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Phosphorylation of Nucleoside Analogues (in Whole Cell assay)
Cell permeability, target specificity and drug metabolism are crucial cellular parameters upon which a drug efficiency depends. The cell metabolism of a drug is of major importance in the case of nucleoside analogues since most, if not all, of them act as prodrugs. To be active, once entered the cell, nucleoside analogues have to be phosphorylated by cell kinases. Mono-, di- and tri-phosphate nucleotide forms, as well as other possible cell-modified derivatives (e.g. deamination) of the corresponding nucleoside analogue act differently on the cell metabolism.
Cell permeability, target specificity and drug metabolism are crucial cellular parameters upon which a drug efficiency depends. The cell metabolism of a drug is of major importance in the case of nucleoside analogues since most, if not all, of them act as prodrugs. To be active, once entered the cell, nucleoside analogues have to be phosphorylated by cell kinases. Mono-, di- and tri-phosphate nucleotide forms, as well as other possible cell-modified derivatives (e.g. deamination) of the corresponding nucleoside analogue act differently on the cell metabolism.
Aim: Identification and quantification of a nucleoside analogue drug and its derivatives produced by the cell metabolism.
IMPORTANT: Client-specified alterations can be accommodated
"Cellular Pharmacology: Phosphorylation of Nucleoside Analogues" is routinely performed on cultured cell extract but the analysis of nucleotide analogue and derivatives can also be done, for instance for pharmacokinetics studies, on other biological samples depending on the feasibility of the analysis.
Nucleotides and nucleosides Analysis: The separation and analytical procedures developed by NOVOCIB are particularly relevant to study the cellular pharmacology of nucleoside analogues. They have been optimized for several nucleoside analogs, particularly with ribavirin. However, the separation and analytical procedures must be specifically adapted to every nucleoside analogue.
Cell culture and treatment: The choice of the cell line and culture conditions has been optimized to get highly reproducible results. Assays are usually done with human hepatoma cell line Huh7. Cells are grown in DMEM supplemented with FCS (5%), glutamine (1mM), sodium pyruvate (1mM) and maintained in exponential phase. Cells are seeded on 10cm-dishes and allowed to adhere overnight. The drug is added next day at the agreed concentration and at a cell confluence of about 50%.
Other samples than cultured cells can be analyzed: blood cells, body fluid...
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IMPORTANT: Client-specified alterations can be accommodated
"Cellular Pharmacology: Phosphorylation of Nucleoside Analogues" is routinely performed on cultured cell extract but the analysis of nucleotide analogue and derivatives can also be done, for instance for pharmacokinetics studies, on other biological samples depending on the feasibility of the analysis.
Nucleotides and nucleosides Analysis: The separation and analytical procedures developed by NOVOCIB are particularly relevant to study the cellular pharmacology of nucleoside analogues. They have been optimized for several nucleoside analogs, particularly with ribavirin. However, the separation and analytical procedures must be specifically adapted to every nucleoside analogue.
Cell culture and treatment: The choice of the cell line and culture conditions has been optimized to get highly reproducible results. Assays are usually done with human hepatoma cell line Huh7. Cells are grown in DMEM supplemented with FCS (5%), glutamine (1mM), sodium pyruvate (1mM) and maintained in exponential phase. Cells are seeded on 10cm-dishes and allowed to adhere overnight. The drug is added next day at the agreed concentration and at a cell confluence of about 50%.
Other samples than cultured cells can be analyzed: blood cells, body fluid...
Do not hesitate to contact us for any matter of feasibility!
| Figure 1: Separation of Ribavirin, Ribavirin mono-, di- and tri-phosphate in cultured cell extracts
Blue: non treated, Red: 3mM RBV |
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| Figure 2: Separation of Gemcitabine (dFdC), Gemcitabine di- and tri-phosphate in cultured cell extracts
Blue: non treated, Red: 200 µM dFdC |
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| Figure 3: Separation of Cytarabine (AraC), Cytarabine mono-, di- and tri-phosphate in cultured cell extracts
Blue: non treated, Red: 130 µM AraC, Green: 260 µM AraC |
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