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12.05.2017 | Original Article | Ausgabe 1/2017

Cancer Chemotherapy and Pharmacology 1/2017

Cellular effect and efficacy of carfilzomib depends on cellular net concentration gradient

Zeitschrift:
Cancer Chemotherapy and Pharmacology > Ausgabe 1/2017
Autoren:
Julia Schäfer, Lukas Welti, Anja Seckinger, Jürgen Burhenne, Dirk Theile, Johanna Weiss
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1007/​s00280-017-3335-4) contains supplementary material, which is available to authorized users.
Dirk Theile and Johanna Weiss contributed equally.

Abstract

Purpose

The cellular interrelation between intracellular concentrations of unbound carfilzomib, a second-generation proteasome inhibitor, and subsequent proteasome inhibition and effect on cell viability are unknown and were evaluated for two different exposure regimens: A high dose bolus regime of 500 nM for 1 h followed by 47 h in drug-free media vs. 48-h continuous exposure to 10 nM.

Methods

Eight multiple myeloma cell lines were exposed to either one of the two exposure regimens. We quantified the intracellular unbound carfilzomib fraction up to 48 h with a new ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS) method. Intracellular concentrations were compared to simultaneously determined cell viability (AlamarBlue® assay) and proteasomal subunit activity (ProGlo™ assay).

Results

Within the first 10 min, the proportional intracellular enrichment of unbound carfilzomib was higher (313 nM; 62.6%) for the exposure to 500 nM compared to 10 nM (1.93 nM; 19.3%). However, after 1 h, an intracellular/extracellular concentration equilibrium was reached with both settings. At low exposure concentrations, drug removal after 1 h diminished carfilzomib efficacy. Moreover, proteasomal activity recovered when exposed to 10 nM for 48 h. However, when exposure concentration was high (500 nM) proteasome inhibition was complete and sustained even with drug removal after 1 h.

Conclusions

We demonstrated that the carfilzomib concentration gradient determines cellular uptake kinetics. The uptake kinetics in turn affects binding, saturation, and activity of the proteasome. Together, these data underscore the importance of steep concentrations for the in vitro efficacy of carfilzomib.

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Zusatzmaterial
Supplementary material 1 (DOCX 684 kb)
280_2017_3335_MOESM1_ESM.docx
Supplementary material 2 (PDF 68 kb)
280_2017_3335_MOESM2_ESM.pdf
Supplementary material 3 (PDF 133 kb)
280_2017_3335_MOESM3_ESM.pdf
Literatur
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