can be increased distinctly in acidified solution or acidified culture medium. Thus, particular attention needs to be paid to the occurring bortezomib degradation in neutral culture medium since correct quantification of intracellular bortezomib can only be achieved in relation to the corresponding extracellular concentration.