Time dependent HPLC analysis of the product ratio of enzymatically reduced prodrug CB1954 by a modified and immobilised nitroreductase.

Ball, Patrick, Thompson, Emma, Anderson, Simon, Gwenin, Vanessa and Gwenin, Chris ORCID: 0000-0003-0363-8168 (2019) Time dependent HPLC analysis of the product ratio of enzymatically reduced prodrug CB1954 by a modified and immobilised nitroreductase. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 127. pp. 217-224.

Text
HPLC Paper European Journal of Pharmaceutical Science Reviewed (003).pdf - Author Accepted Manuscript
Download (709kB) | Preview

Official URL: http://dx.doi.org/10.1016/j.ejps.2018.11.001

Abstract

Directed enzyme prodrug therapy is a chemotherapy strategy that utilises prodrug-activating enzymes to activate prodrugs at the tumour location, thus reducing off-target effects. The most commonly investigated enzyme for use with the CB1954 prodrug is the NfnB nitroreductase from E. coli. Literature states that CB1954 is reduced by NfnB at the 2- or 4-position at a 1:1 ratio; deviation from this ratio has been observed in the literature, but not further investigated. The kinetic parameters for the genetically-modified enzymes; NfnB-his, NfnB-cys and AuNP-NfnB-cys were assessed and HPLC analysis was used to determine the hydroxylamine product ratios formed when reacted with CB1954. Time-dependent HPLC studies were carried out to assess how this ratio changes over time. It was shown that the hydroxylamine ratio formed by the reduction of CB1954 by a nitroreductase changes over time and that this change in ratio relates directly to the kinetics of the reaction. Thus, the hydroxylamine ratio measured using HPLC at a given time point was not a true indication of the preference of the nitroreductase enzymes during catalysis. These results question how nitroreductases are evaluated in terms of the hydroxylamine ratio and it is suspected that this phenomenon may also apply to other enzyme/prodrug combinations.

Item Type:	Article
Uncontrolled Keywords:	HPLC, Prodrug, CB1954, Nitroreductase, DEPT
Depositing User:	Symplectic Admin
Date Deposited:	02 Nov 2020 10:17
Last Modified:	18 Jan 2023 23:25
DOI:	10.1016/j.ejps.2018.11.001
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3105517