Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR‐inhibitors in peripheral human blood

Dieterlen, Maja‐Theresa; Bittner, Hartmuth B.; Klein, S.; Salisch, Sandy von; Mittag, Anja; Tárnok, Attila; Dhein, Stefan; Mohr, Friedrich W.; Barten, Markus J.

doi:10.1002/cyto.b.21005

Cited by 32 publications

(21 citation statements)

References 22 publications

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“…24 In addition, the feasibility of real-time, direct pharmacodynamic monitoring by flow cytometry was demonstrated during clinical trials combining intensive chemotherapy and signal transduction inhibitors, 25 but further validation in patients after solid organ transplantation is urgently needed.…”

Section: Discussionmentioning

confidence: 99%

Development of a Sensitive Phospho-p70 S6 Kinase ELISA to Quantify mTOR Proliferation Signal Inhibition

Hartmann

Keller

et al. 2013

Therapeutic Drug Monitoring

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Background: Drug blood levels can only serve as a surrogate because of the lack of information on the drug's direct pharmacological effects in the individual patient. Measurement of the mammalian target of rapamycin (mTOR) activity dependent on the phosphorylation status of p70 S6 kinase (p70 S6K) offers a practical way for monitoring pharmacodynamic drug activity, with the potential to better assess the state of immunosuppression in individual patients. Material and Methods:Here, we established a novel in vitro model system by treating Jurkat cells and peripheral blood mononuclear cells with different concentrations of sirolimus after stimulation with phorbol 12-myristate 13-acetate.Results: A dose-dependent reduction of the p70 S6K phosphorylation status was demonstrated by Western blot and a newly established enzyme-linked immunosorbent assay (ELISA). Relative phospho-p70 S6K values from ELISA and relative densities from Western blot analysis in peripheral blood mononuclear cells revealed a strong correlation (Spearman correlation coefficient r s = 0.7, P = 0.01). Finally, parallel assays confirmed a sirolimus dosedependent reduction of cytokine production and cell proliferation in the in vitro model. Conclusions:Pharmacodynamic monitoring of mTOR inhibition with a p70 S6K ELISA could guide mTOR inhibitor immunosuppression therapy toward a more individualized therapy. The usage of this technique now has to be evaluated in a clinical series of patients.

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Section: Discussionmentioning

confidence: 99%

Development of a Sensitive Phospho-p70 S6 Kinase ELISA to Quantify mTOR Proliferation Signal Inhibition

Hartmann

Keller

et al. 2013

Therapeutic Drug Monitoring

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show abstract

“…However, the effect could be minimized with leucocyte fixation before lysis of erythrocytes [12]. This concept was addressed by Dieterlen et al, who validated an assay to measure the level of p-S6RP in WBS, and the assay showed satisfactory results regarding specificity, linearity, and precision [14]. Unfortunately, no published data regarding the performance of the assay with samples from patients under immunosuppressive therapy are available yet.…”

Section: Discussionmentioning

confidence: 96%

“…However, the sophisticated protocol, long preparation time and large sample volume limit the use of this assay on a wider scale. Consequently, the use of WBS represents a convinient alternative [14].…”

Section: Discussionmentioning

confidence: 99%

Performance of a phosphoflow assay to determine phosphorylation of S6 ribosomal protein as a pharmacodynamic read out for mTOR inhibition

Abdel-Kahaar

Kabakchiev

Hartmann

et al. 2016

Clinical Biochemistry

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“…The effect of mTOR inhibitors such as sirolimus or everolimus suppresses the phosphorylation of S6RP, and therefore S6RP should be a candidate target to evaluate the pharmacodynamic effects of these drugs on T-cell activation. Dieterlen et al (2012) demonstrated that phosphoflow analysis revealed that sirolimus suppressed p-S6RP in human T cells in a dose-dependent manner with a half-maximal inhibitory concentration (IC(50)) at 19.8 nM and a maximal inhibitory effect (I(max) %) at 91.9 %. Other immunosuppressive agents, such as cyclosporin A, mycophenolic acid, and dexamethasone, are not able to inhibit mTOR-related S6RP phosphorylation.…”

Section: Phosphoflowmentioning

confidence: 99%

Flow Cytometry as Platform for Biomarker Discovery and Clinical Validation

Millán¹,

Brunet²

2015

Biomarkers in Disease: Methods, Discoveries and Applications

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Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR‐inhibitors in peripheral human blood

Cited by 32 publications

References 22 publications

Development of a Sensitive Phospho-p70 S6 Kinase ELISA to Quantify mTOR Proliferation Signal Inhibition

Development of a Sensitive Phospho-p70 S6 Kinase ELISA to Quantify mTOR Proliferation Signal Inhibition

Performance of a phosphoflow assay to determine phosphorylation of S6 ribosomal protein as a pharmacodynamic read out for mTOR inhibition

Flow Cytometry as Platform for Biomarker Discovery and Clinical Validation

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